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  • Sample Preparation
    Parasitic nematodes of the genus Syphacia Seurat, 1916 infecting Muridae in the British Isles, and the peculiar case of Syphacia frederici
    Alex Stewart, Parasitology - 2017
    SUMMARY Syphacia stroma (von Linstow, 1884) Morgan, 1932 and Syphacia frederici Roman, 1945 are oxyurid nematodes that parasitize two murid rodents, Apodemus sylvaticus and Apodemus flavicollis, on the European mainland. Only S. stroma has been recorded previously in Apodemus spp. from the British Isles. Despite the paucity of earlier reports, we identified S. frederici in four disparate British sites, two in Nottinghamshire, one each in Berkshire and Anglesey, Wales. Identification was based on their site in the host (caecum and not small intestine), on key morphological criteria that differentiate this species from S. stroma (in particular the tail of female worms) and by sequencing two genetic loci (cytochrome C oxidase 1 gene and a section of ribosomal DNA). Sequences derived from both genetic loci of putative British S. frederici isolates formed a tight clade with sequences from continental worms known to be S. frederici, clearly distinguishing these isolates from S. stroma which formed a tight clade of its own, distinct from clades representative of Syphacia obvelata from Mus and S. muris from Rattus. The data in this paper therefore constitute the first record of S. frederici from British wood mice, and confirm the status of this species as distinct from both S. obvelata and S. stroma.
  • Reverse Transcription
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    Design of Boron Nitride/Gelatin Electrospun Nanofibers for Bone Tissue Engineering
    Sakthivel Nagarajan, ACS Applied Materials & Interfaces - 2017
    Gelatin is a biodegradable biopolymer obtained by collagen denaturation, which shows poor mechanical properties. Hence, improving its mechanical properties is very essential towards the fabrication of efficient nontoxic material for biomedical applications. For this aim, various methods are employed using external fillers such as ceramics or bioglass. In this report, we introduce boron nitride (BN) reinforced gelatin as a new class of two dimensional biocompatible nanomaterials. The effect of the nanofiller on the mechanical behavior is analyzed. BN is efficiently exfoliated using the biopolymer gelatin as shown through Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The exfoliated BN reinforces gelatin electrospun fibers, which results in an increase in the Young’s modulus. The ESM are stable after the glutaraldehyde cross-linking and the fibrous morphology is preserved. The cross-linked gelatin/BN ESM is highly bioactive in forming bone like hydroxyapatite as shown by scanning electron microscopy. Due to their enhanced mineralization ability, the cross-linked ESM have been tested on human bone cells (HOS osteosarcoma cell line). The cell attachment, proliferation and biocompatibility results show that the ESM are nontoxic and biodegradable. The analysis of osteoblast gene expression and the measurement of alkaline phosphatase activity confirm that these materials are suitable for bone tissue engineering.
    Wnt7a induces a unique phenotype of monocyte-derived macrophages with lower phagocytic capacity and differential expression of pro-and anti-inflammatory cytokines
    Jennillee Wallace, Immunology - 2017
    The variation of macrophage functions suggests the involvement of multiple signaling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or “danger” signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact on monocyte differentiation is not clear. In this study, we assessed the role of Wnt1 and Wnt7a on the differentiation of monocytes and the subsequent phenotype and function of monocyte-derived macrophages (MDMs). We show that Wnt7a decreased the expression of CD14, CD11b, CD163 and CD206 whereas Wnt1 had no effect. Wnt7a effect on CD11b was also observed in the brain and spleen of Wnt7a -/- adult brain mouse tissue and in embryonic Wnt7a -/- tissue. Wnt7a reduced the phagocytic capacity of M-MDM and M1-like MDMs, decreased IL-10 and IL-12 secretion and increased IL-6 secretion. Collectively, these findings demonstrate that Wnt7a generates an MDM phenotype with both pro-inflammatory and alternative MDM cytokine profiles and reduced phagocytic capacity. As such, Wnt7a can have a significant impact on macrophage responses in health and disease. This article is protected by copyright. All rights reserved.
    An amphipathic trans -acting phosphorothioate RNA element delivers an uncharged phosphorodiamidate morpholino sequence in mdx mouse myotubes
    H.V. Jain, RSC Advances - 2017
    An efficient method for the delivery of uncharged polyA-tailed phosphorodiamidate morpholino sequences (PMO) in mammalian cells consists of employing a synthetic 8-mer amphipathic trans-acting poly-2′-O-methyluridylic thiophosphate triester element (2′-OMeUtaPS) as a transfection reagent. Unlike the dTtaPS DNA-based element, this RNA element is potent at delivering polyA-tailed PMO sequences to HeLa pLuc 705 cells or to myotube muscle cells. However, much like dTtaPS, the 2′-OMeUtaPS-mediated internalization of PMO sequences occurs through an energy-dependent mechanism; macropinocytosis appears to be the predominant endocytic pathway used for cellular uptake. The transfected PMO sequences induce alternate splicing of either the pre-mRNA encoding luciferase in HeLa pLuc 705 cells or the excision of exon 23 from the pre-mRNA encoding dystrophin in myotube muscle cells of the mdx mouse model of muscular dystrophy with an efficiency comparable to that of commercial cationic lipid reagents but without detrimental cytotoxicity.
    Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes
    Sangsu Shin, Cancer Genomics - Proteomics - 2017
    Background/Aim: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The ability of HCC to avoid immune detection is considered one of the main factors making it difficult to cure. Abnormal histone deacetylation is thought to be one of the mechanisms for HCC immune escape, making histone deacetylases (HDACs) attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, on HCC (HepG2) gene expression and function. Materials and Methods: A genome wide-transcriptional microarray was used to identify genes regulated by TSA in HepG2 cells. Gene Ontology was used to identify pathways regulated by TSA, and these changes were confirmed by qPCR. The effect of TSA on natural killer (NK) cell-mediated killing of HCC cell lines were analyzed by both flow cytometry and LDH cytotoxicity assay. A study was also conducted in a Balb/c nude mice xenograft model to assess the anti-tumor activity of TSA. Results: TSA regulated the transcription of numerous innate immunity & tumor antigen recognition-associated genes, such as ULBP1 and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell growth in an NK cell-dependent manner. In vitro, TSA treatment of HepG2 cells rendered them more susceptible to NK cell-mediated killing while increasing the expression of NKGD2 ligands, including ULBP1/2/3 and MICA/B. TSA also induced direct killing of HCC cells by stimulating apoptosis. Conclusion: TSA likely increases killing of HCC cells indirectly by increasing NK cell-directed killing and directly by increasing apoptosis.
    Helicobacter pylori infection perturbs iron homeostasis in gastric epithelial cells
    Sebastian E. Flores, PLOS ONE - 2017
    The iron deficiency anaemia that often accompanies infection with Helicobacter pylori may reflect increased uptake of iron into gastric epithelial cells. Here we show an infection-associated increase in total intracellular iron levels was associated with the redistribution of the transferrin receptor from the cell cytosol to the cell surface, and with increased levels of ferritin, an intracellular iron storage protein that corresponded with a significant increase in lysosomal stores of labile iron. In contrast, the pool of cytosolic labile iron was significantly decreased in infected cells. These changes in intracellular iron distribution were associated with the uptake and trafficking of H. pylori through the cells, and enhanced in strains capable of expressing the cagA virulence gene. We speculate that degradation of lysosomal ferritin may facilitate H. pylori pathogenesis, in addition to contributing to bacterial persistence in the human stomach.
    Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival
    Lou-Ella M. M. Alexander, Molecular Immunology - 2017
    B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt’s lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma’s with a germinal center origin.
    In silico analysis and effects of environmental salinity in the expression and activity of digestive α-amylase and trypsins from the euryhaline crab Neohelice granulata
    Antonela Asaro, Canadian Journal of Zoology - 2017
    Studies on molecular characteristics and modulation of expression of α-amylase and trypsin in the hepatopancreas of intertidal euryhaline crabs are lacking. In this work, we cloned and studied by in silico approaches the characteristics of cDNA sequences for α-amylase and two trypsins isoforms, as well as the effect of environmental salinity on gene expression and protein activities in hepatopancreas of Neohelice granulata (Dana,1852), as a good invertebrate model species. The cDNA sequence of -amylase is 1,637 bp long, encoding 459 amino acid residues. Trypsin 1 and 2 are 689 bp and 1,174 bp long, encoding 204 and 151 amino acid residues, respectively. Multiple sequence alignment of deduced protein sequences revealed the presence of conserved motifs found in other invertebrates. In crabs acclimated at 37 psu (hypo-regulation),α-amylase mRNA level and total pancreatic amylase activity were higher than at 10 psu (hyper-regulation) and 35 psu (osmoconformation). Trypsin 1 mRNA levels increased at 37 psu wh...
    Artemisinin permeability via Caco-2 cells increases after simulated digestion of Artemisia annua leaves
    Matthew R. Desrosiers, Journal of Ethnopharmacology - 2017
    Ethnopharmacological relevance Artemisia annua has been used for > 2000 yrs to treat fever and is more recently known for producing the important antimalarial drug, artemisinin. Aim of the study Artemisinin combination therapies (ACTs) are effective for treating malaria, but are often unavailable to those in need. Dried leaves of A. annua (DLA) have recently been studied as a cost effective alternative to traditional ACTs. DLA was shown to dramatically increase oral bioavailability compared to pure artemisinin, so more investigation into the mechanisms causing this increased bioavailability is needed. Materials and methods In this study, we used a simulated digestion system coupled with Caco-2 cell permeability assays to investigate the intestinal permeability of DLA compared to pure artemisinin. We also determined the effects of different phytochemicals (7 flavonoids, 3 monoterpenes, 2 phenolic acids, scopoletin and inulin) and the cytochrome P450 isoform CYP3A4 on artemisinin intestinal permeability. Results Artemisinin permeability, when delivered as digested DLA, significantly increased by 37% (Papp = 8.03 × 10−5 cm s−1) compared to pure artemisinin (Papp = 5.03 × 10−5 cm s−1). However, none of the phytochemicals tested or CYP3A4 had any significant effect on the intestinal permeability of artemisinin. We also showed that essential oil derived from A. annua negatively affected the intestinal permeability of artemisinin, but only after simulated digestion. Finally, we showed that A. annua essential oil reduced the transepithelial electrical resistance of Caco-2 monolayers, but only in the presence of bile. Although also reduced by essential oils, artemisinin Papp subsequently recovered in the presence of plant matrix. Conclusions These results shed light on the mechanisms by which DLA enhances the oral bioavailability of artemisinin.
    Chronic stress targets adult neurogenesis preferentially in the suprapyramidal blade of the rat dorsal dentate gyrus
    Nuno D. Alves, Brain Structure and Function - 2017
    The continuous generation of new neurons and glial cells in the adult hippocampal dentate gyrus (DG) represents an important form of adult neuroplasticity, involved in normal brain function and behavior but also associated with the etiopathogenesis and treatment of psychiatric disorders. Despite the large number of studies addressing cell genesis along the septotemporal axis, data on the anatomical gradients of cytogenesis along the DG transverse axis is scarce, especially after exposure to stress. As such, in this study we characterized both basal proliferation and survival of adult-born neural cells along the transverse axis of the rat dorsal DG, and after stress exposure. In basal conditions, both proliferating cells and newborn neurons and glial cells were preferentially located at the subgranular zone and suprapyramidal blade. Exposure to chronic stress induced an overall decrease in the generation of adult-born neural cells and, more specifically, produced a regional-specific decrease in the survival of adult-born neurons at the suprapyramidal blade. No particular region-specific alterations were observed on surviving adult-born glial cells. This work reveals, for the first time, a distinct survival profile of adult-born neural cells, neurons and glial cells, among the transverse axis of the DG, in both basal and stress conditions. Our results unveil that adult-born neurons are preferentially located in the suprapyramidal blade and suggest a regional-specific impact of chronic stress in this blade with potential repercussions for its functional significance.
    Assessment of Response of Kidney Tumors to Rapamycin and Atorvastatin in Tsc1+/− Mice
    Ming Hong Shen, Translational Oncology - 2017
    Atorvastatin is widely used to lower blood cholesterol and to reduce risk of cardiovascular disease–associated complications. Epidemiological investigations and preclinical studies suggest that statins such as atorvastatin have antitumor activity for various types of cancer. Tuberous sclerosis (TSC) is a tumor syndrome caused by TSC1 or TSC2 mutations that lead to aberrant activation of mTOR and tumor formation in multiple organs. Previous studies have demonstrated that atorvastatin selectively suppressed growth and proliferation of mouse Tsc2 null embryonic fibroblasts through inhibition of mTOR. However, atorvastatin alone did not reduce tumor burden in the liver and kidneys of Tsc2+/− mice as assessed by histological analysis, and no combination therapy of rapamycin and atorvastatin has been tried. In this study, we used T2-weighted magnetic resonance imaging to track changes in tumor number and size in the kidneys of a Tsc1+/− mouse model and to assess the efficacy of rapamycin and atorvastatin alone and as a combination therapy. We found that rapamycin alone or rapamycin combined with atorvastatin significantly reduced tumor burden, while atorvastatin alone did not. Combined therapy with rapamycin and atorvastatin appeared to be more effective for treating renal tumors than rapamycin alone, but the difference was not statistically significant. We conclude that combined therapy with rapamycin and atorvastatin is unlikely to provide additional benefit over rapamycin as a single agent in the treatment of Tsc-associated renal tumors.
    AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment-specific Outcome Under Abiraterone and Enzalutamide
    Anna Katharina Seitz, European Urology - 2017
    Background It has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. Objective To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients. Design, setting, and participants Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n = 56) or enzalutamide (n = 29) were analyzed via droplet digital polymerase chain reaction. Outcome measurements and statistical analysis The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% and with PSA–progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. Results and limitations High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p = 0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs 3.7 mo; p < 0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p < 0.001), and shorter OS (median 4.0 vs. 13.9 mo; p < 0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3–20.7; p < 0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1–4.9; p = 0.02), and shorter OS (HR 3.0, 95% CI 1.4–6.3; p = 0.005). Conclusions Testing of AR-V7 mRNA levels in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or enzalutamide. Patient summary We established a method for determining AR-V7 status in whole blood. This test predicted treatment resistance in patients with metastatic castration-resistant prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective validation is needed before application to clinical practice.
    Variation in the response of tomato (Solanum lycopersicum) breeding lines to the effects of benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) on systemic acquired resistance and seed germination
    Paul H. Goodwin, Journal of Phytopathology - 2017
    Genetic variation may play a major role in how plants respond to activators of systemic acquired resistance. To examine this, the defence activator benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) was applied to seed of different breeding lines of tomato (Solanum lycopersicum) with diverse pedigrees, and the levels of induced resistance against Pseudomonas syringae pv. tomato, changes in defence gene expression and detrimental effects on seed germination and seedling emergence were measured. Two breeding lines, 7007 and 7024, were selected as non-responsive and responsive to BTH. The SAR-associated genes, SlPR1a and SlPR3b, were induced earlier or more strongly over the control prior to inoculation for line 7024 but not for line 7007. This was not observed for the ISR-related genes, SlPin2 and SlPR2b. BTH inhibition of seed germination and seedling emergence was more delayed in line 7024 than 7007. However, applying BTH as a seed or soil drip reduced the delay. Thus, greater levels of BTH response have both positive (i.e., induced resistance and expression of SAR-related gene expression) and negative (i.e., inhibition of seed germination and seedling emergence) effects and can differ significantly between genotypes. Thus, recommendations for use of induced resistance activators should include plant genotype recommendations and consider possible negative impacts of greater responsiveness.
    Fibroblast Growth Factor 15 Deficiency Increases Susceptibility but does not Improves Repair to Acetaminophen-induced Liver Injury in Mice
    Mingxing Huang, Digestive and Liver Disease - 2017
    The leading cause of acute liver failure (ALF) is hepatotoxicity from acetaminophen (APAP) overdose. However, limited options are available to treat this ALF so stimulating liver regeneration maybe a potential treatment. Our previous study has shown that fibroblast growth factor 15 (FGF15) plays a crucial role in liver regeneration, but the roles of FGF15 in liver injury and repair following APAP-overdose are unknown. In this study, treatment of FGF15 knockout (KO) male mice with APAP at 200, 250, or 300 mg/kg significantly increased the degree of liver injury compared to wild type (WT) mice. To determine the effects of FGF15 deficiency on liver repair following APAP overdose, a similar degree of liver injury was first obtained 24 hrs after treatment of WT and Fgf15 KO mice with APAP at different dosage. Fgf15 KO mice did not differ from WT mice in liver repair following similar degree of liver injury. In conclusion, we showed that FGF15 deficiency renders mice more susceptible to APAP-induced liver injury but did not seem to affect liver repair or regeneration. This study suggests that in contrast to the critical role that FGF15 plays in promoting liver regeneration following partial hepatectomy, this intestine factor is less involved in liver repair after APAP-induced liver injury.
    Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors
    Julie W.Reeser, The Journal of Molecular Diagnostics - 2017
    Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan–fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events.
    Chronic traumatic stress impairs memory in mice: Potential roles of acetylcholine, neuroinflammation and corticotropin releasing factor expression in the hippocampus
    Ami Bhakta, Behavioural Brain Research - 2017
    Chronic stress in humans can result in multiple adverse psychiatric and neurobiological outcomes, including memory deficits. These adverse outcomes can be more severe if each episode of stress is very traumatic. When compared to acute or short term stress relatively little is known about the effects of chronic traumatic stress on memory and molecular changes in hippocampus, a brain area involved in memory processing. Here we studied the effects of chronic traumatic stress in mice by exposing them to adult Long Evan rats for 28 consecutive days and subsequently analyzing behavioral outcomes and the changes in the hippocampus. Results show that stressed mice developed memory deficits when assayed with radial arm maze tasks. However, chronic traumatic stress did not induce anxiety, locomotor hyperactivity or anhedonia. In the hippocampus of stressed mice interleukin-1β protein expression was increased along with decreased corticotropin releasing hormone (CRH) gene expression. Furthermore, there was a reduction in acetylcholine levels in the hippocampus of stressed mice. There were no changes in brain derived neurotrophic factor (BDNF) or nerve growth factor (NGF) levels in the hippocampus of stressed mice. Gene expression of immediate early genes (Zif268, Arc, C-Fos) as well as glucocorticoid and mineralocorticoid receptors were also not affected by chronic stress. These data demonstrate that chronic traumatic stress followed by a recovery period might lead to development of resilience resulting in the development of selected, most vulnerable behavioral alterations and molecular changes in the hippocampus.
    An inducible form of Nrf2 confers enhanced protection against acute oxidative stresses in RPE cells
    Khiem T. Vu, Experimental Eye Research - 2017
    Increasing evidence suggests that overt oxidative stress within the retina plays an important role in the progression of age-related retinal decline, and in particular, in the disease age-related macular degeneration (AMD). Nuclear factor erythroid 2-like 2 (Nrf2) is a master transcription factor that upregulates numerous of antioxidant/detoxification genes. Nrf2−/− mice develop progressive retinal degeneration that includes the formation of drusen-like deposits, lipofuscin, and sub-retinal pigment epithelium (RPE) deposition of inflammatory proteins. Furthermore, strategies that promote Nrf2 activation have shown promise for the treatment of cone/rod dystrophies and other forms of retinal degeneration. Herein we explored whether utilizing a small molecule-inducible version of Nrf2 confers additional protection against oxidative stresses when compared to a constitutively expressed version of Nrf2. Stable populations of human ARPE-19 cells were generated that express either constitutive FLAG-tagged (FT) Nrf2 (FT cNrf2) or doxycycline (dox)-inducible FT Nrf2 (FT iNrf2) at low levels (∼4.5 fold vs. endogenous). Expression of either FT cNRF2 or FT iNrf2 upregulated canonical antioxidant genes (e.g., NQO1, GCLC). Both FT cNrf2 and FT iNrf2 ARPE-19 cells were protected from cigarette smoke extract-induced nitric oxide generation to similar extents. However, only FT iNrf2 cells demonstrated enhanced resistance to doxorubicin and cumene hydroperoxide-mediated increases in mitochondrial superoxide and lipid peroxidation, respectively, and did so in a dox-dependent manner. These results suggest that therapeutic approaches which conditionally control Nrf2 activity may provide additional protection against acute oxidative stresses when compared to constitutively expressed Nrf2 strategies.
    A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction
    Ryen MacDonald, Frontiers in Aging Neuroscience - 2017
    Synaptic transmission requires intricate coordination of the components involved in processing of incoming signals, formation and stabilization of synaptic machinery, neurotransmission, and in all related signaling pathways. Changes to any of these components cause synaptic imbalance and disruption of neuronal circuitry. Extensive studies at the neuromuscular junction (NMJ) have greatly aided in the current understanding of synapses and served to elucidate the underlying physiology as well as associated adaptive and homeostatic processes. The heparansulfate proteoglycan agrin is a vital component of the NMJ, mediating synaptic formation and maintenance in both brain and muscle, but very little is known about direct control of its expression. Here, we investigated the relationship between agrin and transcription factor early growth response-1 (Egr-1), as Egr-1 regulates the expression of many genes involved in synaptic homeostasis and plasticity. Using chromatin immunoprecipitation, cell culture with cell lines derived from brain and muscle, and animal models, we show that Egr-1 binds to the AGRN gene locus and suppresses its expression. When compared with wild type (WT), mice deficient in Egr-1 (Egr-1-/-) display a marked increase in AGRN mRNA and agrin full-length and cleavage fragment protein levels, including the 22 kDa, C-terminal fragment in brain and muscle tissue homogenate. Because agrin is a crucial component of the NMJ, we explored possible physiological implications of the Egr-1-agrin relationship. In the diaphragm, Egr-1-/- mice display increased NMJ motor endplate density, individual area, and area of innervation. In addition to increased density, soleus NMJs also display an increase in fragmented and faint endplates in Egr-1-/- versus WT mice. Moreover, the soleus NMJ electrophysiology of Egr-1-/- mice revealed increased quantal content and motor testing showed decreased movement and limb muscle strength compared with WT. This study provides evidence for the potential involvement of a novel Egr-1-agrin pathway in synaptic homeostatic and compensatory mechanisms at the NMJ. Synaptic homeostasis is greatly affected by the process of aging. These and other data suggest that changes in Egr-1 expression may directly or indirectly promote age-related pathologies.
    Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases
    Rui Miao, Metabolic Engineering Communications - 2017
    Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivd without any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.
    Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke
    Ruimin Ge, Journal of Neuroinflammation - 2017
    Choroid plexus (CP) supports the entry of monocyte-derived macrophages (MDMs) to the central nervous system in animal models of traumatic brain injury, spinal cord injury, and Alzheimer’s disease. Whether the CP is involved in the recruitment of MDMs to the injured brain after ischemic stroke is unknown.
    Short-day photoperiods affect expression of genes related to dormancy and freezing tolerance in Norway spruce seedlings
    Elisabeth Wallin, Annals of Forest Science - 2017
    Key MessageGene expression analysis showed that prolonged short day (SD) treatment deepened dormancy and stimulated development of freezing tolerance ofPicea abiesseedlings. Prolonged SD treatment also caused later appearance of visible buds in autumn, reduced risks for reflushing, and promoted earlier spring bud break. ContextShort day (SD) treatment of seedlings is a common practice in boreal forest tree nurseries to regulate shoot growth and prepare the seedlings for autumn planting or frozen storage. AimsThe aim of this study was to examine responses of Norway spruce (Picea abies (L.) Karst.) to a range of SD treatments of different length and evaluate gene expression related to dormancy induction and development of freezing tolerance. MethodsThe seedlings were SD treated for 11 h a day during 7, 14, 21, or 28 days. Molecular tests were performed, and the expression profiles of dormancy and freezing tolerance-related genes were analyzed as well as determination of shoot growth, bud set, bud size, reflushing, dry matter content, and timing of spring bud break. ResultsThe 7-day SD treatment was as effective as longer SD treatments in terminating apical shoot growth. However, short (7 days) SD treatment resulted in later activation of dormancy-related genes and of genes related to freezing tolerance compared to the longer treatments which had an impact on seedling phenology. ConclusionGene expression analysis indicated an effective stimulus of dormancy-related genes when the SD treatment is prolonged for at least 1–2 weeks after shoot elongation has terminated and that seedlings thereafter are exposed to ambient outdoor climate conditions.
    RNAi screen identifies essential regulators of human brain metastasis-initiating cells
    Mohini Singh, Acta Neuropathologica - 2017
    Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein–protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.
    miR-130b directly targets Arhgap1 to drive activation of a metastatic CDC42-PAK1-AP1 positive feedback loop in Ewing sarcoma
    Laura Satterfield, International Journal of Cancer - 2017
    Ewing Sarcoma (ES) is a highly aggressive bone tumor with peak incidence in the adolescent population. It has a high propensity to metastasize, which is associated with dismal survival rates of approximately 25%. To further understand mechanisms of metastasis we investigated microRNA regulatory networks in ES. Our studies focused on miR-130b due to our analysis that enhanced expression of this microRNA has clinical relevance in multiple sarcomas, including ES. Our studies provide insights into a novel positive feedback network involving the direct regulation of miR-130b and activation of downstream signaling events contributing towards sarcoma metastasis. Specifically, we demonstrated miR-130b induces proliferation, invasion, and migration in vitro and increased metastatic potential in vivo. Using microarray analysis of ES cells with differential miR-130b expression we identified alterations in downstream signaling cascades including activation of the CDC42 pathway. We identified Arhgap1, which is a negative regulator of CDC42, as a novel, direct target of miR-130b. In turn, downstream activation of PAK1 activated the JNK and AP-1 cascades and downstream transcriptional targets including IL-8, MMP1 and CCND1. Furthermore, chromatin immunoprecipitation of endogenous AP-1 in ES cells demonstrated direct binding to an upstream consensus binding site within the miR-130b promoter. Finally, small molecule inhibition of PAK1 blocked miR-130b activation of JNK and downstream AP-1 target genes, including primary miR-130b transcripts, and mir-130b oncogenic properties, thus identifying PAK1 as a novel therapeutic target for ES. Taken together, our findings identify and characterize a novel, targetable miR-130b regulatory network that promotes ES metastasis. This article is protected by copyright. All rights reserved.
    Hepatitis C virus NS5A region mutation in chronic hepatitis C genotype 1 patients who are non-responders to two or more treatments and its relationship with response to a new treatment
    Paloma Muñoz de Rueda, World Journal of Gastroenterology - 2017
    AIM To determine the number of mutations in the NS5A region of the hepatitis C virus (HCV) and its relationship to the response to antiviral therapy in patients with chronic hepatitis C genotype 1 who are non-responders to two or more treatments. METHODS Sequences within HCV NS5A [PKR binding domain (PKRBD) and the interferon-sensitivity-determining region (ISDR)] were analysed via direct sequencing in a selected cohort of 72 patients, with a total of 201 treatments [interferon-alpha (IFN-α), n = 49; IFN-α + ribavirin (RBV), n = 75; pegylated (peg) IFN-α + RBV, n = 47; first-generation direct-acting antivirals (DAAs), n = 13; and second-generation DAAs, n = 17]. Of these, 48/201 achieved a sustained virological response (SVR) and 153/201 achieved no virological response (NVR). RESULTS For both regions, treatments resulting in SVR were associated with more baseline mutations than were treatments resulting in NVR (SVR vs NVR; PKRBD: 5.82 ± 3 vs 4.86 ± 2 mutations, P = 0.045; ISDR: 2.65 ± 2 vs 1.51 ± 1.7 mutations, P = 0.005). A decrease or no change in the number of mutations over time between treatments in the PKRBD or ISDR, as shown by sequencing, was associated with patients who usually failed to respond to treatment (PKRBD, P = 0.02; ISDR, P = 0.001). Moreover, patients showing a post-treatment baseline viral load > 600000 IU/mL and increased ISDR mutations with respect to the previous treatment were 9.21 times more likely to achieve SVR (P = 0.001). CONCLUSION The obtained results show that among patients who have shown no response to two or more antiviral treatments, the likelihood of achieving SVR increases with the genetic variability in the ISDR region (≥ 2 mutations or number of substitutions from the HCV-J and HCV-1 prototype), especially when the viral load is greater than 600000 IU/mL.
    The roles of NF-κB and ROS in regulation of pro-inflammatory mediators of inflammation induction in LPS-stimulated zebrafish embryos
    Eun-Yi Ko, Fish & Shellfish Immunology - 2017
    In this study, the roles of reactive oxygen species (ROS) and NF-κB on inflammation induction in lipopolysaccharide (LPS)-stimulated zebrafish embryos were evaluated using N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), specific inhibitors of ROS and NF-κB, respectively. LPS-stimulated zebrafish embryos showed increasing production of NO and ROS and expression of iNOS and COX-2 protein, compared to a control group without LPS. However, NAC significantly inhibited production of NO and ROS and markedly suppressed expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. The mRNA expressions of NF-κB such as p65NF-κB and IκB-A were significantly increased after LPS stimulation, whereas PDTC attenuated mRNA expression of NF-κB. IκB was suppressed by PDTC, but not significantly. PDTC also inhibited production of NO and reduced expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. Taken together, these results indicated that LPS increases pro-inflammatory mediators in zebrafish embryos through ROS and NF-κB regulation.
    Alternative strategy for visceral leishmaniosis control: HisAK70-Salmonella Choleraesuis-pulsed Dendritic Cells
    Gustavo Domínguez-Bernal, Comparative Immunology, Microbiology and Infectious Diseases - 2017
    Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.
    Next Generation Sequencing to characterise the breaking of bud dormancy using a natural biostimulant in kiwifruit (Actinidia deliciosa)
    Frank A. Hoeberichts, Scientia Horticulturae - 2017
    Dormancy occurs in many woody perennials and fruit trees, and is characterised mainly by the temporal absence of growth. This strategy has an ecological role in ensuring plant survival under non-permissive growing conditions. In kiwifruit (Actinidia deliciosa), buds must be exposed to a certain amount of chilling to complete dormancy and achieve optimal bud break and flowering in spring. However, in warmer geographical areas bud break can be limited by insufficient winter chilling, leading to less and uneven flowering and lower productivity. Several bud break-enhancing compounds have been shown to be effective in kiwifruit, overcoming the negative effects of poor winter chilling. However, their mode of action is still largely unknown. In this work, using a molecular approach based on Next Generation Sequencing, we characterised dormancy and its breaking after the application of the biostimulant Erger®. Samples were collected at different time-points and in distinct geographical areas, namely Italy and New Zealand. Our results indicated that the effect of Erger® depends on the moment of application. Furthermore, molecular mechanisms that characterise a biostimulant treatment for bud break, irrespective of the region and seasonality, were identified. This characterisation opens new perspectives to improve bud break-enhancing compound formulations and identify their best moment of application.
    Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains
    Claudia Vuotto, Journal of Applied Microbiology - 2017
    Aims Multi-drug resistant Klebsiella pneumoniae has become a relevant healthcare-associated pathogen. Capsule, type 1 and 3 fimbriae (mrkA gene), type 2 quorum-sensing system (luxS), synthesis of D-galactan I (wbbM), LPS transport (wzm) and poly-beta-1,6-N-acetyl-D-glucosamine (pgaA) seem involved in K. pneumoniae biofilm. Non-enzymatic antibiotic resistance is related to non-expression or mutation of porins (OmpK35 and OmpK36), and efflux pump (acrB) over-expression. The aim of this study was to analyse some virulence factors of K. pneumoniae isolates, and to evaluate possible correlations between their antibiotic resistance profile and ability to form biofilm. Methods and Results Quantitative biofilm production assay, congo red agar test and string test were performed on 120 isolates clustered in 56 extensively drug resistant (XDR, 40 MDR and 24 susceptible (S)). Nine representative strains were analyzed by real-time RT-PCR for the expression of antibiotic resistance (OmpK35, OmpK36, acrB) and biofilm production genes (mrkA, luxS,, pga, wbbM, wzm) during planktonic and sessile growth. XDR isolates showed a higher ability to form biofilm (91.07%) and to produce polysaccharides (78.57%) when compared to MDR and S strains. In biofilm-growing XDR strains, 7 out of 8 genes were upregulated, with the only exception of OmpK36. Conclusions XDR strains exhibited phenotypic and genotypic features supporting a significant growth as biofilm. Significance and Impact of Study this study produces new findings that highlight a positive correlation between antibiotic resistance profile and biofilm-forming ability in XDR K. pneumoniae strains. These new evidences might contribute to the progress in selection of therapeutic treatments of infections caused by Klebsiella pneumoniae resistant also to the “last line of defense” antibiotics, i.e. carbapenems. This article is protected by copyright. All rights reserved.
    The receptor tyrosine kinase AXL promotes migration and invasion in colorectal cancer
    Diana J. Uribe, - 2017
    The receptor tyrosine kinases (RTKs) TYRO3, AXL and MERTK (TAM) have well-described oncogenic functions in a number of cancers. Notwithstanding, TAM RTKs are also potent and indispensable inhibitors of inflammation. The combined deletion of Axl and Mertk in mice enhances chronic inflammation and autoimmunity, including increased inflammation in the gut and colitis-associated cancer. On the other hand, deletion of Tyro3 increases the risk of allergic responses. Therefore, the indiscriminate inhibition of these TAM RTKs could result in undesirable immunological diseases. Here we show that AXL, but not MERTK or TYRO3 expression is enhanced in late stage colorectal cancer (CRC) and AXL expression associates with a cell migration gene signature. Silencing AXL or the inhibition of AXL kinase activity significantly inhibits tumor cell migration and invasion. These results indicate that the selective inhibition of AXL alone might confer sufficient therapeutic benefit in CRC, while preserving at least some of the beneficial, anti-inflammatory effects of MERTK and TYRO3 RTKs.
    Cytokine profiling in the prefrontal cortex of Parkinson's Disease and Multiple System Atrophy patients
    Rasmus Rydbirk, Neurobiology of Disease - 2017
    Parkinson's Disease (PD) and Multiple System Atrophy (MSA) are neurodegenerative diseases characterized neuropathologically by alpha-synuclein accumulation in brain cells. This accumulation is hypothesized to contribute to constitutive neuroinflammation, and to participate in the neurodegeneration. Cytokines, which are the main inflammatory signalling molecules, have been identified in blood and cerebrospinal fluid of PD patients, but studies investigating the human brain levels are scarce. It is documented that neurotrophins, necessary for survival of brain cells and known to interact with cytokines, are altered in the basal ganglia of PD patients. In regards to MSA, no major study has investigated brain cytokine or neurotrophin protein expression. Here, we measured protein levels of 18 cytokines (IL-2, 4–8, 10, 12, 13, 17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and 1β, TNF-α) and 5 neurotrophins (BDNF, GDNF, bFGF, PDGF-BB, VEGF) in the dorsomedial prefrontal cortex in brains of MSA and PD patients and control subjects. We found altered expression of IL-2, IL-13, and G-CSF, but no differences in neurotrophin levels. Further, in MSA patients we identified increased mRNA levels of GSK3β that is involved in neuroinflammatory pathways. Lastly, we identified increased expression of the neurodegenerative marker S100B, but not CRP, in PD and MSA patients, indicating local rather than systemic inflammation. Supporting this, in both diseases we observed increased MHC class II+ and CD45+ positive cells, and low numbers of infiltrating CD3+ cells. In conclusion, we identified neuroinflammatory responses in PD and MSA which seems more widespread in the brain than neurotrophic changes.
    Effect of dietary polyphenols on fructose uptake by human intestinal epithelial (Caco-2) cells
    Nelson Andrade, Journal of Functional Foods - 2017
    Intake of high-fructose products is associated with metabolic syndrome development. We investigated if dietary polyphenols can interfere with fructose absorption by the human intestinal epithelial Caco-2 cell line. Acutely, several polyphenols induced a significant decrease (15–20%) in 14C-fructose uptake. Chronically, 14C-fructose uptake was also affected by polyphenols; quercetin, chrysin and apigenin (100 µM) were the most effective (±25% decrease). These compounds (100 µM) appear to interfere with both GLUT2 and GLUT5 activity; accordingly, they caused a very marked decrease in the mRNA expression levels of GLUT2 (≅90%) and GLUT5 (≅75%). The effect of these compounds on 14C-fructose uptake does not involve interference with PKC, PKA, PI3K and p38 MAPK intracellular signaling pathways. Quercetin, apigenin and chrysin might decrease the intestinal absorption of fructose. Further studies are needed to test for their beneficial effects on metabolic syndrome.
    Mdm2 selectively suppresses DNA damage arising from inhibition of topoisomerase II independent of p53
    Senturk J.C., Oncogene - 2017
    Mdm2 is often overexpressed in tumors that retain wild-type TP53 but may affect therapeutic response independently of p53. Herein is shown that tumor cells with MDM2 amplification are selectively resistant to treatment with topoisomerase II poisons but not other DNA damaging agents. Tumor cells that overexpress Mdm2 have reduced DNA double-strand breaks in response to doxorubicin or etoposide. This latter result is not due to altered drug uptake. The selective attenuation of DNA damage in response to these agents is dependent on both Mdm2 levels and an intact ubiquitin ligase function. These findings reveal a novel, p53-independent activity of Mdm2 and have important implications for the choice of chemotherapeutic agents in the treatment of Mdm2-overexpressing tumors.
    The Absence of Laminin Alpha 4 in Male Mice Results in Enhanced Energy Expenditure and Increased Beige Subcutaneous Adipose Tissue
    Marcella K. Vaicik, Endocrinology - 2017
    Laminin alpha 4 (LAMA4) is located in the extracellular basement membrane that surrounds each individual adipocyte. Here we show that LAMA4 null (Lama4-/-) mice exhibit significantly higher energy expenditure relative to wild type mice at room temperature and when exposed to a cold challenge, despite similar levels of food intake and locomotor activity. The Lama4-/- mice are resistant to age- and diet-induced obesity. Expression of uncoupling protein 1 (UCP1) is higher in subcutaneous white adipose tissue (sWAT) of Lama4-/- mice relative to wild type animals on either a chow diet or a high-fat diet. In constrast, UCP1 expression was not increased in brown adipose tissue (BAT). Lama4-/- mice exhibit significantly improved insulin sensitivity when compared to WT mice, suggesting improved metabolic function. Overall, these data provide critical evidence for a role of the basement membrane in energy expenditure, weight gain, and systemic insulin sensitivity.
    Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells
    Heather L. Caslin, Cellular Immunology - 2017
    While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.
    Cellular zinc homeostasis modulates polarization of THP-1-derived macrophages
    Laura Dierichs, European Journal of Nutrition - 2017
    PurposePolarization of macrophages by environmental stimuli leads to the characteristic of different phenotypes that exhibit distinct functions, ranging in a continuous spectrum from pro-inflammatory M1 up to immunoregulatory and wound-healing M2 macrophages. Diseases like cancer, allergic asthma or diabetes are associated with an M1/M2 imbalance. Owing to the importance of the essential trace element zinc for the immune system and its involvement in signal transduction as a second messenger, we investigated the impact of zinc on M1 and M2 polarization of macrophages in vitro.MethodsA polarization model with human THP-1 cells was established and validated with previously described markers using quantitative real-time PCR, Western blot and flow cytometry. Intracellular free Zn2+ was determined with FluoZin-3-AM.ResultsWhereas pSTAT1 and HLA-DR or pSTAT6 and Dectin-1 distinguish between M1 and M2 macrophages, respectively, CD86 and CD206 failed. Depending on the used markers, both zinc supplementation in physiological dose (50 µM) and zinc deficiency promote M1 polarization of THP-1-derived macrophages. Furthermore, zinc supplementation strongly inhibits M2 polarization.ConclusionFor the first time, we show a modulating effect of zinc for the polarization of human macrophages. The strong inhibitory effect of zinc supplementation on M2 polarization indicates a relevance regarding M2-dominated diseases like allergic asthma or cancer. All in all, zinc achieves a great potential for modulating macrophage polarization.
    Maternal Antiviral Immunoglobulin Accumulates in Neural Tissue of Neonates To Prevent HSV Neurological Disease
    Yike Jiang, mBio - 2017
    While antibody responses to neurovirulent pathogens are critical for clearance, the extent to which antibodies access the nervous system to ameliorate infection is poorly understood. In this study on herpes simplex virus 1 (HSV-1), we demonstrate that HSV-specific antibodies are present during HSV-1 latency in the nervous systems of both mice and humans. We show that antibody-secreting cells entered the trigeminal ganglion (TG), a key site of HSV infection, and persisted long after the establishment of latent infection. We also demonstrate the ability of passively administered IgG to enter the TG independently of infection, showing that the naive TG is accessible to antibodies. The translational implication of this finding is that human fetal neural tissue could contain HSV-specific maternally derived antibodies. Exploring this possibility, we observed HSV-specific IgG in HSV DNA-negative human fetal TG, suggesting passive transfer of maternal immunity into the prenatal nervous system. To further investigate the role of maternal antibodies in the neonatal nervous system, we established a murine model to demonstrate that maternal IgG can access and persist in neonatal TG. This maternal antibody not only prevented disseminated infection but also completely protected the neonate from neurological disease and death following HSV challenge. Maternal antibodies therefore have a potent protective role in the neonatal nervous system against HSV infection. These findings strongly support the concept that prevention of prenatal and neonatal neurotropic infections can be achieved through maternal immunization. IMPORTANCE Herpes simplex virus 1 is a common infection of the nervous system that causes devastating neonatal disease. Using mouse and human tissue, we discovered that antiviral antibodies accumulate in neural tissue after HSV-1 infection in adults. Similarly, these antibodies pass to the offspring during pregnancy. We found that antiviral maternal antibodies can readily access neural tissue of the fetus and neonate. These maternal antibodies then protect neonatal mice against HSV-1 neurological infection and death. These results underscore the previously unappreciated role of maternal antibodies in protecting fetal and newborn nervous systems against infection. These data suggest that maternal immunization would be efficacious at preventing fetal/neonatal neurological infections.
    Polyphenols and tri-terpenoids from Olea europaea L. in alleviation of enteric pathogen infections through limiting bacterial virulence and attenuating inflammation
    Mengfei Peng, Journal of Functional Foods - 2017
    Olea europaea L. was known associated with health benefits, however the role of single bioactive component remains to be elucidated. This study was to investigate the operational feasibility of maslinic/oleanolic acid, hydroxytyrosol, and oleuropein from olive in control and alleviation of enteric pathogen infections. Our results indicated that the growth of Lactobacillus was stimulated by maslinic/oleanolic acid up to 4% (w/v) in dose-dependent manner. Hydroxytyrosol (>0.05%) and oleuropein (>0.5%) significantly reduced Salmonella Typhimurium/EHEC within 12 h, altered their physicochemical properties, influenced host cell-pathogen interactions, and especially 0.6–6.0 folds down-regulated the virulence gene expressions. Further molecular analysis revealed that these olive compounds up-regulated anti-inflammatory cytokine IL-10 gene by 3.0–8.0 folds and down-regulated multiple inflammation related genes at various levels. These results suggest that polyphenols in olive may serve as potential preservatives in post-harvest food processing while tri-terpenoids may assist in modulation of gut microflora and reduction of enteric pathogen colonization.
    Bone marrow mesenchymal stem cell-derived CD63+ exosomes transport Wnt3a exteriorly and enhance dermal fibroblast proliferation, migration and angiogenesis in vitro
    Jeffrey D. McBride, Stem Cells and Development - 2017
    Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies, and found for the first time, that Wnt3a protein is detectable exteriorly on CD63+ exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggests that CD63+ exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63+ exosomes, however, polyethylene glycol-mediated exosome-enrichment prior to exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, as well as endothelial angiogenesis in vitro, was significantly decreased after CD63+-exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63+ exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration and angiogenesis in vitro.
    Dual loss of succinate dehydrogenase (SDH) and complex I activity is necessary to recapitulate the metabolic phenotype of SDH mutant tumors
    Doriane Lorendeau, ResearchGate - 2016
    Mutations in succinate dehydrogenase (SDH) are associated with tumor development and neurodegenerative diseases. Only in tumors, loss of SDH activity is accompanied with the loss of complex I...
    ROS mediates interferon gamma induced phosphorylation of Src, through the Raf/ERK pathway, in MCF-7 human breast cancer cell line
    Kazem Zibara, Journal of Cell Communication and Signaling - 2016
    Interferon gamma (IFN-ɣ) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-ɣ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-ɣ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-ɣ, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-ɣ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-ɣ resulted in ROS generation, through H2O2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-ɣ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.
    Insights into a novel nuclear function for Fascin in the regulation of the amino-acid transporter SLC3A2
    Amine Saad, Scientific Reports - 2016
    Fascin 1 (FSCN1) is a cytoskeleton-associated protein recognized to function primarily in the regulation of cytoskeleton structure and formation of plasma membrane protrusions.
    MicroRNA-126 overexpression rescues diabetes-induced impairment in efferocytosis of apoptotic cardiomyocytes
    Sahana Suresh Babu, Scientific Reports - 2016
    Efferocytosis, a process of clearance of apoptotic cells by phagocytes, is essential for successful resolution of inflammation and maintenance of tissue homeostasis.
    A Ketogenic Diet in Rodents Elicits Improved Mitochondrial Adaptations in Response to Resistance Exercise Training Compared to an Isocaloric Western Diet
    Hayden W. Hyatt, Frontiers in Physiology - 2016
    Purpose: Ketogenic diets (KD) can facilitate weight loss, but their effects on skeletal muscle remain equivocal. In this experiment we investigated the effects of two diets on skeletal muscle mitochondrial coupling, mitochondrial complex activity, markers of oxidative stress, and gene expression in sedentary and resistance exercised rats. Methods: Male Sprague-Dawley rats (9-10 weeks of age, 300-325 g) were fed isocaloric amounts of either a KD (17 g/day, 5.2 kcal/g, 20.2% protein, 10.3% CHO, 69.5% fat, n=16) or a Western diet (WD) (20 g/day, 4.5 kcal/g, 15.2% protein, 42.7% CHO, 42.0% fat, n=16) for 6 weeks. During these six weeks animals were either sedentary (SED, n=8 per diet group) or voluntarily exercised using resistance-loaded running wheels (EXE, n=8 per diet group). Gastrocnemius was excised and used for mitochondrial isolation and biochemical analyses. RESULTS: In the presence of a complex II substrate, the respiratory control ratio (RCR) of isolated gastrocnemius mitochondria was higher (p<0.05) in animals fed the KD compared to animals fed the WD. Complex I and IV enzyme activity was higher (p<0.05) in EXE animals regardless of diet. SOD2 protein levels and GLUT4 and PGC1α mRNA expression were higher (p<0.05) in EXE animals regardless of diet. CONCLUSION: Our data indicate that skeletal muscle mitochondrial coupling of complex II substrates is more efficient in chronically resistance trained rodents fed a KD. These findings may provide merit for further investigation, perhaps on humans.
    Disinhibition of somatostatin-positive GABAergic interneurons results in an anxiolytic and antidepressant-like brain state
    T. Fuchs, Molecular Psychiatry - 2016
    Major depressive disorder (MDD) is associated with reduced concentrations of γ-aminobutyric acid (GABA) that are normalized by antidepressant therapies. Moreover, depressive-like phenotypes of GABAA receptor mutant mice can be reversed by treatment with conventional antidepressants drugs, as well as by subanesthetic doses of ketamine. Thus GABAergic deficits may causally contribute to depressive disorders, while antidepressant therapies may enhance GABAergic synaptic transmission. Here we tested the hypothesis that sustained enhancement of GABAergic transmission alone is sufficient to elicit antidepressant-like behavior, using disinhibition of GABAergic interneurons. We focused on somatostatin-positive (SST+) GABAergic interneurons because of evidence that their function is compromised in MDD. To disinhibit SST+ interneurons, we inactivated the γ2 subunit gene of GABAA receptors selectively in these neurons (SSTCre:γ2f/f mice). Loss of inhibitory synaptic input resulted in increased excitability of SST+ interneurons. In turn, pyramidal cell targets of SST+ neurons showed an increased frequency of spontaneous inhibitory postsynaptic currents. The behavior of SSTCre:γ2f/f mice mimicked the effects of anxiolytic and antidepressant drugs in a number of behavioral tests, without affecting performance in a spatial learning- and memory-dependent task. Finally, brain extracts of SSTCre:γ2f/f mice showed decreased phosphorylation of the eukaryotic elongation factor eEF2, reminiscent of the effects of ketamine. Importantly, these effects occurred without altered activity of the mammalian target of rapamycin pathway nor did they involve altered expression of SST. However, they were associated with reduced Ca2+/calmodulin-dependent auto-phosphorylation of eEF2 kinase, which controls the activity of eEF2 as its single target. Thus enhancing GABAergic inhibitory synaptic inputs from SST+ interneurons to pyramidal cells and corresponding chronic reductions in the synaptic excitation:inhibition ratio represents a novel strategy for antidepressant therapies that reproduces behavioral and biochemical end points of rapidly acting antidepressants.
    A miR-200c/141-BMI1 autoregulatory loop regulates oncogenic activity of BMI1 in cancer cells.
    Manjari Dimri, George Washington University research paper - 2016
    MicroRNAs (miRNAs) are known to function as oncomiRs or tumor suppressors and are important noncoding RNA regulators of oncogenesis. The miR-200c/141 locus on chromosome 12 encodes miR-200c and miR-141, two members of the miR-200 family, which have been shown to function as tumor suppressive miRNAs by targeting multiple oncogenic factors such as polycomb group protein BMI1. Here, we show that BMI1 reciprocally functions as a transcriptional repressor of the miR-200c/141 cluster and that BMI1 inhibitors upregulate expression of miR-200c and miR-141. Our data suggest that BMI1 binds to the miR-200c/141 promoter and regulates it through transcription factor binding motifs E-box 2 and Z-box 1 to repress expression of miR-200c/141 cluster. We also show that PTC-209, a small molecule inhibitor of BMI1 gene expression induces cellular senescence and transcriptionally upregulates expression of miR-200c/141 cluster in breast cancer cells. Furthermore, inhibition of expression of miR-200c or miR-141 overcomes tumor suppressive effects of PTC-209 including induction of cellular senescence and downregulation of breast cancer stem cell phenotype. Therefore, our studies suggest a reciprocal regulation between BMI1 and miR-200c/141 cluster, and that BMI1 inhibitory drugs can further amplify their inhibitory effects on BMI1 via multiple mechanisms including posttranscriptional regulation by upregulating BMI1 targeting miRNAs
    EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models
    Lilian Saryeddine, PLOS ONE - 2016
    EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.
    Relationship between FGF21 and UCP1 levels under time-restricted feeding and high-fat diet
    Nava Chapnik, Journal of Nutritional Biochemistry - 2016
    FGF21 (fibroblast growth factor 21) exhibits a circadian oscillation and its induction is critical during fasting. When secreted by liver and skeletal muscle, FGF21 enhances thermogenic activity in brown adipose tissue (BAT) by utilizing uncoupling protein 1 (UCP1) to dissipate energy as heat. Recently, it has been reported that UCP1 is not required for FGF21-mediated reduction in body weight or improvements in glucose homeostasis. As the relationship between FGF21 and UCP1 induction in tissues other than BAT is less clear, we tested the effect of restricted feeding (RF) and high dietary fat on FGF21 circadian expression and its correlation with UCP1 expression in liver and white adipose tissue (WAT).
    TSAP6 is a novel candidate marker of poor survival in metastatic high-grade serous carcinoma
    Esther Channah Broner, Human Pathology - 2016
    The objective of this study was to analyze the expression and clinical role of molecules involved in exosome synthesis and secretion in high-grade serous carcinoma (HGSC), with focus on malignant effusions. The mRNA expression levels of ARF6, nSMase2, TSAP6, Rab27a and Rab27b by qRT-PCR were analyzed in 103 HGSC effusions and 65 solid specimens (35 ovarian, 30 abdominal metastases). Protein expression of ARF6, nSMase2, TSAP6 and Rab27a by Western blotting was analyzed in 150 specimens (94 effusions, 29 ovarian carcinomas, 27 solid metastases). Secreted ARF6, nSMase2 and Rab27a protein levels in exosomes were analyzed in supernatants from 75 effusions. Expression levels were analyzed for association with anatomic site and clinical parameters, including survival. nSMase2 and TSAP6 mRNA was overexpressed in effusions compared to solid lesions (P < .001 and P = .003, respectively), whereas ARF6, nSMase2, TSAP6 and Rab27a protein was overexpressed in solid specimens (ovarian and peritoneal) compared to effusions (P < .001 for all). Secreted ARF6, nSMase2 and Rab27a levels were found in all effusion supernatants. In univariate survival analysis, higher TSAP6 protein levels in effusions were associated with shorter overall survival (P = .01), a finding which was reproduced in analysis of specimens from patients with pre-chemotherapy effusions tapped at diagnosis (P = .026). Higher levels of exosomal Rab27a protein were significantly related to longer overall survival (P = .025). Molecules which are part of the exosome secretion machinery are differentially expressed in HGSC effusions and solid lesions and are present in effusion supernatant-derived exosomes. TSAP6 and Rab27a may be novel prognostic markers in metastatic HGSC.
    Impact of oral and waterborne administration of rhamnolipids on the susceptibility of channel catfish (Ictalurus punctatus) to Flavobacterium columnare infection
    Dongdong Zhang, Fish & Shellfish Immunology - 2016
    Flavobacterium columnare is the causative agent of columnaris disease and causes tremendous morbidity and mortality of farmed fish globally. Previously, we identified a potential lectin-mediator (a rhamnose-binding lectin; RBL1a) of F. columnare adhesion and showed higher RBL1a expression in susceptible channel catfish under basal conditions and following infection. Exposure of challenged fish to the carbohydrate ligand L-rhamnose just prior to a challenge substantially decreased columnaris mortality and pathogen adherence via the down-regulation of RBL1a. While highly effective in protecting fish from columnaris, L-rhamnose is prohibitively expensive, underscoring the need for alternative cost-effective sources of rhamnose for disease control. One such alternative may be microbially produced glycolipid compounds termed rhamnolipids (RLs), which feature abundant L-rhamnose moieties and are readily available from commercial sources. In the present study, we examined whether commercially available RLs (administered either by immersion or via feed) would function similarly to L-rhamnose in affording host protection against F. columnare. A four-week feeding trial with basal and RL top-coated diets (basal diet + RLs) was conducted in channel catfish fingerlings. Surprisingly, columnaris challenges revealed significantly lower survival following the 10 d challenge period in RL diet fed fish when compared with the basal treatment group (p < 0.001). In fish fed RLs, we observed a rapid and large-scale upregulation of RBL1a immediately after challenge combined with a suppression of mucin and lysozyme transcripts. Similarly, fish that were briefly pre-exposed to RLs by immersion and then challenged exhibited lower survival as compared to unexposed fish during a 4 d trial. In conclusion, RLs do not represent an alternative to rhamnose as an experimental treatment for protecting catfish from columnaris mortality. Further research is needed to find other affordable and efficacious alternative sources of L-rhamnose.
    Carmen Naturil Alfonso, Thesis UNIVERSITAT POLITÈCNICA DE VALÈNCIA - 2016
    The general aim of this thesis was to reproductively characterize females from a rabbit line selected for growth rate (line R) and to evaluate the effect of different nutritional strategies (ad libitum and restricted before reproduction), in order to improve the reproductive performance of these females.In chapter 1, the influence of maternal and embryonic genotype on prenatal survival and fetal growth over gestation was evaluated and contrasted with a maternal line. Prenatal survival, fetal weight and fetal placenta weight were affected by both embryonic and maternal genotype. Firstly, embryonic genotype was a main factor at Days 14 and 24 and maternal genotype contributed at Day 30 of prenatal survival. Secondly, differences in fetal weight were only manifested at Day 14, the embryonic/maternal genotype from the maternal line being the heaviest (0.29±0.01 g vs. 0.19±0.01 g, for line R genotypes). However, while for fetal placenta weight both genotypes showed an effect at Day 24, for maternal placenta weight they were relevant at Day 30. Nevertheless, no differences were detected either at transcriptomic level in fetal placenta or in progesterone and IGF I plasma levels in these females. From this chapter it may be concluded that in rabbit females from paternal lines both embryonic and maternal genotypes are key factors in the reproductive performance of these females.The aim of chapter 2 was to explore the causes of ovulation failures in these rabbit females. Results showed that non ovulated females presented lower LH plasma concentration as well as higher body weight and leptin and BOHB II plasma levels than ovulated females. Thus, ovulation failures in females from line R could be attributed to decreased LH plasma concentrations in these females which may be related with their higher body weight and leptin levels. In the light of the previous results, the following three chapters were focused on improvement of the reproductive performance of these females through a different nutritional strategy: a feed to appetite diet of these females after the rearing period and prior to insemination. The initial hypothesis was that the females are submitted to a restricted nutritional regimen which is not enough to cope with their needs during reproduction, causing long term disturbances of energy balance which leads to the subsequent reproductive problems. Chapter 3 aimed to determine if a feed-to-appetite nutritional strategy would affect the hypothalamus-hypophysis axis and the quality of the produced oocytes, by transcriptomic analysis. While no differences were found in the microarray analysis of the hypothalamus hypophysis, small differences were detected in the transcript expression analysis in oocytes of a group of genes selected. MSY2 was found to be downregulated in oocytes from restricted females. As a key regulator of maternal RNA transcription and translation, changes in this essential gene could explain some of the reproductive problems of these females with high growth potential. Whether the differences found at oocyte level were inherent at embryonic level and so involved in the drop of fertility found in these females was studied in Chapter 4. Although no significant differences were revealed in ovulation, embryo recovery, and implantation rate, higher fetal and gestational losses were found in restricted females, as well as lower fetal growth. Thus, we concluded that the nutritional strategy employed may have an impact on the III oocyte (Chapter 3), but we also demonstrated that these changes were inherited by the embryo, and result in disturbances in gestational losses and fetal growth.For this reason, the final chapter of this thesis was conducted to determine whether these effects on reproductive and metabolic elements were also evident in females following the common semi-intensive farm production system. Although the results obtained showed small variances in NEFAs and BOHB plasma levels, and also in body weight,no differences were detected in global reproductive performance in terms of fertility, prolificacy and productivity. The results obtained established that although differences are found at oocyte level and inherited by embryo and fetus,no improvements are reached with the proposed nutritional strategy in terms of reproductive performance when females selected for growth rate lead a normal semi intensive production system
    Understanding Tamoxifen Resistance In Breast cancer
    Raie Taye Bekele, Department of Biochemistry University of Alberta Thesis - 2016
    Tamoxifen is the accepted therapy for patients with estrogen receptor α (ERα)positive breast cancer. However, clinical resistance to tamoxifen, as demonstrated by recurrence or progression on therapy, is frequent and precedes death from metastases. To improve breast cancer treatment it is vital to understand the mechanisms that result in tamoxifen resistance. The study presented in this thesis shows that concentration of tamoxifen and its metabolites, which accumulate in tumors of patients, killed breast cancer cells by inducing oxidative stress. Breast cancer cells responded to tamoxifen induced oxidation by increasing Nrf2 expression and subsequent activation of the anti-oxidant response element (ARE). This increased the transcription of anti-oxidant genes and multidrug resistance transporters. As a result, breast cancer cells are able to destroy or export toxic oxidation products leading to increased survival from tamoxifen-induced oxidative damage. These responses in cancer cells also occur in breast tumors of tamoxifen treated mice. Additionally, high levels of expression of Nrf2 and its downstream targets in breast tumors of patients at the time of diagnosis were prognostic of poor survival after tamoxifen therapy. The oxidative stress induced by tamoxifen also activated phospholipase D (PLD) and led to the up regulation of the RALBP1 (Ral-binding protein 1). Tamoxifen resistant cells also had a significant increase in both basal and stimulated PLD activity along with increased PLD1 and RALBP1 levels. The activity of PLD provides survival signals to cancer cells, whereas RALBP1 iii exports chemotherapeutic drugs. Thus both RALBP1 and PLD in concert can lead to development of an aggressive and metastatic breast cancers and also contribute to chemo-resistance. In our study, cancerous breast tissues from patients have a significantly higher expression of RALBP1 compared to normal breast tissue. Furthermore, cytotoxic chemotherapy combination offered no significant advantage in patient cohorts with high RALBP1 expression as compared to those patients receiving mono or non-cytotoxic chemotherapies. Moreover, patients with high expression of PLD1 also had poor prognostic outcomes to different treatments. Thus, overcoming adaptive responses to tamoxifen induced oxidative stress could improve the survival of breast cancer patients.
    The DPYSL2 gene connects mTOR and schizophrenia
    X Pham, Translational Psychiatry - 2016
    We previously reported a schizophrenia-associated polymorphic CT di-nucleotide repeat (DNR) at the 5′-untranslated repeat (UTR) of DPYSL2, which responds to mammalian target of Rapamycin (mTOR) signaling with allelic differences in reporter assays. Now using microarray analysis, we show that the DNR alleles interact differentially with specific proteins, including the mTOR-related protein HuD/ELAVL4. We confirm the differential binding to HuD and other known mTOR effectors by electrophoretic mobility shift assays. We edit HEK293 cells by CRISPR/Cas9 to carry the schizophrenia risk variant (13DNR) and observe a significant reduction of the corresponding CRMP2 isoform. These edited cells confirm the response to mTOR inhibitors and show a twofold shortening of the cellular projections. Transcriptome analysis of these modified cells by RNA-seq shows changes in 12.7% of expressed transcripts at a false discovery rate of 0.05. These transcripts are enriched in immunity-related genes, overlap significantly with those modified by the schizophrenia-associated gene, ZNF804A, and have a reverse expression signature from that seen with antipsychotic drugs. Our results support the functional importance of the DPYSL2 DNR and a role for mTOR signaling in schizophrenia.
    AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease
    Allison M.Bradbury, Neuroscience - 2016
    Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16 weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.
    CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence
    N Garg, Oncogene - 2016
    Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.
    Intestinal epithelial injury induced by maternal separation is protected by hydrogen sulfide
    Bo Li, Journal of Pediatric Surgery - 2016
    Oxidative stress has been implicated in the pathogenesis of various neonatal diseases involving the intestine. Hydrogen sulfide (H2S) has been shown to protect against oxidative stress. We hypothesized that administration of sodium hydrosulfide (NaHS), an H2S donor, to neonatal mice can decrease the intestinal epithelial injury associated with maternal separation (MS).
    Osmolality of enteral formula and severity of experimental necrotizing enterocolitis
    Hiromu Miyake, Pediatric Surgery International - 2016
    PurposeAdministration of hyperosmolar formula is regarded as a risk factor for the development of necrotizing enterocolitis (NEC). However, there are limited number of reports about the relationship between formula osmolality and NEC. The aim of this study is to evaluate the effects of formula concentration in an experimental model of NEC.MethodsWe studied experimental NEC in C57BL/6 mice. NEC was induced by giving hypoxia, gavage administration of lipopolysaccharide and gavage formula feeding from postnatal day 5–9. We used two types of formula: (1) hyperosmolar formula (HF): 15 g Similac + 75 ml Esbilac (849 mOsm/kg); (2) diluted formula (DF): dilute hyperosmolar formula with an equal amount of water (325 mOsm/kg). Controls were fed by the mother. On postnatal day 9, the ileum was harvested and evaluated for severity of mucosal injury (hematoxylin/eosin staining) and inflammation (PCR for IL6 and TNFα mRNA expression).ResultsThe incidence of NEC was same in both HF and DF (80%). The intestinal inflammatory response was similar between HF and DF (IL6: p = 0.26, TNFα: p = 0.69).ConclusionsThis study indicates the osmolality of enteral formula does not affect incidence of experimental NEC. This experimental study provides new insights into the relationship between formula feeding and NEC.
    Muscle injury and impaired function, and insulin resistance in Chromogranin A knockout mice
    Kechun Tang, Journal of Endocrinology - 2016
    Chromogranin A (CgA) is widely expressed in endocrine and neuroendocrine tissues as well as in the central nervous system. We observed CgA expression (mRNA and protein) in the gastrocnemius (GAS) muscle and found that performance of CgA-deficient Chga-KO mice in treadmill exercise was impaired. Supplementation with CgA in Chga-KO mice restored exercise ability suggesting a novel role for endogenous CgA in skeletal muscle function. Chga-KO mice display (i) lack of exercise-induced stimulation of pAKT, pTBC1D1 and phospho-p38 kinase signaling, (ii) loss of GAS muscle mass, (iii) extensive formation of tubular aggregates (TA), (iv) disorganized cristae architecture in mitochondria, (v) increased expression of the inflammatory cytokines Tnfα, Il6 and Ifnɣ, and fibrosis. The impaired maximum running speed and endurance in the treadmill exercise in Chga-KO mice correlated with decreased glucose uptake and glycolysis, defects in glucose oxidation and decreased mitochondrial cytochrome C oxidase activity. The lack of adaptation to endurance training correlated with the lack of stimulation of p38MAPK that is known to mediate the response to tissue damage. Since CgA sorts proteins to the regulated secretory pathway, we speculate that lack of CgA could cause misfolding of membrane proteins inducing aggregation of sarcoplasmic reticulum (SR) membranes and formation of tubular aggregates that is observed in Chga-KO mice. In conclusion, CgA deficiency renders the muscle energy deficient, impairs performance in treadmill exercise and prevents regeneration after exercise-induced tissue damage.
    A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)
    Divya Varun, Acta Biomaterialia - 2016
    Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular ‘raw material’ needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.
    Whey protein-derived exosomes increase protein synthesis and hypertrophy in C2C12 myotubes
    C. Brooks Mobley, Journal of Dairy Science - 2016
    We sought to examine potential amino acid independent mechanisms whereby hydrolyzed whey protein (WP) affects muscle protein synthesis (MPS) and anabolism in vitro. Specifically, we tested (1) whether 3-h and 6-h treatments of WP, essential amino acids, or l-leucine (Leu) affected MPS, and whether 6-h treatments with low-, medium-, or high doses of WP versus Leu affected MPS; (2) whether knockdown of the primary Leu transporter affected WP- and Leu-mediated changes in MPS, mammalian target of rapamycin (mTOR) signaling responses, or both, following 6-h treatments; (3) whether exosomes isolated from WP (WP-EXO) affected MPS, mTOR signaling responses, or both, compared with untreated (control) myotubes, following 6-h, 12-h, and 24-h treatments, and whether they affected myotube diameter following 24-h and 48-h treatments.
    Therapeutic reversal of food allergen sensitivity by mature retinoic acid–differentiated dendritic cell induction of LAG3+CD49b−Foxp3− regulatory T cells
    Wojciech Dawicki, Journal of Allergy and Clinical Immunology - 2016
    Anaphylaxis is a life-threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients.
    Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs
    Antonia Piazzesi, Cell Reports - 2016
    Summary Chromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms.
    Yaarit Adamovich, Cell Metabolism - 2016
    Summary The mammalian circadian system consists of a master clock in the brain that synchronizes subsidiary oscillators in peripheral tissues. The master clock maintains phase coherence in peripheral cells through systemic cues such as feeding-fasting and temperature cycles. Here, we examined the role of oxygen as a resetting cue for circadian clocks. We continuously measured oxygen levels in living animals and detected daily rhythms in tissue oxygenation. Oxygen cycles, within the physiological range, were sufficient to synchronize cellular clocks in a HIF1α-dependent manner. Furthermore, several clock genes responded to changes in oxygen levels through HIF1α. Finally, we found that a moderate reduction in oxygen levels for a short period accelerates the adaptation of wild-type but not of HIF1α-deficient mice to the new time in a jet lag protocol. We conclude that oxygen, via HIF1α activation, is a resetting cue for circadian clocks and propose oxygen modulation as therapy for jet lag.
    Protein S Regulates Neural Stem Cell Quiescence and Neurogenesis
    Katya Zelentsova, STEM CELLS - 2016
    Neurons are continuously produced in brains of adult mammalian organisms throughout life—a process tightly regulated to ensure a balanced homeostasis. In the adult brain, quiescent Neural Stem Cells (NSCs) residing in distinct niches engage in proliferation, to self-renew and to give rise to differentiated neurons and astrocytes. The mechanisms governing the intricate regulation of NSC quiescence and neuronal differentiation are not completely understood. Here, we report the expression of Protein S (PROS1) in adult NSCs, and show that genetic ablation of Pros1 in neural progenitors increased hippocampal NSC proliferation by 47%. We show that PROS1 regulates the balance of NSC quiescence and proliferation, also affecting daughter cell fate. We identified the PROS1-dependent downregulation of Notch1 signaling to correlate with NSC exit from quiescence. Notch1 and Hes5 mRNA levels were rescued by reintroducing Pros1 into NCS or by supplementation with purified PROS1, suggesting the regulation of Notch pathway by PROS1. Although Pros1-ablated NSCs show multilineage differentiation, we observed a 36% decrease in neurogenesis, coupled with a similar increase in astrogenesis, suggesting PROS1 is instructive for neurogenesis, and plays a role in fate determination, also seen in aged mice. Rescue experiments indicate PROS1 is secreted by NSCs and functions by a NSC-endogenous mechanism. Our study identifies a duple role for PROS1 in stem-cell quiescence and as a pro-neurogenic factor, and highlights a unique segregation of increased stem cell proliferation from enhanced neuronal differentiation, providing important insight into the regulation and control of NSC quiescence and differentiation. Stem Cells 2016
    Development of TRACER: tissue roll for analysis of cellular environment and response
    Darren Rodenhizer, Biofabrication - 2016
    The tumour microenvironment is heterogeneous and consists of multiple cell types, variable extracellular matrix (ECM) composition, and contains cell-defined gradients of small molecules, oxygen, nutrients and waste. Emerging in vitro cell culture systems that attempt to replicate these features often fail to incorporate design strategies to facilitate efficient data collection and stratification. The tissue roll for analysis of cellular environment and response (TRACER) is a novel strategy to assemble layered, three-dimensional tumours with cell-defined, graded heterogeneous microenvironments that also facilitates cellular separation and stratification of data from different cell populations from specific microenvironments. Here we describe the materials selection and development of TRACER. We find that cellulose fibre scaffolding is an ideal support to generate tissue constructs having homogenous cell seeding and consistent properties. We explore ECM remodeling and long-term cell growth in the scaffold, and characterize the tumour microenvironment in assembled TRACERs using multiple established analysis methods. Finally, we confirm that TRACERs replicate small molecule gradients of glucose and lactate, and explore cell phenotype associated with these gradients using confocal microscopy, flow cytometry, and quantitative PCR analysis. We envision this technology will provide a platform to create complex, yet controlled tumour microenvironments that can be easily disassembled for snapshot analysis of cell phenotype and response to therapy in relation to microenvironment properties.
    HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells
    Steffen Jørgensen, PLOS ONE - 2016
    The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis.
    Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence
    C Yang, Oncogene - 2016
    Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.
    A novel FRET-based screen in high-throughput format to identify inhibitors of malarial and human glucose transporters
    Thomas E. Kraft, Antimicrobial Agents and Chemotherapy - 2016
    The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose-sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z'-factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counter screen PfHT hits against the human orthologues GLUT1, 2, 3 and 4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and ortholog selectivity, thereby successfully validating our assay for anti-malarial screening.
    An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery
    Aline Koch, PLOS Pathog - 2016
    Author Summary RNA interference has emerged as a powerful genetic tool for scientific research. The demonstration that agricultural pests, such as insects and nematodes, are killed by exogenously supplied RNA targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNA signals. We show that spraying barley with a 791 nt long dsRNA ( CYP3 -dsRNA) targeting the three fungal ergosterol biosynthesis genes ( CYP51A , CYP51B , CYP51C ), whose respective proteins also are known as azole fungicide targets, efficiently inhibited the necrotrophic fungus Fusarium graminearum in directly sprayed and systemic leaf tissue. Strong inhibition of fungal growth required an operational fungal RNA interference mechanism as demonstrated by the fact that a Fusarium DICER-LIKE-1 mutant was insensitive to CYP3 -dsRNA in systemic, non-sprayed leaf areas. Our findings will help in the efficient design of RNAi-based plant disease control. We provide essential information on a fundamentally new plant protection strategy, thereby opening novel avenues for improving crop yields in an environmentally friendly and sustainable manner.
    Farnesoid X Receptor Signaling Shapes the Gut Microbiota and Controls Hepatic Lipid Metabolism
    Limin Zhang, mSystems - 2016
    The gut microbiota modulates obesity and associated metabolic phenotypes in part through intestinal farnesoid X receptor (FXR) signaling. Glycine-β-muricholic acid (Gly-MCA), an intestinal FXR antagonist, has been reported to prevent or reverse high-fat diet (HFD)-induced and genetic obesity, insulin resistance, and fatty liver; however, the mechanism by which these phenotypes are improved is not fully understood. The current study investigated the influence of FXR activity on the gut microbiota community structure and function and its impact on hepatic lipid metabolism. Predictions about the metabolic contribution of the gut microbiota to the host were made using 16S rRNA-based PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), then validated using 1H nuclear magnetic resonance-based metabolomics, and results were summarized by using genome-scale metabolic models. Oral Gly-MCA administration altered the gut microbial community structure, notably reducing the ratio of Firmicutes to Bacteroidetes and its PICRUSt-predicted metabolic function, including reduced production of short-chain fatty acids (substrates for hepatic gluconeogenesis and de novo lipogenesis) in the ceca of HFD-fed mice. Metabolic improvement was intestinal FXR dependent, as revealed by the lack of changes in HFD-fed intestine-specific Fxr-null (FxrΔIE) mice treated with Gly-MCA. Integrative analyses based on genome-scale metabolic models demonstrated an important link between Lactobacillus and Clostridia bile salt hydrolase activity and bacterial fermentation. Hepatic metabolite levels after Gly-MCA treatment correlated with altered levels of gut bacterial species. In conclusion, modulation of the gut microbiota by inhibition of intestinal FXR signaling alters host liver lipid metabolism and improves obesity-related metabolic dysfunction. IMPORTANCE The farnesoid X receptor (FXR) plays an important role in mediating the dialog between the host and gut microbiota, particularly through modulation of enterohepatic circulation of bile acids. Mounting evidence suggests that genetic ablation of Fxr in the gut or gut-restricted chemical antagonism of the FXR promotes beneficial health effects, including the prevention of nonalcoholic fatty liver disease in rodent models. However, questions remain unanswered, including whether modulation of FXR activity plays a role in shaping the gut microbiota community structure and function and what metabolic pathways of the gut microbiota contribute in an FXR-dependent manner to the host phenotype. In this report, new insights are gained into the metabolic contribution of the gut microbiota to the metabolic phenotypes, including establishing a link between FXR antagonism, bacterial bile salt hydrolase activity, and fermentation. Multiple approaches, including unique mouse models as well as metabolomics and genome-scale metabolic models, were employed to confirm these results.
    Lack of effects of ooplasm transfer on early development of interspecies somatic cell nuclear transfer bison embryos
    L. Antonio González-Grajales, BMC Developmental Biology - 2016
    BackgroundSuccessful development of iSCNT (interspecies somatic cell nuclear transfer) embryos depends on complex interactions between ooplasmic and nuclear components, which can be compromised by genetic divergence. Transfer of ooplasm matching the genetic background of the somatic cell in iSCNT embryos is a valuable tool to study the degree of incompatibilities between nuclear and ooplasmic components. This study investigated the effects of ooplasm transfer (OT) on cattle (Bos taurus) and plains bison (Bison bison bison) embryos produced by iSCNT and supplemented with or without ooplasm from cattle or plains bison oocytes.ResultsEmbryos in all groups were analysed for developmental competence that included cleavage rates, ATP content, and expression of nuclear- and mitochondrial- encoded genes at 8–16 cell stage. Interestingly, no significant differences were observed in embryo development, ATP content, and expression of nuclear respiratory factor 2 (NRF2), mitochondrial transcription factor A (TFAM) and mitochondrial subunit 2 of cytochrome c oxidase (mt-COX2) among groups. Thus, although OT did not result in any detrimental effects on the reconstructed embryos due to invasive manipulation, significant benefits of OT were not observed up to the 8–16 cell stage.ConclusionsThis study showed that a viable technique for OT + SCNT is possible, however, further understanding of the effects of OT on blastocyst development is necessary.
    Conjugates of HA2 with octaarginine-grafted HPMA copolymer offer effective siRNA delivery and gene silencing in cancer cells
    Moran Golan, Science Direct - 2016
    The key for successful gene silencing is to design a safe and efficient siRNA delivery system for the transfer of therapeutic nucleic acids into the target cells. Here, we describe the design of hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer displaying multiple copies of octaarginine (R8) and its use in promoting the effective delivery of small interfering RNA (siRNA) molecules intracellularly. Fluorescein-5-isothiocyanate (FITC)-labeled HPMA copolymer-bound R8 (P-R8-FITC) was synthesized with increasing R8 molar ratios (4–9.5 mol-%) to define the optimal R8 content that allowed the polymer to serve both as a siRNA-binding domain and as an intracellular transduction moiety mediating improved cellular delivery. A subunit of the influenza virus hemagglutinin (HA2), known for its ability to disrupt endosomal membranes, was further conjugated to P-R8-FITC copolymer to promote endosomal escape. Of the different P-(R8)-FITC conjugates considered, only that polymer containing the highest mol-% of R8 (P-(R8)9.5-FITC) was able to encapsulate siRNA molecules into nano-sized polyion complexes (PICs) presenting positive surface charge, low in vitro cytotoxicity, and high serum stability. P-(R8)9.5-FITC/cy5-siRNA complexes can efficiently deliver siRNA molecules into cells, while naked siRNA or siRNA encapsulated within polymers with lower R8 mol-% were unable to transfect the same cells. Conjugation of HA2 fusogenic peptide to P-(R8)-FITC significantly decreased the oncogenic RAC1 mRNA levels in cancer cells. This indicates that P-(R8)-(HA2)-FITC can deliver siRNA into target cells, and that the siRNA can reach the perinuclear region where it interacts with the RNA-induced silencing complex.
    An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy
    Mitsuo Kato, Nature Communications - 2016
    Nephropathy is a common and hard-to-treat consequence of diabetes. Here Kato et al. show that a megacluster of microRNAs regulates early development of diabetic nephropathy in mice, and that inhibition of the cluster's host long non-coding RNA transcript attenuates disease symptoms, suggesting a new therapy for diabetic nephropathy.
    microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure
    Gesche K.Gerresheim, Cellular and Molecular Life Sciences - 2016
    We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3′ untranslated region (3′UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3′UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3′UTR target site inhibits translation directed by the HCV 5′UTR. Thus, the miR-122 target sites in the 3′-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.
    Acute Loss of Cited2 Impairs Nanog Expression and Decreases Self-Renewal of Mouse Embryonic Stem Cells
    Kranc, KR, The University of Edinburgh - 2016
    Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While over-expression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.
    Dongmei Cheng, Journal of Biological Chemistry - 2016
    Hepatic apolipoprotein A-IV (apoA-IV) expression is correlated with hepatic triglyceride (TG) content in mouse models of chronic hepatosteatosis and steatosis-induced hepatic apoA-IV gene expression is regulated by nuclear transcription factor cAMP responsive element-binding protein H (CREBH) processing. To define what aspects of TG homeostasis regulates hepatic CREBH processing and apoA-IV gene expression, several mouse models of attenuated VLDL particle assembly were subjected to acute hepatosteatosis induced by an overnight fast or short-term ketogenic diet feeding. Compared with chow-fed C57BL/6 mice, fasted or ketogenic diet-fed mice displayed increased hepatic TG content, which was highly correlated (r2=0.95) with apoA-IV gene expression, and secretion of larger, TG-enriched VLDL, despite a lower rate of TG secretion and similar or reduced rate of apoB100 secretion. When VLDL particle assembly and secretion was inhibited by hepatic shRNA-induced apoB silencing or genetic or pharmacologic reduction in microsomal triglyceride transfer protein (MTP) activity, hepatic TG content increased dramatically; however, CREBH processing and apoA-IV gene expression was attenuated compared to controls. Adenovirus-mediated reconstitution of MTP expression proportionately restored CREBH processing and apoA-IV expression in liver-specific MTP knockout mice. These results reveal that hepatic TG content per se, does not regulate CREBH processing. Instead TG mobilization into the ER for nascent VLDL particle assembly activates CREBH processing and enhances apoA-IV gene expression in the setting of acute steatosis. We conclude that VLDL assembly and CREBH activation play key roles in the response to hepatic steatosis by upregulating apoA-IV and promoting assembly and secretion of larger, more TG-enriched VLDL particles.
    Low concentrations of bromodichloromethane induce a toxicogenomic response in porcine embryos in vitro
    Florence Pagé-Larivièrea,, Reproductive Toxicology - 2016
    Bromodichloromethane (BDCM) is one of the trihalomethanes present in chlorinated water. Humans are thus daily exposed. Previous contradictory results failed to clearly establish the adverse effects of low concentrations of BDCM. By using the porcine preimplantation embryo as a sensitive model, we showed that exposure to low concentrations of BDCM (10 and 100 ppb) during the first week of embryo development induced adverse effect on the blastocyst rate and alteration of the estradiol pathway. Our results also suggest that blastocysts exposed to BDCM present transcriptomic and epigenomic adaptive modifications compatible with the cardiac anomalies observed by previous studies of newborns exposed to BDCM during gestation. Thus, phenotypic observations and toxicogenomic adaptations of embryo to low concentration of BDCM provide insights for BDCM risk assessment. Indeed, our results support the use of sensitive toxicogenomic models using environmentally relevant concentrations to which humans are exposed in order to conduct the risk assessment.
    Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity
    Maria D. Purice, Nature Communications - 2016
    Advanced age is the greatest risk factor for neurodegenerative disorders, but the mechanisms that render the senescent brain vulnerable to disease are unclear. Glial immune responses provide neuroprotection in a variety of contexts. Thus, we explored how glial responses to neurodegeneration are altered with age. Here we show that glia–axon phagocytic interactions change dramatically in the aged Drosophila brain. Aged glia clear degenerating axons slowly due to low phosphoinositide-3-kinase (PI3K) signalling and, subsequently, reduced expression of the conserved phagocytic receptor Draper/MEGF10. Importantly, boosting PI3K/Draper activity in aged glia significantly reverses slow phagocytic responses. Moreover, several hours post axotomy, early hallmarks of Wallerian degeneration (WD) are delayed in aged flies. We propose that slow clearance of degenerating axons is mechanistically twofold, resulting from deferred initiation of axonal WD and reduced PI3K/Draper-dependent glial phagocytic function. Interventions that boost glial engulfment activity, however, can substantially reverse delayed clearance of damaged neuronal debris.
    Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells
    Erik C. Hansen, eLife - 2016
    Human immunodeficiency virus type 1 (HIV-1) infects and kills immune cells known as CD4+ T cells, leading to the disease AIDS. Current drug treatments enable HIV-1 infected patients to live relatively long and healthy lives. However, no cure for HIV-1 exists because the virus lives indefinitely in a resting state within the genetic material – or genome – of the infected cell, where it is not susceptible to drug treatments. Most HIV-1 research focuses on T cells, but another type of immune cell – the macrophage – may also harbor resting HIV-1 in its genome. Compared to other cells, macrophages are unusual because they produce large amounts of a molecule called deoxyuridine triphosphate (dUTP). Most cells, including T cells, keep dUTP levels very low because it closely resembles molecules that are used to make DNA and so it can be accidentally incorporated into the cell’s DNA. When this happens, the cell removes the dUTP from the DNA using enzymes in a process called uracil base excision repair (UBER). To hide inside the cell’s genome, HIV-1 needs to produce a DNA copy of its own genome, but it was not known what happens when HIV-1 tries to do this within a macrophage that contains high levels of dUTP and UBER enzymes. Here, Hansen et al. reveal that about 90% of macrophages have exceptionally high levels of dUTP and are poorly infected by HIV-1. The high levels of dUTP result in the virus incorporating dUTP into its DNA, which is then attacked and fragmented by UBER enzymes. However, about one in a hundred viral DNA molecules do manage to successfully integrate into the genome of the macrophage. This viral DNA later gives rise to new virus particles through an error-prone process that, by introducing new mutations into the virus genome, may help HIV-1 to evolve and persist. Further experiments examined cells that give rise to macrophages from infected patients who had been on anti-HIV drug therapy for several years. Hansen et al. found that there was lots of dUTP in the DNA sequences of HIV-1 viruses found in these “precursor” cells. These precursor cells only live for several days before being eliminated, so the presence of viruses containing dUTP suggests these cells were infected recently. A future challenge will be to identify new anti-HIV drugs that specifically target macrophages and to understand the role of error-prone production of new viral genomes.
    A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture
    Tom Levy, Marine Biotechnology - 2016
    Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.
    A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture
    Tom Levy, Marine Biotechnology - 2016
    Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.
    Sex-related factors influence expression of mood-related genes in the basolateral amygdala differentially depending on age and stress exposure | Biology of Sex Differences | Full Text
    Rachel Puralewski, BioMed Central - 2016
    Women are twice as likely to be diagnosed with major depressive disorder (MDD) compared to men, but the molecular mechanisms underlying this sex difference are unclear. Previous studies in the human postmortem brain suggest dysfunction in basolateral amygdala (BLA) inhibitory gamma-aminobutyric acid (GABA) signaling and brain-derived neurotrophic factor (BDNF) function, specifically in females with MDD.
    Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease
    Ellis Y. Kim, Skeletal Muscle - 2016
    Cellular models of muscle disease are taking on increasing importance with the large number of genes and mutations implicated in causing myopathies and the concomitant need to test personalized therapies. Developing cell models relies on having an easily obtained source of cells, and if the cells are not derived from muscle itself, a robust reprogramming process is needed. Fibroblasts are a human cell source that works well for the generation of induced pluripotent stem cells, which can then be differentiated into cardiomyocyte lineages, and with less efficiency, skeletal muscle-like lineages. Alternatively, direct reprogramming with the transcription factor MyoD has been used to generate myotubes from cultured human fibroblasts. Although useful, fibroblasts require a skin biopsy to obtain and this can limit their access, especially from pediatric populations.
    The Kaposi's-sarcoma-associated herpesvirus orf35 gene product is required for efficient lytic virus reactivation
    Shir Bergson, Virology - 2016
    Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.
    Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes
    Caroline H. Diep, Science Direct - 2016
    Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10 nM-10 μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR-B and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10 μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated).
    Denervation drives mitochondrial dysfunction in skeletal muscle of octogenarians
    Sally Spendiff, The Journal of Physiology - 2016
    Experimental denervation modulates mitochondrial function, where changes in both reactive oxygen species (ROS) and sensitivity to permeability transition are implicated in the resultant muscle atrophy. Notably, although denervation occurs sporadically in ageing muscle, its impact on ageing muscle mitochondria is unknown. As this information has important therapeutic implications concerning targeting the mitochondrion in ageing muscle, we examined mitochondrial function in skeletal muscle from four groups of humans, comprising two active (mean age 23.7 (SD 2.7) yr & 71.2 (4.9) yr) and two inactive groups (64.8 (3.1) yr & 82.5 (4.8) yr), and compared this to a murine model of sporadic denervation. We tested the hypothesis that while some alterations of mitochondrial function in aged muscle are attributable to a primary organelle defect, in advanced age, mitochondrial dysfunction would be impacted by persistent denervation. Both ageing in humans, and sporadic denervation in mice, increased mitochondrial sensitivity to permeability transition (Humans P = 0.004; Mice P = 0.01). To ascertain the contribution of sporadic denervation to mitochondrial function we pharmacologically inhibited the denervation-induced ROS response. This reduced ROS emission by 60% (P = 0.02) in sporadically denervated mouse muscle, similar to what was seen in humans older than 75 years (-66%, P = 0.02) but not those younger than 75 years. We conclude that an increased sensitivity to permeability transition is a primary mitochondrial defect in ageing muscle. However, at more advanced age when muscle atrophy becomes more clinically severe, mitochondrial function changes are markedly impacted by persistent sporadic denervation, making the mitochondrion a less viable therapeutic target. This article is protected by copyright. All rights reserved
    PLOS ONE: Ezrin Is Associated with Disease Progression in Ovarian Carcinoma
    Vered Horwitz, PLOS ONE - 2016
    Objective Ezrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC). Methods OC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression. Results Higher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome. Conclusions The 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The in vitro results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas.
    β-Catenin signaling positively regulates glutamate uptake and metabolism in astrocytes
    Victoria Lutgen, Journal of Neuroinflammation - 2016
    Neurological disorders have been linked to abnormal excitatory neurotransmission. Perturbations in glutamate cycling can have profound impacts on normal activity, lead to excitotoxicity and neuroinflammation, and induce and/or exacerbate impairments in these diseases. Astrocytes play a key role in excitatory signaling as they both clear glutamate from the synaptic cleft and house enzymes responsible for glutamate conversion to glutamine. However, mechanisms responsible for the regulation of glutamate cycling, including the main astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2 or GLT-1 in rodents) and glutamine synthetase (GS) which catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine, remain largely undefined.
    Failed reinnervation in aging skeletal muscle
    Sudhakar Aare, Skeletal Muscle - 2016
    Skeletal muscle displays a marked accumulation of denervated myofibers at advanced age, which coincides with an acceleration of muscle atrophy.
    High fat diet drives obesity regardless the composition of gut microbiota in mice
    Sylvie Rabot, Scientific Reports - 2016
    The gut microbiota is involved in many aspects of host physiology but its role in body weight and glucose metabolism remains unclear. Here we studied the compositional changes of gut microbiota in diet-induced obesity mice that were conventionally raised or received microbiota transplantation. In conventional mice, the diversity of the faecal microbiota was weakly associated with 1st week weight gain but transferring the microbiota of mice with contrasting weight gain to germfree mice did not change obesity development or feed efficiency of recipients regardless whether the microbiota was taken before or after 10 weeks high fat (HF) feeding. Interestingly, HF-induced glucose intolerance was influenced by microbiota inoculation and improved glucose tolerance was associated with a low Firmicutes to Bacteroidetes ratio. Transplantation of Bacteroidetes rich microbiota compared to a control microbiota ameliorated glucose intolerance caused by HF feeding. Altogether, our results demonstrate that gut microbiota is involved in the regulation of glucose metabolism and the abundance of Bacteroidetes significantly modulates HF-induced glucose intolerance but has limited impact on obesity in mice. Our results suggest that gut microbiota is a part of complex aetiology of insulin resistance syndrome, individual microbiota composition may cause phenotypic variation associated with HF feeding in mice.
    Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report
    Xavier Tekpli, Cellular Oncology - 2016
    PurposeIn the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells.MethodsERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9).ResultsUsing RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts.ConclusionsTaken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.
    Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation
    Tierra R. Farris, Infection and Immunity - 2016
    Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing, as primary TRP32 targets. TRP32 bound genes (n=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.
    Dependence-induced increase of alcohol self-administration and compulsive drinking mediated by the histone methyltransferase PRDM2
    E. Barbier, Molecular Psychiatry - 2016
    Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the specific molecular mechanisms mediating dependence-induced neuroadaptations remain largely unknown. Here, we found that a history of alcohol dependence persistently decreased the expression of Prdm2, a histone methyltransferase that monomethylates histone 3 at the lysine 9 residue (H3K9me1), in the rat dorsomedial prefrontal cortex (dmPFC). Downregulation of Prdm2 was associated with decreased H3K9me1, supporting that changes in Prdm2 mRNA levels affected its activity. Chromatin immunoprecipitation followed by massively parallel DNA sequencing showed that genes involved in synaptic communication are epigenetically regulated by H3K9me1 in dependent rats. In non-dependent rats, viral-vector-mediated knockdown of Prdm2 in the dmPFC resulted in expression changes similar to those observed following a history of alcohol dependence. Prdm2 knockdown resulted in increased alcohol self-administration, increased aversion-resistant alcohol intake and enhanced stress-induced relapse to alcohol seeking, a phenocopy of postdependent rats. Collectively, these results identify a novel epigenetic mechanism that contributes to the development of alcohol-seeking behavior following a history of dependence.
    Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report
    Xavier Tekpli, Cellular Oncology - 2016
    PurposeIn the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells.MethodsERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9).ResultsUsing RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts.ConclusionsTaken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.
    Investigating the physiological role of HDAC1 and HDAC2 in embryonic stem cells
    Shereen Jamal Jamaladdin, University of Leicester Thesis - 2016
    Histone deacetylases 1 and 2 (HDAC1/2) are highly similar proteins (83% identical) that form the core catalytic components of corepressor complexes that modulate gene expression. Germline deletion of Hdac1 in mice results in early embryonic lethality and conditional deletion of Hdac1 but not Hdac2 causes precocious differentiation in ES cells. Therefore to further investigate the role of HDAC1/2 during the early embryogenesis, we have generated a compound conditional knockout ES cell line Hdac1ko; Hdac2Het in which HDAC1/2 activity is reduced but not entirely lost. Hdac1ko; Hdac2He cells have a significant reduction in total deacetylase activity and disruption of core pressor complex integrity. The prolifration capicity of Hdac1ko; Hdac2He cells is not inhibited, however, upon differentiation they were predisposed to toward the cardiomyocyte lineage. In most cell types, deletion of both Hdac1 and Hdac2 is required to produce a henotype, suggesting their activity is redundant. To circumvent this functional edundancy, we generated a double conditional knockout (DKO) cells in which both dac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 results in a 0% reduction in total HDAC activity and a loss of cell viability, which is associated ith increased abnormal mitotic spindle, chromatin bridges and miconuclei, suggesting that HDAC1/2 are necessary for accurate chromosome segregation. Transcriptome analysis reveals 1,708 differentially expressed genes in DKO cells including a reduction in the expression of the ES cells core pluripotent factors. HDAC1/2 activity can be regulated in vitro through the binding of inositol tetraphosphate (IP4). By rescuing the viability of DKO cells using wt and mutant forms of HDAC1, we demonstrated that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. We have also shown that treatment of DKO ES cells with RA results in reduces induction of HOX genes, suggesting a positive role of HDAC1/2 in gene activation as well as gene repression.
    Osteopontin has a protective role in prostate tumor development in mice
    Keiko Danzaki, European Journal of Immunology - 2016
    Osteopontin (OPN) is a protein, generally considered to play a pro-tumorigenic role, whereas several reports have demonstrated the anti-tumorigenic function of OPN during tumor development. These opposing anti- and pro-tumorigenic functions are not fully understood. Here, we report that host-derived OPN plays an anti-tumorigenic role in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model and a TRAMP tumor transplant model. Tumor suppression mediated by OPN in Rag2−/− mice suggests that OPN is dispensable in the adaptive immune response. We found that host-derived OPN enhanced infiltration of natural killer (NK) cells into TRAMP tumors. The requirement of OPN in NK cell migration towards TRAMP cells was confirmed by an ex vivo cell migration assay. In contrast to TRAMP cells, in vivo B16 tumor development was not inhibited by OPN, and B16 tumors did not show OPN-mediated cell recruitment. It is possible that low levels of chemokine expression by B16 cells do not allow OPN to enhance immune cell recruitment. In addition to demonstrating the anti-tumorigenic role of OPN in TRAMP tumor development, this study also suggests that the contribution of OPN to tumor development depends on the type of tumor as well as the source and isoform of OPN. This article is protected by copyright. All rights reserved
    Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
    Purvi C. Trivedi, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids - 2016
    Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function is regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2 A, Hsc70, Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and supressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.
    Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing
    Kevin A. Henry, Protein Engineering Design and Selection - 2016
    The epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-β3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VhH library against TGF-β3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-β3 binding and neutralized TGF-β3 in in vitro cellular assays. In contrast, all other sdAbs identified using conventional panning approaches (i.e., without regard to epitope specificity) did not target the site of receptor:cytokine interaction. We expect this strategy to be generally applicable for identifying epitope-specific sdAbs when binding reagents directed against the epitope of interest are available. The sdAbs identified here are of potential interest as cancer immunotherapeutics.
    Cell type specific modulation of innate immune signalling by vitamin D in human mononuclear phagocytes
    Rhiannon Kundu, Immunology - 2016
    Vitamin D is widely reported to inhibit innate immune signalling and dendritic cell (DC) maturation as a potential immunoregulatory mechanism. It is not known whether vitamin D has global or gene-specific effects on transcriptional responses downstream of innate immune stimulation, or whether vitamin D inhibition of innate immune signalling is common to different cells. We confirmed vitamin D inhibition of NFκB and p38 mitogen activated protein kinase (MAPK) signalling in monocyte derived DC (MDDC) stimulated with lipopolysaccharide (LPS). This was associated with global but modest attenuation of LPS-induced transcriptional changes at genome-wide level. Surprisingly, vitamin D did not inhibit innate immune NFκB activation in monocyte derived macrophages. Consistent with our findings in MDDC, ex vivo vitamin D treatment of primary peripheral blood myeloid DC also lead to significant inhibition of LPS inducible NFκB activation. Unexpectedly, in the same samples, vitamin D enhanced activation of both NFκB and MAPK signalling in primary peripheral blood monocytes. In a cross sectional clinical cohort, we found no relationship between peripheral blood vitamin D levels and LPS inducible activation of NFκB and MAPK pathways in monocytes of myeloid DC. Remarkably however, in vivo supplementation of vitamin D deficient people in this clinical cohort also enhanced LPS inducible MAPK signalling in peripheral blood monocytes. Therefore, we report that vitamin D differentially modulates the molecular response to innate immune stimulation in monocytes, macrophages and dendritic cells. These results are of importance in the design of studies on vitamin D supplementation in infectious and immunological diseases. This article is protected by copyright. All rights reserved.
    Inhibition of adhesion, migration and of α5β1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A
    Chiara Pelillo, Journal of Inorganic Biochemistry - 2016
    NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5β1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5β1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5β1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5β1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5β1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration.
    Effect of Roughness on in Situ Biomineralized CaP-Collagen Coating on the Osteogenesis of Mesenchymal Stem Cells
    Xingjie Zan, Langmuir - 2016
    Because of its outstanding osteo-conductive property, a calcium phosphate (CaP) coating has been used as an implant coating for bone tissue engineering. Nevertheless, the issues, such as harsh fabrication conditions, long-term stability and biocompatibility, and the requirement for expensive instruments, still exist in current coating techniques. To address these issues, the CaP coatings doped with collagen (CaP-Col) were in situ generated on polyelectrolyte multilayers (PEMs) by incubating PEMs in a mixture of the collagen, phosphate, and calcium ions. The resulting coatings have controllable physical properties (chemical composition, crystallinity, and roughness) and good stability before and after incubation with cell culture medium. We also found that both the cellular viability and osteogenesis of mesenchymal stem cells (MSCs) were closely related to the roughness of PEMs/CaP-Col, one of the easily ignored physical factors in current coating designs but very critical. The existed roughness window (between 18 ± 1.2 and 187 ± 7.3 nm) suitable for MSC proliferation on PEMs/CaP-Col coating and the optimal roughness (∼98 ± 3.5 nm) for MSC osteogenesis further demonstrated that the roughness was a critical factor for bone formation. Therefore, we envision that our exploration of the effects of surface roughness on MSC behaviors would provide better guidance for the future design of material coating and eventual medical success.
    The Piwi P athway in Bovine Gametes and Preimplantation Embryo s
    Stewart James Russell, The University of Guelph A Thesis - 2016
    Epigenetic reprogramming is required during both embryo and gamete development to restore the potential for cellular pluripotency. Changes in DNA methylation during reprogramming can allow the expression of integrated endogenous transposable elements (TEs) that may lead to genomic instability should widespread reintegration occur. Small RNA mediated mechanisms have therefore evolved to constrain TE expression during reprogramming. PIWI proteins are a subfamily of the Argonaute clade of proteins which have functions in small RNA (sRNA) mediated TE and gene silencing. PIWI proteins direct silencing through RNA-mediated interactions with complementary nucleic acid targets. TE expression has been documented in early bovine embryos, although mechanisms regulating their expression are not widely studied. This period is further complicated by extensive targeted decay of maternally-derived mRNAs which may utilize related mechanisms of gene silencing. Based on these observations, it was hypothesized that the PIWI pathway is present in bovine gametes and embryos, and regulates endogenous TEs and mRNAs during reprogramming. PIWIL1 expression was characterized in bovine gonads, oocytes, and embryos. The testis-specific expression of full-length PIWIL1 showed localization to spermatocytes and spermatids, and association with piRNAs of 29-32 nucleotides in length. Additionally, two shorter, 3ʹtruncated PIWIL1 transcript isoforms were found to be expressed in testis, oocytes and embryos, and showed dynamic regulation throughout embryogenesis. Full-length PIWIL2 and PIWIL3 transcripts were also quantified throughout embryo genesis. To address potential roles for the PIWI pathway, sRNAs from oocytes and zygotes were profiled through next-generation sequencing and compared to those from testis, ovary and sperm. An analysis “pipeline” was developed to identify putative piRNAs from the sequencing libraries. The piRNA-“like” RNAs (pilRNAs) were characterized based on their size and canonical biogenesis features and were abundant in oocytes and embryos. When mapped to bovine repeats, up to one third of embryonic pilRNAs targeted TE classes including LINEs, SINEs, and ERVs. Through comparisons with published embryonic mRNA transcriptomes this analysis also revealed the potential for maternally-deposited, zygotic pilRNAs to direct mRNA degradation. These data provide one of the first characterizations of the PIWI pathway outside of classical models, and provide evidence for novel functions in embryo TE and gene regulation.
    Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma
    A. Bergeron, Andrology - 2016
    The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5′AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/μg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2 ± 14.8 fmoles/min/μg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.
    Role of the orexin (hypocretin) system in contextual fear conditioning in rats
    Huiying Wang, Behavioural Brain Research - 2016
    Orexin (hypocretin) neurons located in the posterior hypothalamus send projections to multiple areas of the brain involved in arousal and experimental evidence indicates that these neurons play a role in the physiological and behavioral responses to stress. This study was done to determine if the orexin system was involved in mediating the fear associated with shock context (5 × 2 s of 1.5 mA). First, real-time RT-PCR was used to examine changes in the mRNA levels for prepro-orexin (ppOX), the orexin-1 receptor (OX1R) and the orexin-2 receptor (OX2R) at two weeks post-shock. We found that the mRNA levels for ppOX and OX1R were increased in the posterior hypothalamus of shocked rats. In contrast, no significant difference was found in the midline thalamus or the locus coeruleus/parabrachial region. Second, the study examined if systemic injections of antagonists for orexin receptors attenuated the freezing related to contextual fear. The OX1R antagonist SB334867 (20 or 30 mg/kg; i.p.) decreased freezing while the same doses of the OX2R antagonist TCSOX229 had no effect. The dual orexin antagonist TCS1102 (20 mg/kg; i.p.) also decreased the freezing to the shock context. The results of the present study show upregulation of orexin activity and of the OX1R in the hypothalamus following exposure of rats to footshocks and highlight a specific role of OX1R in contextual fear.
    The antiandrogen flutamide is a novel aryl hydrocarbon receptor ligand that disrupts bile acid homeostasis in mice through induction of Abcc4
    Xiaoxia Gao, Biochemical Pharmacology - 2016
    Flutamide (FLU), an oral, nonsteroidal antiandrogen drug used in the treatment of prostate cancer, is associated with idiosyncratic hepatotoxicity that sometimes causes severe liver damage, including cholestasis, jaundice, and liver necrosis. To understand the mechanism of toxicity, a combination of aryl hydrocarbon receptor (Ahr)-deficient (Ahr−/−) mice, primary hepatocytes, luciferase reporter gene assays, in silico ligand docking and ultra-performance chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics was used. A significant increase of liver weights, and liver and serum bile acid levels was observed after FLU treatment, indicating hepatomegaly and disrupted bile acid homeostasis. Expression of the AhR gene battery was markedly increased in livers of wild-type mice Ahr+/+ treated with FLU, while no change was noted in Ahr−/− mice. Messenger RNAs encoded by AhR target genes were induced in primary mouse hepatocytes cultured with FLU, which confirmed the in vivo results. Ligand-docking analysis further predicted that FLU is an AhR agonist ligand which was confirmed by luciferase reporter gene assays. Multivariate data analysis showed that bile acids were responsible for the separation of vehicle- and FLU-treated Ahr+/+ mice, while there was no separation in Ahr−/− mice. Expression of mRNA encoding the bile acid transporter ABCC4 was increased and farnesoid X receptor signaling was inhibited in the livers of Ahr+/+ mice, but not in Ahr−/− mice treated with FLU, in agreement with the observed downstream metabolic alterations. These findings provide new insights into the mechanism of liver injury caused by FLU treatment involving activation of AhR and the alterations of bile acid homeostasis, which could guide clinical application.
    Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility
    Bronwyn G. Butcher, BMC Microbiology - 2016
    Pseudomonas syringae infects diverse plant species and is widely used in the study of effector function and the molecular basis of disease. Although the relationship between bacterial metabolism, nutrient acquisition and virulence has attracted increasing attention in bacterial pathology, there is limited knowledge regarding these studies in Pseudomonas syringae. The aim of this study was to investigate the function of the carA gene and the small RNA P32, and characterize the regulation of these transcripts.
    Inhibition of IL-13-induced periostin in airway epithelium attenuates cellular protein expression of MUC5AC
    Isao Suzaki, Respirology - 2016
    Background and objective Serum periostin is increased in asthma and serves as a surrogate marker for IL-13 activity in the lung. Serum levels of periostin are the most robust biomarker predicting a favourable response to the anti-IL-13 drug, lebrikizumab. We investigated the mechanisms of IL-13 stimulation of periostin, the polarized secretion of periostin and whether periostin would have a direct effect on mucin secretion by airway cells. Methods Normal human bronchial epithelial (NHBE) cells were cultured at air–liquid interface (ALI) in the presence of IL-13, and we evaluated the effect of the specific inhibitors, leflunomide (Janus kinase (JAK)/signal transducer and activator of transcription factor 6 (STAT6) inhibitor) or PD98059 (MEK/extracellular regulated protein kinase (ERK) inhibitor), on periostin production. We examined MUC5AC secretion from NHBE cells exposed to recombinant human (rh) periostin or IL-13 in the presence and absence of OC-20, a periostin-neutralizing antibody. Results IL-13 induced periostin protein which was predominantly secreted towards the basal surface of the cells. Periostin production was much greater from goblet cells than ciliated cells (P < 0.001). Periostin production after exposure to IL-13 was attenuated by both leflunomide (P < 0.001) and PD98059 (P < 0.001). The addition of exogenous periostin modestly increased MUC5AC secretion (P < 0.01), but did not visibly change cell morphology. IL-13-induced MUC5AC secretion was attenuated by OC-20 (P < 0.01). Conclusion Periostin production in differentiated airway cells is mediated by JAK/STAT6 and MEK/ERK pathways. Periostin secretion is much greater from immunologically active goblet cells. IL-13-driven mucin production is partially inhibited by OC-20.
    Reinforcement of poly-l-lactic acid electrospun membranes with strontium borosilicate bioactive glasses for bone tissue engineering
    João S. Fernandes, Acta Biomaterialia - 2016
    Herein, for the first time, we combined poly-l-lactic acid (PLLA) with a strontium borosilicate bioactive glass (BBG-Sr) using electrospinning to fabricate a composite bioactive PLLA membrane loaded with 10% (w/w) of BBG-Sr glass particles (PLLA-BBG-Sr). The composites were characterised by scanning electron microscopy (SEM) and microcomputer tomography (μ-CT), and the results showed that we successfully fabricated smooth and uniform fibres (1–3 μm in width) with a homogeneous distribution of BBG-Sr microparticles (<45 μm). Degradation studies (in phosphate buffered saline) demonstrated that the incorporation of BBG-Sr glass particles into the PLLA membranes increased their degradability and water uptake with a continuous release of cations. The addition of BBG-Sr glass particles enhanced the membrane’s mechanical properties (69% higher Young modulus and 36% higher tensile strength). Furthermore, cellular in vitro evaluation using bone marrow-derived mesenchymal stem cells (BM-MSCs) demonstrated that PLLA-BBG-Sr membranes promoted the osteogenic differentiation of the cells as demonstrated by increased alkaline phosphatase activity and up-regulated osteogenic gene expression (Alpl, Sp7 and Bglap) in relation to PLLA alone. These results strongly suggest that the composite PLLA membranes reinforced with the BBG-Sr glass particles have potential as an effective biomaterial capable of promoting bone regeneration. Statement of Significance PLLA membranes were reinforced with 10% (w/w) of strontium-bioactive borosilicate glass microparticles, and their capacity to induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was evaluated. These membranes presented an increased: degradability, water uptake, Young modulus and tensile strength. We also demonstrated that these membranes are non-cytotoxic and promote the attachment of BM-MSCs. The addition of the glass microparticles into the PLLA membranes promoted the increase of ALP activity (under osteogenic conditions), as well as the BM-MSCs osteogenic differentiation as shown by the upregulation of Alpl, Sp7 and Bglap gene expression. Overall, we demonstrated that the reinforcement of PLLA with glass microparticles results in a biomaterial with the appropriate properties for the regeneration of bone tissue.
    Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient salinities
    I. Ruiz-Jarabo, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology - 2016
    Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55 ppt) for 14 days, and plasma free thyroid hormones (fT3, fT4), outer ring deiodination and Na+/K+-ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5 ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, natural salinity conditions in the Bay of Cádiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit β (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15 ppt). The high salinity (55 ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (dio1 and dio2) and activity did not change and were similar to controls (40 ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity.
    Testosterone inhibits expression of lipogenic genes in visceral fat by an estrogen-dependent mechanism
    A. Maleah Holland, Journal of Applied Physiology - 2016
    The influence of the aromatase enzyme on the chronic fat-sparing effects of testosterone requires further elucidation. Our purpose was to determine whether chronic anastrozole (AN; aromatase inhibitor) treatment alters testosterone-mediated lipolytic/lipogenic gene expression in visceral fat. 10 month old F344 rats (n=6/group) received SHAM surgery, orchiectomy(ORX), ORX+testosterone-enanthate(TEST; 7.0mg/week), or ORX+TEST+AN(0.5mg/day), with drug treatment beginning 14 days post-surgery. At day 42, ORX animals exhibited nearly undetectable serum testosterone and 29% higher retroperitoneal fat mass versus SHAM (p<0.001). Testosterone treatment produced a ~380-415% higher serum testosterone versus SHAM (p<0.001) and completely prevented ORX-induced visceral fat gain (p<0.001). Retroperitoneal fat was 21% and 16% lower in ORX+TEST versus SHAM (p<0.001) and ORX+TEST+AN (p=0.007), while serum estradiol was 62% (p=0.024) and 87% (p=0.010) higher, respectively. ORX stimulated lipogenic-related gene expression in visceral fat, demonstrated by ~84-154% higher SREBP-1 (p=0.023), FASN (p=0.01), and LPL mRNA (p<0.001) versus SHAM, effects that were completely prevented in ORX+TEST animals (p<0.01 versus ORX for all). FASN (p=0.061, trend) and LPL mRNA (p=0.043) were lower in ORX+TEST+AN versus ORX and not different than SHAM, but remained higher than ORX+TEST (p<0.05). In contrast, the ORX-induced elevation in SREBP-1 mRNA was not prevented by TEST+AN, with SREBP-1 expression remaining ~117-171% higher than SHAM and ORX+TEST (p<0.01). Across groups, visceral fat mass and lipogenic-related gene expressions were negatively associated with serum testosterone, but not estradiol. Aromatase inhibition constrains testosterone-induced visceral fat loss and the down-regulation of key lipogenic genes at the mRNA level, indicating that estradiol influences the visceral fat-sparing effects of testosterone.
    Molecular and Kinetic Characterization of The Fermentative Behavior of Saccharomyces cerevisiae strains Isolated from North Patagonia
    Adriana Beatriz Simes, Scientific Research Publishing - 2016
    At present, Argentina does not count with a production of indigenous yeast strains with suitable technological and oenological features to be used in the regional winery industry. Isolation and molecular characterization of these microorganisms and its fermentation attributes would be relevant to the sustainable development of the activity in the country and to recognize and preserve the biodiversity of the region. Eight strains isolated from grapes and musts from the North Patagonian region and genetically identified as Saccharomyces cerevisiae, were Studied for their fermentation behavior, emphasizing in hexose transport through the plasma membrane, which is the limiting step of the process. Thus, Sugar consumption profiles were analyzed in different media at laboratory scale, to be subsequently applied to the fermentation of natural musts. Three Of the eight initial strains were selected, named ÑNM10, ÑIF 8 and ÑMN16 According to their fermentation profiles. The Expression of hexose transporters during fermentations revealed interesting differences in the response of each strain to sugar consumption, where transporters HXT 2 and HXT 5 showed Significant changes in expression in Patagonian strains, which are normally associated to endurance to culture stress conditions. The Results obtained by combining the characteristics studied, at molecular and physiological level, are extremely encouraging. Native Strain ÑMN16, showed a high potential for application in local winemaking. Assays Carried out on a pilot scale will determine the feasibility of applying this strain with promising technological features at industrial scale
    N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis
    Kenan C Murphy, UMass - 2016
    The rising incidence of antimicrobial resistance (AMR) makes it im- perative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was rep orted to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the put ative DNA-binding and dimeriza- tion domains of rv2887 , a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S -adenosyl- L - methionine-dependent methyltransferase that N -methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl- β - D -ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.
    Transcriptomic evaluation of the American oyster, Crassostrea virginica, deployed during the Deepwater Horizon oil spill: Evidence of an active hydrocarbon response pathway
    Matthew J. Jenny, Marine Environmental Research - 2016
    Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.
    IL-6 blockade reprograms the lung tumor microenvironment to limit the development and progression of K-ras mutant lung cancer
    Mauricio S. Caetano, Cancer Research - 2016
    Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL-6, and activation of its responsive transcription factor STAT3 in K-ras mutant lung tumors, which was further amplified by the tumor enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL-6 using a monoclonal anti-IL-6 antibody in a K-ras mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL-6 blockade significantly inhibited lung cancer promotion, tumor cell intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL-6 inhibition reduced expression of pro-tumor type 2 molecules (Arginase 1, Fizz 1, Mgl, and IDO), number of M2 type macrophages and G-MDSCs, and pro-tumor T-regulatory/T helper 17 cell responses. This was accompanied by increased expression of anti-tumor type 1 molecule (Nos2), and anti-tumor T helper 1/CD8 T cell responses. Our study demonstrates that IL-6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also re-educates the lung microenvironment toward an anti-tumor phenotype by altering the relative proportion between pro-tumor and anti-tumor immune cells. This information introduces IL-6 as a potential druggable target for prevention and treatment of K-ras mutant lung tumors.
    The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme
    Vamsi K. Moparthi, Biochimica et Biophysica Acta (BBA) - Bioenergetics - 2016
    Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.
    Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA
    Cherie Blenkiron, PLOS ONE - 2016
    Background Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Methods and Results Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC . It was estimated that 1% of MV RNA cargo is delivered into cultured cells. Conclusions These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.
    Development of an Injectable Calcium Phosphate/Hyaluronic Acid Microparticles System for Platelet Lysate Sustained Delivery Aiming Bone Regeneration
    Pedo Babo, Macromolecular Bioscience - 2016
    Despite the biocompatibility and osteoinductive properties of calcium phosphate (CaP) cements their low biodegradability hampers full bone regeneration. Herein the incorporation of CaP cement with hyaluronic acid (HAc) microparticles loaded with platelet lysate (PL) to improve the degradability and biological performance of the cements is proposed. Cement formulations incorporating increasing weight ratios of either empty HAc microparticles or microparticles loaded with PL (10 and 20 wt%) are developed as well as cements directly incorporating PL. The direct incorporation of PL improves the mechanical properties of the plain cement, reaching values similar to native bone. Morphological analysis shows homogeneous particle distribution and high interconnectivity between the HAc microparticles. The cements incorporating PL (with or without the HAc microparticles) present a sustained release of PL proteins for up to 8 d. The sustained release of PL modulates the expression of osteogenic markers in seeded human adipose tissue derived stem cells, thus suggesting the stimulatory role of this hybrid system toward osteogenic commitment and bone regeneration applications.
    CKIP-1 silencing promotes the new bone formation in the rat mandibular distraction osteogenesis
    Zi-chao Zhou, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology - 2016
    Objective This study investigated the effects and possible molecular mechanism of CKIP-1 silencing on bone regeneration during rat mandibular distraction osteogenesis (DO).Study Design CKIP-1 silencing by chitosan/si-CKIP-1 was employed and analyzed both in the rat mandibular DO models in vivo and in cultured rat mandible BMSCs in vitro. Results Gross observation, MicroCT analysis, and H&E staining revealed that the new bone formation in the distraction gap of the chitosan/si-CKIP-1-treated group was better compared with the chitosan/si-NC- or PBS-treated groups both in quantity and quality. Proliferation assay, flow cytometry, and alizarin red staining indicated that CKIP-1 silencing significantly inhibited apoptosis, but promoted the osteogenic differentiation of cultured BMSCs. Additionally, CKIP-1 silencing significantly promoted the expression of Wnt3a, β-catenin, and OCN both in the new bone formation of DO models in vivo and in the osteogenic differentiation process of BMSCs in vitro. ConclusionsThe promotion of bone formation after CKIP-1 silencing in the rat mandibular distraction osteogenesis appears to be mediated through the Wnt3a/β-catenin signaling pathway.
    A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex
    Margaret L. Rodgers, Oxford Journals - 2016
    The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.
    PLOS Genetics: Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function
    Yichang Chen, PLOS ONE - 2016
    Concerns about the safety of Bisphenol A, a chemical found in plastics, receipts, food packaging and more, have led to its replacement with substitutes now found in a multitude of consumer products. However, several popular BPA-free alternatives, such as Bisphenol S, share a high degree of structural similarity with BPA, suggesting that these substitutes may disrupt similar developmental and reproductive pathways. We compared the effects of BPA and BPS on germline and reproductive functions using the genetic model system Caenorhabditis elegans. We found that, similarly to BPA, BPS caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast data mining all indicate that these effects are partly achieved via mechanisms distinct from BPAs. These findings therefore raise new concerns about the safety of BPA alternatives and the risk associated with human exposure to mixtures.
    PLOS ONE: Nutritional Supplement of Hatchery Eggshell Membrane Improves Poultry Performance and Provides Resistance against Endotoxin Stress
    S.K Makkar, PLOS ONE - 2016
    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of chickens to endotoxin.
    The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation
    Jixiu Shan,, Oxford Journals - 2016
    The response to amino acid (AA) limitation of the entire aminoacyl-tRNA synthetase (ARS) gene family revealed that 16/20 of the genes encoding cytoplasmic-localized enzymes are transcriptionally induced by activating transcription factor 4 (Atf4) via C/ebp-Atf-Response-Element (CARE) enhancers. In contrast, only 4/19 of the genes encoding mitochondrial-localized ARSs were weakly induced. Most of the activated genes have a functional CARE near the transcription start site (TSS), but for others the CARE is downstream. Regardless of the location of CARE enhancer, for all ARS genes there was constitutive association of RNA polymerase II (Pol II) and the general transcription machinery near the TSS. However, for those genes with a downstream CARE, Atf4, C/ebp-homology protein (Chop), Pol II and TATA-binding protein exhibited enhanced recruitment to the CARE during AA limitation. Increased Atf4 binding regulated the association of elongation factors at both the promoter and the enhancer regions, and inhibition of cyclin-dependent kinase 9 (CDK9), that regulates these elongation factors, blocked induction of the AA-responsive ARS genes. Protein pull-down assays indicated that Atf4 directly interacts with CDK9 and its associated protein cyclin T1. The results demonstrate that AA availability modulates the ARS gene family through modulation of transcription elongation.
    Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis
    Sandrine Sallï-Lefort, International Journal of Oncology - 2016
    Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway.
    Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation
    Kai Fu, eLife - 2016
    Cells use signaling pathways to detect and respond to harmful conditions by switching on genes that keep the cell healthy. One important pathway is the nuclear factor kappa B (NF-κB) signaling pathway, which is activated by many stimuli. These stimuli may come from infections from outside the cell or may originate inside the cell, as seen for DNA damage caused by irradiation, chemicals or rapid DNA replication in cancer cells. Most of a cell’s DNA is located in the cell nucleus. However, NF-κB proteins are normally located outside the nucleus, in the cell’s cytoplasm. Damage to DNA triggers a signal from the nucleus to the cytoplasm. This signal activates the NF-κB proteins, which move into the nucleus and turn on genes that help the cell to recover from the damage. These genes include those that prevent the cell from self-destructing. In one step of the NF-κB activation process, chain-like molecules called polymers are made from a compound called poly(ADP-ribose), or PAR for short. However, few other details are known about how the damaged DNA in the nucleus signals to the cytoplasm. A protein called Sam68, which is found in the cell nucleus, has been linked to DNA damage signaling. Fu, Sun et al. now present evidence that suggests that if mouse cells lack Sam68, they do not produce PAR polymers in response to DNA damage. In addition, these cells could not trigger the PAR-dependent signaling cascade that is essential for activating NF-κB and for turning on the protective genes. Consequently, cells that lacked Sam68 were extremely sensitive to agents that cause DNA damage, such as chemicals and irradiation. The NF-κB pathway is regulated incorrectly in some cancers, but is also activated by DNA damage caused by cancer treatments. Therefore, Fu, Sun et al. also explored the role of Sam68 in cancer. Reducing the levels of Sam68 made human colon cancer cells more likely to self-destruct when they were exposed to DNA-damaging agents. Furthermore, removing Sam68 from mice that spontaneously grow colon cancer caused their tumors to develop more slowly than mice that retained Sam68 in their cells. Overall, the findings presented by Fu, Sun et al. suggest that Sam68 regulates the signal from the nucleus to the cytoplasm that activates NF-κB proteins in response to DNA damage. Sam68 also appears to be important for helping colon cancer cells grow and survive. Future challenges will be to understand how Sam68 regulates the production of the PAR polymer in this response and to explore whether Sam68 can be targeted for treating cancer.
    Jane Azizkhan-clifford, United States Patent - 2016
    The present invention is directed toward the use of non-thermal plasma-treated liquids as treatment options for herpes keratitis.
    CD84 mediates CLL-microenvironment interactions
    A. Marom, Oncogene - 2016
    Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.
    Adipocyte nuclei captured from VAT and SAT | BMC Obesity | Full Text
    Suresh Ambati, BioMed Central - 2016
    Obesity-related comorbidities are thought to result from the reprogramming of the epigenome in numerous tissues and cell types, and in particular, mature adipocytes within visceral and subcutaneous adipose tissue, VAT and SAT. The cell-type specific chromatin remodeling of mature adipocytes within VAT and SAT is poorly understood, in part, because of the difficulties of isolating and manipulating large fragile mature adipocyte cells from adipose tissues.
    Interleukin (IL)‐1 in rat parturition: IL‐1 receptors 1 and 2 and accessory proteins abundance in pregnant rat uterus at term – regulation by progesterone
    Tomohito Ishiguro, Physiological Reports - 2016
    The role of interleukin‐1 (IL‐1), a pro‐inflammatory cytokine, in parturition is typically noted by changes in its concentrations. Studying the expression of its receptor family, IL‐1 receptor (IL‐1R) 1, IL‐1R2, IL‐1R accessory protein (IL‐1RAcP), and its predominantly brain isoform, IL‐1RAcPb, during late gestation in the uterus in the Long‐Evans rat is another. We assessed changes in their mRNA and protein relative abundance in the uterus and compared IL‐1RAcP and IL‐1RAcPb mRNA abundance in uterus, cervix, ovaries, placenta, and whole blood of Long‐Evans rats during late gestation or in RU486 and progesterone‐treated dams using quantitative real‐time PCR and western immunoblotting. IL‐1R1, IL‐1RAcP, and IL‐1RAcPb mRNA abundance significantly increased in the uterus at delivery whereas IL‐1R2 mRNA abundance significantly decreased. IL‐1R1 protein increased at term and IL‐1R2 protein decreased at term compared to nonpregnant uteri. IL1‐RAcPb mRNA abundance was less than IL‐1RAcP, but in the lower uterine segment it was the highest of all tissues examined. RU486 stimulated preterm delivery and an increase in IL‐1R1 mRNA abundance whereas progesterone administration extended pregnancy and suppressed the increase in IL‐1R1. These data suggest that changes in uterine sensitivity to IL‐1 occur during late gestation and suggest another level of regulation for the control of delivery. The roles for IL‐1RAcP and IL‐1RAcPb need to be determined, but may relate to different intracellular signaling pathways.
    Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo
    Leo Boneschansker, The Journal of Immunology - 2016
    Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4+ T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4+ T cells, but it had no demonstrable effects on the migration of purified CD4+CD25+CD127dim T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3+ T cell infiltrates. Neogenin was also observed on CD3+ T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4+ T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.
    Plasmalogen Deficit: A New and Testable Hypothesis for the Etiology of Alzheimer’s Disease - wood_2011.pdf
    Tara Smith, Phenomenome Discoveries, - 2011
    Alzheimer’s disease (AD) is a complex cognitive disorder for which the single greatest risk factor is age. The pathophysiological basis for AD is still a matter of debate with no current hypothesis explaining all of the complex pathological changes observed. These include neurofibrillary tangles, amyloid plaques, neuroinflammation, hypomyelination, neuronal shrinkage (eg. N- basalis cholinergic neurons, resulting in a dramatic cholinergic deficit), ocular pathology, microvascular pathology and liver peroxisomal dysfunction. The hypothesis that we are presenting, namely peroxisomal dysfunction resulting in decreased supply of critical ethanolamine plasmalogens to the brain, eye and vascular endothelium, is the first hypothesis that can potentially explai n all of these complex pathologies in AD. The value of this hypothesis is that it is imminently testable via resupply of critical ether lipid precursors of plasmalogens. PPI-1011 is such a drug candidate and will be presented after a review of the basis of this hypothesis.
    The ER membrane-anchored ubiquitin ligase Hrd1 is a positive regulator of T-cell immunity
    Yuanming Xu, Nature Communications - 2016
    Identification of positive regulators of T-cell immunity induced during autoimmune diseases is critical for developing novel therapies. The endoplasmic reticulum resident ubiquitin ligase Hrd1 has recently emerged as a critical regulator of dendritic cell antigen presentation, but its role in T-cell immunity is unknown. Here we show that genetic deletion of Hrd1 in mice inhibits T-cell proliferation, production of IL-2, and differentiation of Th1 and Th17 cells, and consequently protects mice from experimental autoimmune encephalomyelitis. Hrd1 facilitates T-cell proliferation by the destruction of cyclin-dependent kinase inhibitor p27kip1, and deletion of p27kip1 in Hrd1-null T-cells rescues proliferative capacity but not the production of cytokines, including IL-2, IFN-γ and IL-17. T-cell expression of Hrd1 is higher in patients with multiple sclerosis than in healthy individuals, and knockdown of Hrd1 in human CD4+ T cells inhibits activation and differentiation to Th1 and Th17 cells. Our study identifies Hrd1 as a previously unappreciated positive regulator of T cells and implies that Hrd1 is a potential therapeutic target for autoimmune diseases.
    Responses of bovine early embryos to S-adenosyl methionine supplementation in culture
    Habib A Shojaei Saadi, Epigenomics - 2016
    Aim: There is a growing concern about the potential adverse effects of high dose folic acid (FA) supplementation before and during pregnancy. FA metabolism generates S-adenosyl methionine (SAM) which is an important cofactor of epigenetic programming. We sought to assess the impact of a large dose of SAM on early embryo development. Materials & methods: In vitro cultured bovine embryos were treated with SAM from the eight-cell stage to the blastocyst stage. In addition to the phenotype, the genome-wide epigenetic and transcription profiles were analyzed. Results: Treatment significantly improved embryo hatching and caused a shift in sex ratio in favor of males. SAM caused genome-wide hypermethylation mainly in exonic regions and in CpG islands. Although differentially expressed genes were associated with response to nutrients and developmental processes, no correspondence was found with the differentially methylated regions, suggesting that cellular responses to SAM treatment during early embryo development may not require DNA methylation-driven changes. Conclusion: Since bovine embryos were not indifferent to SAM, effects of large-dose FA supplements on early embryonic development in humans cannot be ruled out.
    Rare novel variants in the ZIC3 gene cause X-linked heterotaxy
    Aimee D. C. Paulussen, European Journal of Human Genetics - 2016
    Variants in the ZIC3 gene are rare, but have demonstrated their profound clinical significance in X-linked heterotaxy, affecting in particular male patients with abnormal arrangement of thoracic and visceral organs. Several reports have shown relevance of ZIC3 gene variants in both familial and sporadic cases and with a predominance of mutations detected in zinc-finger domains. No studies so far have assessed the functional consequences of ZIC3 variants in an in vivo model organism. A study population of 348 patients collected over more than 10 years with a large variety of congenital heart disease including heterotaxy was screened for variants in the ZIC3 gene. Functional effects of three variants were assessed both in vitro and in vivo in the zebrafish. We identified six novel pathogenic variants (1,7%), all in either male patients with heterotaxy (n=5) or a female patient with multiple male deaths due to heterotaxy in the family (n=1). All variants were located within the zinc-finger domains or leading to a truncation before these domains. Truncating variants showed abnormal trafficking of mutated ZIC3 proteins, whereas the missense variant showed normal trafficking. Overexpression of wild-type and mutated ZIC protein in zebrafish showed full non-functionality of the two frame-shift variants and partial activity of the missense variant compared with wild-type, further underscoring the pathogenic character of these variants. Concluding, we greatly expanded the number of causative variants in ZIC3 and delineated the functional effects of three variants using in vitro and in vivo model systems.
    HLA peptides derived from tumor antigens induced by inhibition of DNA methylation for development of drug-facilitated immunotherapy
    Bracha Shraibman, Molecular & Cellular Proteomics - 2016
    Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137.
    Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system
    Atul K. Singh, Nucleic Acids Research - 2016
    Despite many methodological advances that have facilitated investigation of Mycobacterium tuberculosis pathogenesis, analysis of essential gene function in this slow-growing pathogen remains difficult. Here, we describe an optimized CRISPR-based method to inhibit expression of essential genes based on the inducible expression of an enzymatically inactive Cas9 protein together with gene-specific guide RNAs (CRISPR interference). Using this system to target several essential genes of M. tuberculosis, we achieved marked inhibition of gene expression resulting in growth inhibition, changes in susceptibility to small molecule inhibitors and disruption of normal cell morphology. Analysis of expression of genes containing sequences similar to those targeted by individual guide RNAs did not reveal significant off-target effects. Advantages of this approach include the ability to compare inhibited gene expression to native levels of expression, lack of the need to alter the M. tuberculosis chromosome, the potential to titrate the extent of transcription inhibition, and the ability to avoid off-target effects. Based on the consistent inhibition of transcription and the simple cloning strategy described in this work, CRISPR interference provides an efficient approach to investigate essential gene function that may be particularly useful in characterizing genes of unknown function and potential targets for novel small molecule inhibitors.
    An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells : Scientific Reports
    Franka Klatte-Schulz, Scientific Reports - 2016
    The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.
    Metformin elicits antitumor effects and downregulates the histone methyltransferase multiple myeloma SET domain (MMSET) in prostate cancer cells
    Nicole M. A. White-Al Habeeb, The Prostate - 2016
    BACKGROUND This study explored the biological effects of metformin on prostate cancer (PCa) cells and determined molecular pathways and epigenetic regulators implicated in its mechanism of action. METHODS We performed mRNA expression profiling in 22Rv1 cells following 2.5 mM and 5 mM metformin treatment. Genes significantly modified by metformin treatment were ranked based on altered expression, involvement with cancer-related processes, and reported dysregulation in PCa. The effects of the top ranked gene, MMSET, on the proliferative and invasive capabilities of PCa cells were investigated via siRNA knockdown alone and also combined with metformin treatment. RESULTS Metformin treatment decreased cell growth of PCa cell line 22Rv1 and stalled cells at the G1/S checkpoint in a time- and dose-dependent manner, resulting in increased cells in G1 (P < 0.05) and decreased cells in S (P < 0.05) phase. Metformin activated the AMPK/mTOR signaling pathway as shown by increased p-AMPK and decreased p-p70S6K. mRNA expression profiling following metformin treatment identified significant changes in 136 chromatin-modifying genes. The top ranked gene, multiple myeloma SET domain (MMSET) showed increased expression in PCa cell lines (22Rv1 and DU145) when compared to the benign prostate epithelium-derived cell-line RWPE-1, and its expression was decreased upon metformin treatment. siRNA-mediated knockdown of MMSET showed decreased cellular migration and invasion in DU-145 cells. MMSET knockdown in combination with metformin treatment resulted in further reduction in the capacity of PCa cells to migrate and invade. CONCLUSIONS These data suggest MMSET may play a role in the inhibitory effect of metformin on PCa and could serve as a potential novel therapeutic target for PCa. Prostate © 2016 Wiley Periodicals, Inc.
    Gluten-Free Diet Only during Pregnancy Efficiently Prevents Diabetes in NOD Mouse Offspring
    Julie C. Antvorskov,, Journal of Diabetes Research - 2016
    Gluten exposure is an important factor in the development of type 1 diabetes (T1D) [1]. Both in nonobese diabetic (NOD) mice and in Biobreeding- (BB-) rats a gluten-free (GF) [2, 3] or hydrolyzed [4] diet lowers the incidence of the diseasemarkedly. In humans, a GF, low carbohydrate diet was recently reported to induce remission in a newly diagnosed T1D patient [5], and improved insulin secretion has been observed after 6 months of GF diet [6]. The appearance of beta cell autoimmunity may be related to the age at introduction of cereals into the infant diet [7, 8]. Further, a high dietary content of monosaccharides increases the risk of developing T1D [9, 10]. The mechanism by which gliadin influences disease development is not known. It is likely that gut immunity is involved since we [11] and others [12, 13] have reported that gluten intake confers a proinflammatory cytokine profile in multiple regulatory T cell populations, including 𝛾𝛿 T cells, inmucosal lymphoid tissues. It is likely that these changes are facilitated by or even dependent on gluten peptides being able to transverse the epithelium,whichwe recently demonstrated in NOD and BALB/c mice (unpublished) and which was previously reported in T1D patients [14]. It is also possible that a direct effect of gluten peptides on the beta cells can influence disease development. We have shown that a 33-mer gliadin peptide can directly close the K channel and induce insulin secretion [15]. Although it is not clear at the present time whether gliadin interacts directly with the K channel or the signal is relayed through other receptors, such as TLR4, this cellular activation might increase the risk of disease development through beta cell stress [16]. Previous experiments with GF diet in animal models of autoimmune diabetes [2, 3, 17] were all carried out in animals exposed to GF diet both during pregnancy and during the neonatal period. However, the potential of gluten to affect the diabetogenic process seems to be dependent on the time of gluten introduction, both in animals [12, 13] and in humans [7, 8]. It is therefore not known if there is a critical period, during which the effect of a GF diet is most efficient, or if both time slots are equally important. This was investigated in this study, which showed that GF diet, exclusively during pregnancy, far exceeds the efficiency of other treatment periods.
    Transcriptome meta-analysis of three follicular compartments and its correlation with ovarian follicle maturity and oocyte developmental competence in cows
    Daulat Raheem Khan, Physiological Genomics - 2016
    Oocyte developmental competence in super-stimulated cows is dependent in part on the duration of the FSH coasting. FSH coasting refers to super-stimulation with FSH (two days of endogenous FSH following follicle ablation and three days of FSH injections) followed by no FSH for a specific duration. The optimal duration varies among individuals. FSH coasting appears to modulate the transcriptome of different follicular compartments, which cooperate as a single functional unit. However, the integrative effects of FSH coasting on different follicular compartments remain ambiguous. Meta-analysis of three independent transcriptome studies each focused on a single cell type (granulosa, cumulus and oocyte) during FSH coasting allowed the identification of 12 gene clusters with similar time-course expression patterns in all three compartments. Network analysis identified HNF4A (involved in metabolic functions) and ELAVL1 (an RNA-binding protein) as hub genes regulated respectively upwards and downwards in the clusters enriched at the optimal coasting time, and APP (involved in mitochondrial functions) and COPS5 (a member of the COP9 signalosome) as hub genes regulated respectively upwards and downwards in the clusters enriched progressively throughout the coasting period. We confirmed the effects on HNF4A downstream targets (TTR, PPL) and other hub genes (ELAVL1, APP, MYC and PGR) in 30 cows using RT-qPCR. The correlation of hub gene expression levels with FSH coasting indicated that a combination of these genes could predict oocyte competence with 83% sensitivity, suggesting that they are potential biomarkers of follicle differentiation. These findings could be used to optimize FSH coasting on an individual basis.
    MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma (PDF Download Available)
    t Tine Norman Alver, Oncotarget - 2016
    The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway is frequently hyper-activated upon vemurafenib treatment of melanoma. We have here investigated the relationship between SRY-box 10 (SOX10), forkhead box 3 (FOXD3) and microphthalmia-associated transcription factor (MITF) in the regulation of the receptor tyrosine-protein kinase ERBB3, and its cognate ligand neuregulin 1-beta (NRG1-beta). We found that both NRG1-beta and ERBB3 mRNA levels were elevated as a consequence of MITF depletion, induced by either vemurafenib or MITF small interfering RNA (siRNA) treatment. Elevation of ERBB3 receptor expression after MITF depletion caused increased activation of the PI3K pathway in the presence of NRG1-beta ligand. Together, our results suggest that MITF may play a role in the development of acquired drug resistance through hyper-activation of the PI3K pathway.
    Influence of energy level and nicotinic acid supplementation on apoptosis of blood leukocytes of periparturient dairy cows
    S. Bühlera, Science Direct - 2016
    The periparturient period of dairy cows is accompanied by an immunosuppression that leaves the animal more susceptible to infections and metabolic disorders. Non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB) which peak shortly after parturition due to lipolysis are known to impair immune cell functions. Niacin with its well-known anti-lipolytic effect may have the ability to ameliorate this situation. Additionally, niacin shows also anti-inflammatory effects that may be beneficial to the immune status of the cow. To address this 29 multiparous and 18 primiparous German Holstein cows were subjected to four different feeding groups. They were fed either a ration with a high concentrate proportion of 60% (HC), or a low concentrate proportion of 30% (LC). After parturition both concentrate levels were reduced to 30% and increased again to 50% either within 16 days (LC-group) or within 24 days (HC-group). Half of the animals received either 24 g per day of nicotinic acid from 42 days prepartum until 24 days postpartum (LC-NA, HC-NA) or no supplement (LC-CON, HC-CON). Apoptosis in polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) was examined with an Annexin V and propidium iodide (PI) based fluorescence flow cytometry assay and distinguished into early apoptotic (Annexin V positive and PI negative) and late apoptotic (Annexin V and PI positive) cells. Additionally, the pro-apoptotic gene BAX, the effector caspase CASP3, and the anti-apoptotic genes BCL2 and BCL-xL, as well as the NFκB subunit RELA were quantified by real-time PCR in blood leukocytes. All variables showed time dependencies that were mainly related to parturition (p < 0.01). Early apoptotic PBMC were significantly affected by concentrate level showing higher numbers of apoptotic cells in the HC groups (p = 0.029). PBMC were characterized by a more pronounced apoptosis than PMN and seemed to be more susceptible to the changes that occur around parturition. The genes BAX and CASP3 were positively correlated (0.631) and their peak preceded the apoptotic peak around parturition in the blood leukocytes. The LC animals showed a decrease in BCL2 expression before parturition, whereas the HC animals showed a continuous increase in BCL2 mRNA abundance (p = 0.059). RELA correlated stronger with the pro-apoptotic genes (0.715 and 0.650 with BAX and CASP3 respectively) and its expression was higher in primiparous than in multiparous cows (p = 0.011). Nicotinic acid supplementation did show some influence in increasing numbers of early apoptotic PMN and late apoptotic PBMC between 42 and 100 DIM.
    Adam Giangreco, United States Patent - 2016
    The present disclosure relates to a therapeutic agent for use in a method for modulating the function and/or growth of a thymus in a subject, wherein the therapeutic agent comprises an HER2 or HER1 pathway antagonist or agonist, and/or a CCR/CCL5 antagonist the method involving administering the therapeutic agent to the subject. Also disclosed herein is a therapeutic agent for use in a method for treating a disorder in a subject, the disorder selected from systemic autoimmunity, peripheral autoimmunity and Systemic Lupus Erythematosus,
    Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival
    Taslima T. Lina, mBio - 2016
    Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. IMPORTANCE E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.
    PTBP1 modulation of MCL1 expression regulates cellular apoptosis induced by antitubulin chemotherapeutics
    J. Cui, Cell Death & Differentiation - 2016
    Myeloid cell leukemia sequence 1 (MCL1), an anti-apoptotic BCL2 family protein, is a key regulator of intrinsic apoptosis. Normal cells require strict control over MCL1 expression with aberrant MCL1 expression linked to the emergence of various diseases and chemoresistance. Previous studies have detailed how MCL1 expression is regulated by multiple mechanisms both transcriptionally and translationally. However, characterization of the post-transcriptional regulators of MCL1 mRNA is limited. Polypyrimidine tract binding protein 1 (PTBP1) is a known regulator of post-transcriptional gene expression that can control mRNA splicing, translation, stability and localization. Here we demonstrate that PTBP1 binds to MCL1 mRNA and that knockdown of PTBP1 upregulates MCL1 expression in cancer cells by stabilizing MCL1 mRNA and increasing MCL1 mRNA accumulation in cytoplasm. Further, we show that depletion of PTBP1 protects cancer cells from antitubulin agent-induced apoptosis in a MCL1-dependent manner. Taken together, our findings suggest that PTBP1 is a novel regulator of MCL1 mRNA by which it controls apoptotic response to antitubulin chemotherapeutics.
    Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome
    Ronghui Li, PLOS Genet - 2016
    Author Summary Rett syndrome (RTT) is a debilitating neurodevelopmental disorder with no cure or effective treatment. To fully understand the disease mechanism and develop therapies, it is necessary to study all aspects of the molecular function of methyl-CpG binding protein 2 (MeCP2), mutations in which have been identified as the genetic cause of RTT. Over the years, MeCP2 has been shown to maintain DNA methylation, regulate transcription and chromatin structure, control microRNA processing, and modulate RNA splicing. Among these known functions, the role of MeCP2 in modulating RNA splicing is less well understood. We took several unbiased approaches to investigate the how MeCP2 may regulate splicing, what splicing changes are caused by the loss of MeCP2, and what functional consequences are caused by altered splicing. We discovered that MeCP2 interacts with splicing factors to regulated the splicing of glutamate receptor genes, which mediate the vast majority of excitatory synaptic transmission in the brain; and linked the altered splicing of glutamate receptor genes to specific synaptic changes in a RTT mouse model. Our findings not only advance the understanding of RTT disease mechanism, but also reveal a potential drug target for future development of therapies.
    Paternal age and diet: The contributions of a father’s experience to susceptibility for post-concussion symptomology
    Harleen Hehar, Neuroscience - 2016
    In an attempt to improve current understanding of risk factors that influence individual susceptibility to poor outcomes following mild traumatic brain injury (mTBI) or concussion, this project investigated whether modifications to paternal experiences (Advanced Age (AA) or High-Fat Diet (HFD)) affected offspring susceptibility to behavioral symptomology and changes in gene expression following pediatric concussion in a rodent model. The study demonstrated that paternal treatment prior to conception altered behavioral outcomes and molecular characterization of offspring. Offspring of AA fathers demonstrated abnormal behavioral performance when compared to offspring of control fathers. Similarly, paternal HFD altered pathophysiological outcomes for offspring, contributing to the heterogeneity in post-concussion syndrome. Additionally, this study provided insight into the mechanisms that mediate non-genetic paternal inheritance. Paternal treatment and the mTBI significantly influenced expression of a majority of the genes under examination in the prefrontal cortex, hippocampus, and nucleus accumbens, with changes being dependent upon sex and the brain region examined. These epigenetic changes may have contributed to the differences in offspring susceptibility to concussion.
    Intraarticular slow-release triamcinolone acetate reduces allodynia in an experimental mouse knee osteoarthritis model
    Jeffrey S. Kroina, Redearch Paper - 2016
    Intraarticular steroid injection has been the mainstay of short-term treatment of knee osteoarthritis (OA) pain. However, the duration of therapeutic effect from a single injection is not as long as desired. In this study we use a viscous formulation of triamcinolone acetate (TCA) in hyaluronic acid to prolong the anti-allodynia effect of that steroid. OA was induced in mice by a partial medial meniscectomy. Over time the animals' developed a mechanical allodynia in the injected leg. Mice were then given a single intraarticular injection of TCA in a short-acting DMSO formulation, or a standard commercial suspension, or the drug formulated in 5% hyaluronic acid for slow-release. Control injections in OA mice were PBS or 5% hyaluronic acid vehicle. Mechanical allodynia was then monitored over the therapeutic period. Organotypic spinal cord slices and DRG culture were performed to assess whether TCA attenuates expressions of pain mediators induced by interleukin 1β. TCA 40 μg in a fast-releasing DMSO formulation produced relief from mechanical allodynia for a few days compared to PBS control injections (P = 0.007). Similarly, the commercial suspension of TCA 40 μg also produced relief from mechanical allodynia for a few days compared to PBS control injections (P = 0.001). However, TCA 100 μg in 5% hyaluronic acid produced relief from mechanical allodynia for at least 28 days compared to PBS control or 5% hyaluronic acid vehicle injections (P = 0.0005). Furthermore, TCA significantly suppressed expression of pain mediators induced by interleukin 1β in spinal cord and DRG organotypic culture. Intraarticular TCA in a sustained release formulation of viscous 5% hyaluronic acid will produce a long-term attenuation of mechanical allodynia in the OA knees of mice.
    Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters
    María Cabello-Donayre, Molecular Microbiology - 2016
    Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.
    The Effects of Fortetropin Supplementation on Body Composition, Strength, and Power in Humans and Mechanism of Action in a Rodent Model
    Matthew H. Sharp, Journal of the American College of Nutrition - 2016
    Objective: The purpose of this study was to investigate the effects of Fortetropin on skeletal muscle growth and strength in resistance-trained individuals and to investigate the anabolic and catabolic signaling effects using human and rodent models.Methods: In the rodent model, male Wistar rats (250 g) were gavage fed with either 1.2 ml of tap water control (CTL) or 0.26 g Fortetropin for 8 days. Then rats participated in a unilateral plantarflexion exercise bout. Nonexercised and exercised limbs were harvested at 180 minutes following and analyzed for gene and protein expression relative to mammalian target of rapamycin (mTOR) and ubiquitin signaling. For the human model, 45 (of whom 37 completed the study), resistance-trained college-aged males were divided equally into 3 groups receiving a placebo macronutrient matched control, 6.6 or 19.8 g of Fortetropin supplementation during 12 weeks of resistance training. Lean mass, muscle thickness, and lower and upper body strength were measured before and after 12 weeks of training.Results: The human study results indicated a Group × Time effect (p ≤ 0.05) for lean mass in which the 6.6 g (+1.7 kg) and 19.8 g (+1.68 kg) but not placebo (+0.6 kg) groups increased lean mass. Similarly, there was a Group × Time effect for muscle thickness (p ≤ 0.05), which increased in the experimental groups only. All groups increased equally in bench press and leg press strength. In the rodent model, a main effect for exercise (p ≤ 0.05) in which the control plus exercise but not Fortetropin plus exercise increased both ubiquitin monomer protein expression and polyubiquitination. mTOR signaling was elevated to a greater extent in the Fortetropin exercising conditions as indicated by greater phosphorylation status of 4EBP1, rp6, and p70S6K for both exercising conditions.Conclusions: Fortetropin supplementation increases lean body mass (LBM) and decreases markers of protein breakdown while simultaneously increasing mTOR signaling.
    Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli
    Steven B. Lewis, Frontiers in Cellular and Infection Microbiology - 2016
    Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8.
    DNA methylation in lung fibroblasts and its role in pulmonary fibrosis
    Ian Matthew Garner, UCL thesis - 2015
    Altered methylation and subsequent changes in gene expression have been implicated in several fibroses including lung however, the full extent and role of altered DNA methylation in fibrotic lung fibroblasts is unknown. Emerging evidence also suggests gender specific methylation differences are common in disease and could elucidate why diseases characterised by pulmonary fibrosis including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) have a sex biased prevalence.Using a Genome wide array based approach, this thesis investigates differentially methylated and expressed genes in fibrotic compared to control lung fibroblasts, gender specific methylation and expression differences and the effects of modulating DNA methylation using a DNA methyltransferase (DNMT)inhibitor, 5 Aza 2’deoxycytidine(5Aza). Data show primary human IPF and SSc lung fibroblasts have multiple genes with altered DNA methylation and expression compared to control lung fibroblasts. Multiple biological processes were enriched in these genes, many of which are relevant to fibrosis including, transcriptional regulation, extracellular matrix (ECM) organisation, Wnt signalling and apoptosis. Using siRNA knockdown and collagen gel contraction assays, novel genes including Tenascin XB (TNXB), which encodes the ECM glycoprotein TenasicnX (TNX), were identified as having potential functional significance in the pathogenesis of pulmonary fibrosis. Furthermore, multiple genes including TNXB had altered methylation and expression in IPF compared to SSc lung fibroblasts and may distinguish IPF from other diseases associated with pulmonary fibrosis. Multiple genes were identified with gender specific differences in methylation and expression in lung fibroblasts.Interestingly, multiple genes with altered methylation in IPF males compared to control males were not the same genes with altered methylation in IPF females compared to control females, which may in part explain why IPF predominates in males. The final chapter of my thesis shows 5 Aza treatment alters the methylation and expression of multiple genes in primary human lung fibroblasts. Strong correlation between changes in methylation and changes in expression were identified suggesting DNA methylation can directly regulate the expression of multiple genes in lung fibroblasts
    Sarah Hernandez, United States Patent - 2016
    The invention features compositions and methods for a pre-cancer prognostic screen
    AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000
    Erik Markel, Journal of Bacteriology - 2016
    Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and genes involved with resisting osmotic and oxidative stress. AlgU is active while these bacteria are associated with plants where its presence supports bacterial growth and disease symptoms. We found that AlgU is an important virulence factor for P. syringae pv. tomato DC3000 but that alginate production is dispensable for disease in host plants. This implies that AlgU regulates additional genes that facilitate bacterial pathogenesis. We used RNA-seq to characterize the AlgU regulon and ChIP-seq to identify AlgU-regulated promoters associated with genes directly controlled by this sigma factor. We found that in addition to genes involved with alginate and osmotic and oxidative stress responses, AlgU regulates genes with known virulence functions including components of the Hrp type III secretion system, virulence effectors and the hrpL and hrpRS transcription regulators. These data suggest that P. syringae pv. tomato DC3000 has adapted to use signals that activate AlgU to induce expression of important virulence functions that facilitate survival and disease in plants. Importance Plant immune systems produce antimicrobial and bacteriostatic conditions in response to bacterial infection. Plant pathogenic bacteria are adapted to suppress and/or tolerate these conditions, however, the mechanisms controlling these bacterial systems are largely uncharacterized. The work presented here provides a mechanistic explanation for how P. syringae pv. tomato DC3000 coordinates expression of multiple genetic systems, including those dedicated to pathogenicity, in response to environmental conditions. This work demonstrates the scope of AlgU regulation in P. syringae pv. tomato DC3000, and characterizes the promoter sequence regulated by AlgU in these bacteria.
    The serine protease, dipeptidyl peptidase IV as a myokine: dietary protein and exercise mimetics as a stimulus for transcription and release
    Leslie E. Neidert, Physiological Reports - 2016
    Dipeptidyl‐peptidase IV (DPP‐IV) is an enzyme with numerous roles within the body, mostly related to regulating energy metabolism. DPP‐IV is also a myokine, but the stimulus for its release is poorly understood. We investigated the transcription and release of DPP‐IV from skeletal muscle in a three‐part study using C2C12 myotube cultures, an acute rat exercise and postexercise feeding model, and human feeding or human exercise models. When myotubes were presented with leucine only, hydrolyzed whey protein, or chemicals that cause exercise‐related signaling to occur in cell culture, all caused an increase in the mRNA expression of DPP‐IV (1.63 to 18.56 fold change, P < 0.05), but only whey protein caused a significant increase in DPP‐IV activity in the cell culture media. When rats were fed whey protein concentrate immediately following stimulated muscle contractions, DPP‐IV mRNA in both the exercised and nonexercised gastrocnemius muscles significantly increased 2.5‐ to 3.7‐fold (P < 0.05) 3–6 h following the exercise/feeding bout; of note exercise alone or postexercise leucine‐only feeding had no significant effect. In humans, plasma and serum DPP‐IV activities were not altered by the ingestion of whey protein up to 1 h post consumption, after a 10 min bout of vigorous running, or during the completion of three repeated lower body resistance exercise bouts. Our cell culture and rodent data suggest that whey protein increases DPP‐IV mRNA expression and secretion from muscle cells. However, our human data suggest that DPP‐IV is not elevated in the bloodstream following acute whey protein ingestion or exercise.
    A critical postnatal period of heightened vulnerability to lipopolysaccharide
    Kyle S. Rourke, Respiratory Physiology & Neurobiology - 2016
    Evidence of respiratory abnormalities and vulnerability to infection during a critical period of development have been implicated in Sudden Infant Death Syndrome (SIDS). Here we investigated whether the acute hypoxic ventilatory response (HVR) exhibits a heightened vulnerability to the endotoxin lipopolysaccharide (LPS) during a critical period of development. The acute HVR was measured 2 h after an i.p. injection of saline or LPS (0.1 mg/kg) at various postnatal (P) ages (P5, P10, or P20 days). LPS attenuated the early (1–2 min) and late (4–6 min) phase of the acute HVR in P10 but not P5 or P20 rats. The P10 age group exhibited the largest increase in brainstem TNFα and iNOS mRNA expression following LPS. LPS also caused a higher mortality rate in P10 rats (48%) compared to P5 (12%) and P20 (0%) age groups. After stratifying LPS treated P10 rats into survivors vs non-survivors, only the latter exhibited an attenuated HVR (specifically the early phase). Thus, the heightened vulnerability to endotoxin exposure during this critical period of development is characterized by a depression of the ventilatory response to acute hypoxia in association with an increased incidence of mortality. These data share similarities with some of the circumstances surrounding a SIDS scenario, including evidence of infection, increased brainstem cytokine expression, a disturbance in respiratory control, and a peak incidence of mortality during a critical period of development.
    GLUT3 is present in Clone 9 liver cells and translocates to the plasma membrane in response to insulin
    Danielle M. Defries, Biochemical and Biophysical Research Communications - 2016
    Clone 9 cells have been reported to express only the GLUT1 facilitative glucose transporter; however, previous studies have not examined Clone 9 cells for GLUT3 content. The current study sought to profile the presence of glucose transporters in Clone 9 cells, H4IIE hepatoma cells, and L6 myoblasts and myotubes. While the other cell types contained the expected complement of transporters, Clone 9 cells had GLUT3 which was previously not reported. Interestingly, both GLUT3 mRNA and protein were detected in Clone 9 cells, but only mRNA for GLUT1 was detected. Glucose transport in Clone 9 cells was insulin-sensitive in a concentration-dependent manner, concomitant with the presence of GLUT3 in the plasma membrane after insulin treatment. Although basal glucose uptake was unaffected, insulin-stimulated glucose uptake was abolished with siRNA-mediated GLUT3 knockdown. These results contradict previous reports that Clone 9 cells exclusively express GLUT1 and suggest GLUT3 is a key insulin-sensitive glucose transporter required for insulin-stimulated glucose uptake by Clone 9 cells.
    Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells
    Maria K. Herndon, Molecular and Cellular Endocrinology - 2016
    FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 h, and conducted RNA deep sequencing. Results show that FSH regulates 3772 genes ≥2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs.
    CHARACTERIZATION OF DEFB103 Gene Structure, Gene Expression and its haplotype Association with three Common Diseases in Beef and Holstein Cattle
    Ali Mirabzadeh-Ardakani, M.Sc., University of Saskatchewan Thesis - 2016
    The DEF B103 gene is a member of the β defensin gene family, and it has not been well studied in cattle. In this study DEFB103 gene expression, gene structure and its haplotypes association with three diseases were studied. DEFB103 gene expression was profiled for 27 tissues in nine two week old calves and the transcript was most abundant in tissues with stratified squamous epithelium. An age dependent decrease (P <0.05) in DEFB103 gene expression was observed in buccal epithelium when comparing healthy two week old and 10 to 12 Month Old calves. A bovine herpes virus 1 respiratory infection did, however, significantly (P < 0.05) up regulate DEFB103 Gene expression in the buccal epithelium of 6 to 8 Month old calves. Immunohistochemistry was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums to confirm the DEFB103 gene expression results. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells before birth. A close association was observed between the dendritic cells and epithelial cells expressing DEFB103 in both the fetus and newborn calf, and this is consistent with the β defensin regulatory effect on dendritic cell responses.
    Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light
    Violetta Pilorz, PLOS Biol - 2016
    Light can produce either sleep or arousal in mice. This study reveals that these opposing effects depend upon the wavelength of light and appear to involve separate pathways, both modulated by the photopigment melanopsin.
    Using Fluorescence Imaging to Track Drug Delivery and Guide Treatment Planning In Vivo
    Qiaoya Lin, In Vivo Fluorescence Imaging - 2016
    Imaging has become an indispensable tool in both clinical medicine and preclinical sciences. It enables doctors to locate sites of cancer/disease, track drug delivery, and guide operative planning, thus enhancing the treatment efficacy. Recently, we developed a multimodal theranostic lipid nanoparticles, named HPPS(NIR)-chol-siRNA with its built-in near-infrared (NIR) fluorescent probe core as a useful surrogate for tracking small interfering RNA (siRNA) delivery. By using the image co-registration of computed tomography (CT) and fluorescence molecular tomography (FMT), we achieved noninvasive assessment and treatment planning of siRNA delivery into the orthotopic tumor, thus enabling efficacious RNA interference (RNAi) therapy. In this chapter, we introduce this method to illustrate the use of CT-FMT co-registration for tracking drug delivery and guiding treatment planning in vivo.
    c-Jun is Required for NF-κB-dependent, LPS-stimulated Fos-related Antigen-1 Transcription in Alveolar Macrophages
    Rakesh K Mishra, American Journal of Respiratory Cell and Molecular Biology - 2016
    Previously, we have reported that Fos-related antigen-1 (Fra-1) transcription factor promotes LPS-induced acute lung injury and mortality, and LPS-induced Fra-1 expression in the lung occurs predominantly in alveolar macrophages. NF-κB and c-Jun transcription factors play key roles in modulating inflammatory and immune responses induced by infectious and non-infectious insults. Here, we report that NF-κB and c-Jun co-regulate Fra-1 induction by LPS in alveolar macrophages, and this regulation occurs through both NF-κB and extracellular signal-regulated protein kinase (ERK) signaling pathways. Transient transfections with Fra-1 promoter-reporter constructs and inhibitor studies revealed that the transcriptional activation of Fra-1 by LPS in alveolar macrophages mediated by NF-κB and ERK1/2 signaling. Importantly, chromatin immunoprecipitation assays revealed the recruitment of c-Jun and NF-κB to the endogenous Fra-1 promoter following LPS stimulation. We found that inhibition of ERK1/2 signaling reduces LPS-stimulated c-Jun and NF-κB recruitment to the promoter. Likewise, NF-κB inhibitor blocked LPS-induced NF-κB and c-Jun binding to the promoter. ERK1/2 inhibition had no effect on c-Jun activation, but suppressed LPS-stimulated NF-κB phosphorylation. Finally, functional assays show reduced levels of LPS-stimulated NF-κB regulated pro-inflammatory IL-1β and MIP1-α expression and increased anti-inflammatory IL-10 expression in lung alveolar macrophages of Fra-1-null mice in vivo. Thus, our studies define that NF-κB and c-Jun co-regulate LPS-induced Fra-1 transcription, and that Fra-1 selectively modulates LPS-stimulated inflammatory cytokine expression in lung alveolar macrophages during inflammatory lung injury.
    Osteopontin regulates proliferation, apoptosis, and migration of murine claudin-low mammary tumor cells
    S. Saleh, BMC Cancer - 2016
    Osteopontin is a secreted phosphoglycoprotein that is expressed by a number of normal cells as well as a variety of tumor cells. With respect to breast cancer, osteopontin has been implicated in regulating tumor cell proliferation and migration/metastasis and may serve as a prognostic indicator. However it remains unclear whether osteopontin has the same impact in all breast cancer subtypes and in particular, osteopontin’s effects in claudin-low breast cancer are poorly understood.
    Nir Shpak, Aquaculture - 2016
    The recent introduction into aquaculture of RNA interference (RNAi) for producing the preferred all-male monosex cultures, which give improved yields, has raised awareness of the need to investigate the consequences of such novel biotechnological manipulations. Here, we present meta-analysis style study on data from observations of three consecutive cultured all-male (ZZ) generations of the giant freshwater prawn Macrobrachium rosenbergii (De Man). Each consecutive generation comprised the progeny of RNAi-manipulated sex-reversed males. The manipulation was achieved through the administration of dsRNA encoding the insulin-like androgenic hormone into males (which transformed them into ‘neofemales,’ ZZ) of the previous all-male generation. Each generation was cultured in a separate earthen pond for a short (~ 4–5 months) growout period. At harvest of each of the generations, the typical M. rosenbergii population structure comprising three male morphotypes was obtained. An anatomical examination of the male reproductive system of a representative specimen of the third all-male generation showed normal reproductive outputs, even though the prawns had been grown without the presence of females (WZ) for three generations. At the molecular level, expression of vital male-specific genes in the third generation of all-male M. rosenbergii culture was demonstrated. Thus, the present study showing the lack of any overtly apparent long-term consequences of the RNAi-based biotechnology provides support for the responsible use of temporal RNAi in aquaculture. Finally, the absence of the W chromosome for three generations raises questions with regard to its role and content with respect to crustacean sexual determination and differentiation.
    HIF1A and EPAS1 mRNA and protein expression during in vitro culture of human primary term cytotrophoblasts and effect of oxygen tension on their expression
    Christophe Louis Depoix, Reproductive Biology - 2016
    During the first trimester of pregnancy, placenta formation probably occurs in a low-oxygen environment necessary to protect cytotrophoblasts from oxidative stress and to allow proper gene regulation. Transcription factors involved in gene regulation under low oxygen tension are the hypoxia-inducible factors, mainly HIF1A, EPAS1 and their dimerization partner HIF1B. Little is known about their expression during in vitro culture of cytotrophoblasts under chronic hypoxia. We assessed HIF1A and EPAS1 expression in a 4-day in vitro culture of primary term cytotrophoblasts under 21% O2 and 2.5% O2. Copy numbers and relative mRNA expression were assessed by real-time quantitative polymerase chain reaction. Protein levels were quantified by immunoblot and densitometric analysis. In undifferentiated cytotrophoblasts, EPAS1 transcripts were four times more abundant than HIF1A transcripts (2.14e7 and 5e6 copies/μg total RNA, respectively). During cell culture, HIF1A mRNA expression increased after 24 h and then decreased to stay stable. The expression was even lower when cells were grown under 2.5% O2. EPAS1 mRNA expression increased during cytotrophoblast differentiation. The expression was higher when cells were under 21% O2 than when they were under 2.5% O2. Interestingly, HIF1A, but not EPAS1, was detected in the nuclei of undifferentiated cytotrophoblasts, and in the nuclei of cytotrophoblasts that grew under 21% O2. During cytotrophoblast differentiation, no variation in HIF1A protein levels was detected. To the contrary, EPAS1 protein level increased during differentiation, and oxygen tension had no effect on EPAS1 protein level. In conclusion, HIF1A and EPAS1 expression was not inhibited by chronic hypoxia during in vitro cytotrophoblast differentiation.
    Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum
    Verónica Gómez Pérez, International Journal for Parasitology: Drugs and Drug Resistance - 2016
    Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin) in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime®, 100 mg/kg/day for 28 days). After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to SbIII for promastigotes and >3-fold to SbIII and 3-fold to SbV for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.
    KAT8 Regulates Androgen Signaling in Prostate Cancer Cells
    Ji-Young Kim, Original Research - 2016
    Androgen receptor (AR) plays pivotal roles in prostate cancer. Upon androgen stimulation, AR recruits the Protein kinase N1 (PKN1), which phosphorylates histone H3 at threonine 11, with subsequent recruitment of tryptophan, aspartic acid (WD) repeat-containing protein 5 (WDR5) and the su(var)3–9, enhancer of zeste, trithorax/mixed-lineage leukemia (SET1/MLL) histone methyltransferase complex to promote AR target gene activation and prostate cancer cell growth. However, the underlying mechanisms of target gene activation and cell growth subsequent to WDR5 recruitment are not well understood. Here, we demonstrate an epigenetic cross talk between histone modifications and AR target gene regulation. We discovered that K(lysine) acetyltransferase 8 (KAT8), a member of the MOZ, YBF2/SAS2, and TIP 60 protein 1 (MYST) family of histone acetyltransferases that catalyzes histone H4 lysine 16 acetylation, colocalized with WDR5 at AR target genes, resulting in hormone-dependent gene activation in prostate cancer cells. PKN1 or WDR5 knockdown severely inhibited KAT8 association with AR target genes and histone H4 lysine 16 acetylation upon androgen treatment. Knockdown of KAT8 significantly decreased AR target gene expression and prostate cancer cell proliferation. Collectively, these data describe a transhistone modification pathway involving PKN1/histone H3 threonine 11 phosphorylation followed by WDR5/MLL histone methyltransferase and KAT8/histone acetyltransferase recruitment to effect androgen-dependent gene activation and prostate cancer cell proliferation.(Molecular Endocrinology30:925–936, 2016)
    High alpha-methylacyl-CoA racemase (AMACR) is associated with ERG expression and with adverse clinical outcome in patients with localized prostate cancer
    Adrian Box, Tumor Biology - 2016
    Alpha-methylacyl-CoA racemase (AMACR) is a well-characterized marker extensively utilized in prostate cancer (PCA) diagnosis. However, the prognostic value of AMACR expression and its relation to TMPRSS2-ERG gene rearrangement as one of the most common molecular alterations in PCA is not fully explored. AMACR expression was investigated in a cohort of 218 men with localized PCA treated by radical prostatectomy and correlated with ERG and various clinical and pathological parameters. In vitro studies assessed AMACR changes to ERG knockdown and other related genes. In addition, bioinformatics validated the significance of AMACR/ERG expression and assessed relevant genetic signatures in relation to AMACR/ERG expression. AMACR expression was significantly associated with disease progression and with ERG (p ∼0). Seventeen percent of cancer foci showed negative/weak AMACR expression while being ERG positive. High AMACR expression was significantly associated with positive surgical margins (p = 0.01), specifically in tumors with lower Gleason score <7, with ∼95 % exhibiting positive surgical margin (p = 0.008). High AMACR showed marginal association with PSA biochemical recurrence (BCR) (p = 0.06) which was slightly more pronounced in ERG-positive tumors (p = 0.04). This was validated in other public cohorts. However, in this cohort, the association with BCR was not statistically significant in multivariate analysis (p = 0.09). Using in vitro cellular models, AMACR messenger RNA (mRNA) expression, but not protein levels, showed an association with ERG expression. We report for the first time a significant association between AMACR and ERG with prognostic implication. Patients with high AMACR/ERG-positive PCA may be at higher risk for disease progression, and additional studies in larger cohorts are needed to confirm the above findings. Functional studies investigating the molecular pathways connecting AMACR and ERG may provide an additional insight into PCA progression pathways.
    Christian S. Hinrichs, United States Patent - 2016
    Disclosed are methods of preparing an isolated population of human papillomavirus (HPV)-specific T cells comprise dividing an HPV-positive tumor sample into multiple fragments; separately culturing the multiple fragments; obtaining T cells from the cultured multiple fragments; testing the T cells for specific autologous HPV-positive tumor recognition; selecting the T cells that exhibit specific autologous HPV-positive tumor recognition; and expanding the number of selected T cells to produce a population of HPV-specific T cells for adoptive cell therapy. Related methods of treating or preventing cancer using the T cells are also disclosed.
    Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells
    Lara Pasovic, Current Eye Research - 2016
    Purpose: The development of a suitable storage method for retinal pigment epithelium (RPE) is necessary in the establishment of future RPE replacement therapy, and storage temperature has proven to be pivotal for cell survival. ARPE-19, a widely used model for RPE, has been shown to yield the greatest number of viable cells when stored at 16°C compared to other storage temperatures. In this study, we analyze the gene expression profile of cultured ARPE-19 cells after seven days of storage at different temperatures in an effort to predict the gene-level consequences of storage of RPE transplants.Materials and Methods: ARPE-19 cells were cultured until confluence and then stored in minimum essential medium at 4°C, 16°C, and 37°C for seven days. The total RNA was isolated and the gene expression profile was determined using DNA microarrays. The Results were validated using qPCR.Results: Principal component and hierarchical clustering analyses show that the gene expression profiles of cell cultures stored at different temperatures cluster into separate groups. Cultures stored at 4°C cluster closest to the control cultures that were not stored and display the least change in gene expression after storage (157 differentially expressed genes). Cultures stored at 16°C and 37°C display a much larger change in differential gene expression (1787 and 1357 differentially expressed genes, respectively). At 16°C, the expression of several genes with proposed tumor suppressor functions was markedly increased. Changes in regulation of several known signaling pathways and of oxidative stress markers were discovered at both 16°C and 37°C, and activation of the angiogenesis marker vascular endothelial growth factor (VEGF) was discovered at 37°C. There was no evidence of the activation of inflammatory processes in stored cell cultures.Conclusion: ARPE-19 cultures stored at 16°C show the greatest propensity to modulate their gene expression profile in a manner that supports cell survival during storage.
    Hypoxia and Reactive Oxygen Species Homeostasis in Mesenchymal Progenitor Cells Define a Molecular Mechanism for Fracture Nonunion
    Emma Muinos-López, STEM CELLS - 2016
    Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5′Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1α independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1α-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression. Stem Cells 2016.
    Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1
    Martina Maywald, European Journal of Nutrition - 2016
    PurposeThe essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms.MethodsCell proliferation and pro-inflammatory cytokine production were monitored by [3H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression.ResultsZinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+ T cells (p < 0.05). The effect is based on zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1.ConclusionIn summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.
    Characterization of catecholamine receptors and transporters in murine macrophages - viewcontent.cgi
    Elizabeth Gon, University of Richmon Student Research - 2016
    Macrophages are a critical part of the immune response. When circulating monocytes move into tissues they differentiate into macrophages to mount the first line of defense against pathogens. Macrophages are primarily phagocytic cells, physically engulfing pathogens and cell debris at the site of an infection (Abbas et al. 2009). They play a key role in the innate immune system, initiating and maintaining the inflammatory response and acting as antigen presenting cells to active adaptive immunity (Abbas et al. 2009). Macrophages are activated in two different ways: the binding of a pro-inflammatory cytokine or by the recognition of a pathogen-associated molecular pattern (PAMP) by a receptor on the macrophage's surface (Abbas et al. 2009, Chi et al. 2003). Lipopolysaccharide (LPS) is a component of gram-negative bacterial cell walls and is a common activating PAMP that triggers NF-KB mediated pro-inflammatory effects (Abbas et al. 2009). When an immune response is mounted, macrophages act by phagocytosing pathogens and by releasing nitric oxide (NO) and other reactive intermediates as well as by secreting pro-inflammatory cytokines (Abbas et al. 2009). Cytokine release enhances the activation of phagocytes and promotes the recruitment of other immune cells to the site of infection (Abbas et al. 2009). Macrophages are able to control the spread of the pathogen through these non-specific destructive effects as well as by recruiting other immune cells to initiate a targeted immune response.
    Detection of Murine TRPA1 Transcripts in Keratinocytes
    Kelvin Y Kwan, Journal of Skin and Stem Cell - 2016
    Background: Transient receptor potential ankyrin 1 (TRPA1) is expressed by nociceptive neurons of the dorsal root ganglia (DRGs) and trigeminal ganglia, but its expression in other cell types is largely unexplored. TRPA1 contributes to chemical, mechanical and cold nociception and expression of TRPA1 in keratinocytes may extend the detection of chemical and physical stimuli from the nerve endings to the surface of the skin. Results: Comparison of TRPA1 genes from multiple inbred mouse lines revealed a large number of genetic variations in the TRPA1 locus. These genetic variations may affect annealing of primers to detect TRPA1 transcript when using nucleic acid-based methods. To consistently detect TRPA1 in genetically diverse mouse strains and in different tissue types, an optimized qPCR protocol for TRPA1 detection was created. Conclusions: TRPA1 transcripts are consistently detected in murine keratinocytes.
    Testosterone and trenbolone enanthate increase mature myostatin protein expression despite increasing skeletal muscle hypertrophy and satellite cell number in rodent muscle
    V.J Dalbo, Andrologia - 2016
    The androgen-induced alterations in adult rodent skeletal muscle fibre cross-sectional area (fCSA), satellite cell content and myostatin (Mstn) were examined in 10-month-old Fisher 344 rats (n = 41) assigned to Sham surgery, orchiectomy (ORX), ORX + testosterone (TEST; 7.0 mg week−1) or ORX + trenbolone (TREN; 1.0 mg week−1). After 29 days, animals were euthanised and the levator ani/bulbocavernosus (LABC) muscle complex was harvested for analyses. LABC muscle fCSA was 102% and 94% higher in ORX + TEST and ORX + TREN compared to ORX (p < .001). ORX + TEST and ORX + TREN increased satellite cell numbers by 181% and 178% compared to ORX, respectively (p < .01), with no differences between conditions for myonuclear number per muscle fibre (p = .948). Mstn protein was increased 159% and 169% in the ORX + TEST and ORX + TREN compared to ORX (p < .01). pan-SMAD2/3 protein was ~30–50% greater in ORX compared to SHAM (p = .006), ORX + TEST (p = .037) and ORX + TREN (p = .043), although there were no between-treatment effects regarding phosphorylated SMAD2/3. Mstn, ActrIIb and Mighty mRNAs were lower in ORX, ORX + TEST and ORX + TREN compared to SHAM (p < .05). Testosterone and trenbolone administration increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Several genes and signalling proteins related to myostatin signalling were differentially regulated by ORX or androgen therapy.
    Axonal localization and mitochondrial association of precursor microRNA 338
    Jose Norberto S.Vargas, Cellular and Molecular Life Sciences - 2016
    MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.
    Autonomous osteogenic differentiation of hASCs encapsulated in methacrylated gellan-gum hydrogels
    Mariana B. Oliveira,, Science Direct - 2016
    Methacrylated gellan-gum (GG-MA) alone and combined with collagen type I (Coll) is suggested here for the first time as a cell-laden injectable biomaterial for bone regeneration. On-chip high-throughput studies allowed rapidly assessing the suitability of 15 biomaterials/media combinations for the osteodifferentiation of human adipose stem cells (hASCs). Hydrogels composed solely of GG-MA (GG100:0Coll) led hASCs from three different donors into the osteogenic lineage after 21 days of cell culture, in the absence of any osteogenic or osteoconductive factors. Hydrogels containing more than 30% of Coll promoted increased cellular proliferation and led hASCs into osteogenic differentiation under basal conditions. Studies using isolated individual hydrogels – excluding eventual on-chip crosstalk – and standard biochemical assays corroborated such findings. The formation of focal adhesions of hASCs on GG100:0Coll hydrogels was verified. We hypothesize that the hydrogels osteogenic effect could be guided by mechanotransduction phenomena. Indeed, the hydrogels showed elastic modulus in ranges previously reported as osteoinductive and the inhibition of the actin-myosin contractility pathway impaired hASCs’ osteodifferentiation. GG-MA hydrogels also did not promote hASCs’ adipogenesis while used in basal conditions. Overall, GG-MA showed promising properties as an innovative and off-the shelf self-inducing osteogenic injectable biomaterial.
    Acrolein enhances epigenetic modifications, FasL expression and hepatocyte toxicity induced by anti-HIV drug Zidovudine
    Smita S. Ghare, Toxicology in Vitro - 2016
    Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity.
    Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes
    Nava Chapnik, The International Journal of Biochemistry & Cell Biology - 2016
    In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6 h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes.
    Effect of ovarian stimulation treatments on the embryo quality in rabbit
    José Isaí Cedano Castro, Original Paper - 2016
    Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to support both genetic improvement programs and ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, its administration mode, the donor and the environment. Nowadays the use of gonadotropins such as FSH, LH, hCG and eCG has enabled us to achieve the superovulation in different species successfully. However,the posology and the effect of both gonadotropins when used simultaneously (e.g. FSH and LH) it is not yet clearly defined. The superovulation in rabbit does and in other species, has had a great advance in recent years, changing the eCG use by the recombinant gonadotropins and jumping the morphological classification to the molecular analysis of embryos. The objective of this study was to evaluate the effect of long acting FSH CTP and rhFSH alone or supplemented with rhLH on ovarian stimulation in rabbit does and determine the impact of its stimulation on in vitro and in vivo development of fresh and cryopreserved embryos.
    Experimental Infection of Calves by Two Genetically-Distinct Strains of Rift Valley Fever Virus
    William C. Wilson, Viruses - 2016
    Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves.
    Epithelial-mesenchymal transition and FOXA genes during tobacco smoke carcinogen induced transformation of human bronchial epithelial cells
    Audun Bersaas, Toxicology in Vitro - 2016
    Lung cancer is largely an environmentally caused disease with poor prognosis. An in vitro transformation model of human bronchial epithelial cells (HBEC) was used to study long-term effects of tobacco smoke carcinogens on epithelial-mesenchymal transition (EMT) and the forkhead box transcription factors FOXA1 and FOXA2. CDK4 and hTERT immortalized HBEC2 and HBEC12 cell lines were exposed weekly to either cigarette smoke condensate (CSC), benzo[a]pyrene, or methylnitrosourea. Transformed cell lines were established from soft-agar colonies after 12 weeks of exposure. HBEC12 was transformed by all exposures while HBEC2 was only transformed by CSC. Untransformed HBEC2 showed little invasive capacity, whereas transformed cell lines completely closed the gap in a matrigel scratch wound assay. CDH1 was down-regulated in all of the transformed cell lines. In contrast, CDH2 was up-regulated in both HBEC2 and one of the HBEC12 transformed cell lines. Furthermore, transformed cells showed activation of EMT markers including SNAI1, ZEB1, VIM, and MMP2. All transformed cell lines had significant down-regulation of FOXA1 and FOXA2, indicating a possible role in cell transformation and EMT. ChIP analysis showed increased binding of Histone-H3 and macroH2A in FOXA1 and FOXA2 in the transformed HBEC2 cell lines, indicating a compact chromatin. In conclusion, long-term carcinogen exposure lead to down-regulation of FOXA1 and FOXA2 concomitantly with the occurrence of EMT and in vitro transformation in HBEC cells.
    Molecules Associated with Fatty Acid Biosynthetic Pathways and Uses Thereof - Synthetic Genomics, Inc.
    Gerardo V.Toledo, US Patent - 2016
    The present disclosure relates in part to recombinant microorganisms that include non-native genes encoding PUFA-PKS polypeptides, and to methods of making and using such microorganisms for producing at least one PUFA. In particular, the disclosure further relates to methods and related materials useful for the production of at least one PUFA by heterologous expression of the nucleic acid sequences disclosed herein encoding PUFA-PKS polypeptides.
    Effects of Arachidonic Acid Supplementation on Acute Anabolic Signaling and Chronic Functional Performance and Body Composition Adaptations
    Eduardo O. De Souza, PLOS ONE - 2016
    Background The primary purpose of this investigation was to examine the effects of arachidonic acid (ARA) supplementation on functional performance and body composition in trained males. In addition, we performed a secondary study looking at molecular responses of ARA supplementation following an acute exercise bout in rodents. Methods Thirty strength-trained males (age: 20.4 ± 2.1 yrs) were randomly divided into two groups: ARA or placebo (i.e. CTL). Then, both groups underwent an 8-week, 3-day per week, non-periodized training protocol. Quadriceps muscle thickness, whole-body composition scan (DEXA), muscle strength, and power were assessed at baseline and post-test. In the rodent model, male Wistar rats (~250 g, ~8 weeks old) were pre-fed with either ARA or water (CTL) for 8 days and were fed the final dose of ARA prior to being acutely strength trained via electrical stimulation on unilateral plantar flexions. A mixed muscle sample was removed from the exercised and non-exercised leg 3 hours post-exercise. Results Lean body mass (2.9%, p<0.0005), upper-body strength (8.7%, p<0.0001), and peak power (12.7%, p<0.0001) increased only in the ARA group. For the animal trial, GSK-β (Ser9) phosphorylation (p<0.001) independent of exercise and AMPK phosphorylation after exercise (p-AMPK less in ARA, p = 0.041) were different in ARA-fed versus CTL rats. Conclusions Our findings suggest that ARA supplementation can positively augment strength-training induced adaptations in resistance-trained males. However, chronic studies at the molecular level are required to further elucidate how ARA combined with strength training affect muscle adaptation.
    ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation
    Petrus R. de Jong, Nature Communications - 2016
    The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.
    Functional reprogramming of human prostate cancer to promote local attraction of effector CD8+ T cells
    Ravikumar Muthuswamy, The Prostate - 2016
    BACKGROUND Local infiltration of CD8+ T cells (CTLs) in tumor lesions predicts overall clinical outcomes and the clinical benefit of cancer patients from immune checkpoint blockade. In the current study, we evaluated local production of different classes of chemokines in prostate cancer lesions, and the feasibility of their modulation to promote selective entry of CTLs into prostate tumors. METHODS Chemokine expression in prostate cancer lesion was analyzed by TaqMan-based quantitative PCR, confocal fluorescence microscopy and ELISA. For ex vivo chemokine modulation analysis, prostate tumor explants from patients undergoing primary prostate cancer resections were cultured for 24 hr, in the absence or presence of the combination of poly-I:C, IFNα, and celecoxib (PAC). The numbers of cells producing defined chemokines in the tissues were analyzed by confocal microscopy. Chemotaxis of effector CD8+ T cells towards the untreated and PAC-treated tumor explant supernatants were evaluated in a standard in vitro migration assays, using 24 well trans-well plates. The number of effector cells that migrated was enumerated by flow cytometry. Pearson (r) correlation was used for analyzing correlations between chemokines and immune filtrate, while paired two tailed students t-test was used for comparison between treatment groups. RESULTS Prostate tumors showed uniformly low levels of CTL/NK/Th1-recruiting chemokines (CCL5, CXCL9, CXCL10) but expressed high levels of chemokines implicated in the attraction of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg): CCL2, CCL22, and CXCL12. Strong positive correlations were observed between CXCL9 and CXCL10 and local CD8 expression. Tumor expression levels of CCL2, CCL22, and CXCL12 were correlated with intratumoral expression of MDSC/Treg markers: FOXP3, CD33, and NCF2. Treatment with PAC suppressed intratumoral production of the Treg-attractant CCL22 and Treg/MDSC-attractant, CXCL12, while increasing the production of the CTL attractant, CXCL10. These changes in local chemokine production were accompanied by the reduced ability of the ex vivo-treated tumors to attract CD4+ FOXP3+ Treg cells, and strongly enhanced attraction of the CD8+Granzyme B+ CTLs. CONCLUSIONS Our data demonstrate that the chemokine environment in prostate cancer can be reprogrammed to selectively enhance the attraction of type-1 effector immune cells and reduce local attraction of MDSCs and Tregs. Prostate 76:1095–1105, 2016. © 2016 Wiley Periodicals, Inc.
    Physico-chemical properties and biological effects of diesel and biomass particles
    Eleonora Longhin, Environmental Pollution - 2016
    Diesel combustion and solid biomass burning are the major sources of ultrafine particles (UFP) in urbanized areas. Cardiovascular and pulmonary diseases, including lung cancer, are possible outcomes of combustion particles exposure, but differences in particles properties seem to influence their biological effects. Here the physico-chemical properties and biological effects of diesel and biomass particles, produced under controlled laboratory conditions, have been characterized. Diesel UFP were sampled from a Euro 4 light duty vehicle without DPF fuelled by commercial diesel and run over a chassis dyno. Biomass UFP were collected from a modern automatic 25 kW boiler propelled by prime quality spruce pellet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of both diesel and biomass samples showed aggregates of soot particles, but in biomass samples ash particles were also present. Chemical characterization showed that metals and PAHs total content was higher in diesel samples compared to biomass ones. Human bronchial epithelial (HBEC3) cells were exposed to particles for up to 2 weeks. Changes in the expression of genes involved in xenobiotic metabolism were observed after exposure to both UFP already after 24 h. However, only diesel particles modulated the expression of genes involved in inflammation, oxidative stress and epithelial-to-mesenchymal transition (EMT), increased the release of inflammatory mediators and caused phenotypical alterations, mostly after two weeks of exposure. These results show that diesel UFP affected cellular processes involved in lung and cardiovascular diseases and cancer. Biomass particles exerted low biological activity compared to diesel UFP. This evidence emphasizes that the study of different emission sources contribution to ambient PM toxicity may have a fundamental role in the development of more effective strategies for air quality improvement.
    FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct - me.2016-1027
    Jumpei Terakawa, Original Research - 2016
    Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of Np63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express Np63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morpho genetic protein 4 and activin A (ActA) was sufficient to induce Np63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.(Molecular Endocrinology30: 783–795, 2016
    Effect of Aronia melanocarpa (Black Chokeberry) supplementation on the development of obesity in mice fed a high-fat diet
    Jamie Baum, Journal of Berry Research - 2016
    BACKGROUND: Products derived from black chokeberry are claimed to be beneficial in treating chronic diseases, such as obesity and diabetes. OBJECTIVE: The objective of this study was to determine if supplementation with Aronia melanocarpa (black chokeberry) juice concentrate (AJC) has anti-obesity properties in mice fed obesogenic diets. METHODS: Male C57BL/6J mice (n = 10/dietary treatment) were placed on either a low-fat, high-sucrose (LFHS; 5% fat), LFHS+AJC (1.44 g AJC/kg diet), high-fat (HF; 30% fat), or HF+AJC for 12-weeks. RESULTS: Final body weight was lower in LFHS+AJC compared to LFHS, HF and HF+AJC (∼14, 20% and ∼16%, respectively; P < 0.05). Mice receiving LFHS and LFHS+AJC had significantly higher (P = 0.001) energy intake than HF and HF+AJC. LFHS-fed mice had less (–30%) epididymal fat (p < 0.05) than HF-fed mice, however mice on the LFHS+AJC had less epididymal fat per gram body weight than LFHS controls. There was no effect of diet or AJC on adipose tissue gene expression. There was no difference in plasma insulin, glucose or triglycerides between groups, however there was a positive effect of AJC on adiponectin (P = 0.059). There was also a significant effect of diet (LFHS versus HF) on HOMA-IR (P = 0.004) and HOMA-BCF (P = 0.002). CONCLUSIONS: The results from this study demonstrate that AJC supplementation has the potential to prevent weight gain and markers of obesity. Further research is needed to determine mechanisms of action.
    Josep Bassaganya-riera,, US Patent - 2016
    Provided are compounds that target the lanthionine synthetase C-like protein 2 pathway. The compounds can be used to treat a number of conditions, including infectious disease, autoimmune disease, diabetes, and a chronic inflammatory disease.
    Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein
    Cason King, PLOS Pathog - 2016
    Author Summary Studies of human adenovirus (HAdV), a small DNA tumor virus, illustrate the profound impact of viral proteins on multiple host functions. The multifunctional E1A proteins of HAdV are particularly adept at targeting key cellular regulators. Mechanistically, E1A alters or inhibits the normal function of the cellular proteins that it targets, and also establishes new connections in the cellular protein interaction network. Through these interactions, E1A creates a cellular milieu more conducive for replication. Here we show that HAdV E1A mimics cellular A-kinase anchoring proteins (AKAPs) in both appearance and function. We found that the protein kinase A (PKA) regulatory subunits are conserved targets of most HAdV E1A species. Structural modeling and a docking analysis predict a remarkable similarity between the binding of E1A and cellular AKAPs to PKA, which was confirmed experimentally. In addition, we observed E1A-mediated relocalization of PKA subunits and competition between E1A and cellular AKAPs during infection that contribute to HAdV gene expression and overall viral replication. Together, our studies identify E1A as the first known viral AKAP, and reveal a unique example of viral subversion of the PKA pathway via structural mimicry.
    DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes
    Natalie M.Hohos, Journal of Immunological Methods - 2016
    Background Peripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease-related stress. We considered the hypothesis that the various classes of peripheral leukocytes differentially regulate the synthesis of 5-methylcytosine (5mCG) and its removal via Ten-Eleven Translocation (TET) dioxygenase catalyzed hydroxymethylation to 5-hydroxymethylcytosine (5hmCG), reflecting their responsiveness to environment. Although it is known that reductions in TET1 and/or TET2 activity lead to the over-proliferation of various leukocyte precursors in bone marrow and in development of chronic myelomonocytic leukemia and myeloproliferative neoplasms, the role of 5mCG hydroxymethylation in peripheral blood is less well studied. Results We developed simplified protocols to rapidly and reiteratively isolate non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B), the turnover of 5mC by demethylation (TET1, 2, 3), and DNA repair (GADD45A, B, G) and in the global and gene-region-specific levels of DNA 5hmCG (CD4 + T cells ≫ CD14 + monocytes > CD16 + neutrophils > CD19 + B cells > CD56 + NK cells > Siglec8 + eosinophils > CD8 + T cells). Conclusions Our data taken together suggest a potential hierarchy of responsiveness among classes of leukocytes with CD4 +, CD8 + T cells and CD14 + monocytes being the most distinctly poised for a rapid methylome response to physiological stress and disease.
    Ye Li, University of Kentucky - 2016
    . Circadian rhythm is a biological rhythm with period of about 24 hours. Circadian rhythm is universal in phyla from bacteria to mammals and exist in different level from gene expression to behavior. Circadian system consists of three components: 1) a self-sustained oscillator; 2) an input pathway which can alter the phase of the oscillator; and 3) an output such as gene expression, enzyme activity, hormone production, heart rate, body temperature or loco motor activities. The way the oscillator regulates its outputs is complicated, in that on one hand usually the oscillator is not the only factor affecting the outputs, and on the other, the oscillator itself is incorporated in intricate pathways. Chicken pineal cell culture is a well-established in vitro model to study circadian rhythm. It contains a self-sustained oscillator which can be phase-shifted by light as input and rhythmically releases melatonin as an output. Here I have characterized the role of norepinephrine (NE), the sympathetic regulatory input of pineal gland, and the microenvironment of pineal cells in melatonin rhythmicity of cultured chicken pineal cells. Chapter 1 of this dissertation provides a review of circadian rhythm with a focus on melatonin regulation in pineal gland. Chapter 2 describes the methods to build up a fraction collector which offers high time resolution of sampling for a super fusion system. Chapter 3 is a technical report of a melatonin enzyme-linked immunosorbent assay suitable for high throughput measurement of melatonin. Chapter 4 presents data demonstrating that daily administration of NE recovers damped melatonin rhythm in constant darkness. In addition, NE does not change the expression of clock genes but the recovery effect of NE depends on the internal clock. Furthermore, the data indicates that NE administration stimulates the gene expression of phosphodiesterase 4D (PDE4D) and adenylate cyclase 1 (AC1) in a time order, potentially corresponding to the trough and peak of recovered melatonin rhythm. Chapter 5 presents data showing that the amplitude of melatonin rhythm in cultured pineal cells is affected by microenvironments of the cell culture and connexin plays a role in this effect. Finally, in Chapter 6I discuss how the results of each chapter demonstrate multiple regulatory mechanism of the melatonin rhythm of chicken pineal cells. Furthermore, I discuss the implications of this work in the field of developmental biology and how the current data will shape future investigations. My dissertation incorporates engineering, immunocytochemistry, chicken genetics, and biochemical analyses, and will help in better understanding the regulation mechanism of output in a circadian system.
    Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea
    Azadeh Jadali, Biology Open - 2016
    Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP) cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.
    Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis
    Rémi Labrecque, Molecular Reproduction and Development - 2016
    Follicle size is recognized as a predictor of the potential for the enclosed oocyte to yield an embryo following in vitro maturation and in vitro fertilization. Oocytes from larger follicles are more likely to reach the blastocyst stage than those from smaller follicles. A growing oocyte accumulates all the transcripts needed to ensure development until the maternal embryonic transition, and this accumulation must be completed before the period of transcriptional arrest. Accordingly, the transcriptomes of bovine germinal-vesicle-stage oocytes collected from follicles of increasing sizes (<3, 3–5, >5–8, and >8 mm) were evaluated, using the EmbryoGENE bovine transcriptomic platform (custom Agilent 4 × 44 K), to better understand transcriptional modulation in the oocyte as the follicle becomes larger. Microarray analyses revealed very few differences between oocytes from small follicles (<3 vs. 3–5 mm), whereas an important number of differences were detected at the mRNA level between oocytes from larger follicles. Weighted gene correlation network analysis allowed for the identification of several hub genes involved in crucial functions such as transcriptional regulation (TAF2), chromatin remodeling (PPP1CB), energy production (SLC25A31), as well as transport of key molecules within the cell (NAGPA, CYHR1, and SLC3A12). The results presented here thus reinforce the hypothesis that developmental competence acquisition cannot be seen as a simple one-step process, especially in regards to the modulation of mRNA. Mol. Reprod. Dev. 83: 558–569, 2016. © 2016 Wiley Periodicals, Inc.
    Non-canonical role of matrix metalloprotease (MMP) in activation and migration of hepatic stellate cells (HSCs)
    Mirza S. Baiga,, Science Direct - 2016
    Aims Matrix metalloproteinases (MMPs) that degrade extracellular matrix (ECM) and help to resolve the excess matrix are considered to be under-expressed in fibrosis. MMPs are generally anti-fibrotic, however others can have pro-fibrotic functions. Therefore, the aim of this study was to find out the mechanism of pro-fibrotic function of MMPs in hepatic stellate cells' (HSC's) activation and migration. Main methods Human MMP Antibody Array from Abcam was used to profile MMPs in macrophages. Gelatin or casein zymography was performed using 10% SDS-polyacrylamide gels (SDS-PAGE) containing gelatin (1 mg/ml) or Casein (1 mg/ml) as substrate. HSCs migration assay was performed using Boyden chamber as described previously (Guo et al., 2007, McGarrigle et al., 2006, Shan et al., 2006 and Yang and Huang, 2005). Real-time PCR with SYBR green was performed using iTaq™ universal SYBR® Green supermix (BIO-RAD) and a 7500 Real-Time PCR System (Applied Biosystems). Collagen, type I, alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA) expression was determined by immunoblot analysis. Key findings We first profiled the expression of all MMPs in primary murine bone marrow-derived macrophages (BMDMs) and differentiated THP-1 cells and found that MMP-8, -10, & -13, were significantly overexpressed after 12 h of lipopolysaccharide (LPS) treatment. Based on this pattern of expression, we speculated that macrophage MMP-8,-10, &-13 might play a non-canonical role in HSCs activation. Further, we found that exogenous active MMP-8 (Collagenase-2) treated HSC shows markedly increased migration and COL1A1 expression as compared to MMP-10 and MMP-13 treated HSCs. Thus, macrophage MMP-8 (Collagenase-2) expression in macrophages emerges as an important moderator of HSC cell migration and invasion.
    The bright side of plasmonic gold nanoparticles; activation of Nrf2, the cellular protective pathway
    Alona Goldstein, Nanoscale - 2016
    Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.
    Transcriptome Analysis of Piperlongumine-Treated Human Pancreatic Cancer Cells Reveals Involvement of Oxidative Stress and Endoplasmic Reticulum Stress Pathways
    Harsharan Dhillon, Journal of Medicinal Food - 2016
    Piperlongumine (PL), an alkaloid obtained from long peppers, displays antitumorigenic properties for a variety of human cell- and animal-based models. The aim of this study was to identify the underlying molecular mechanisms for PL anticancer effects on human pancreatic cancer cells. RNA sequencing (RNA-seq) was used to identify the effects of PL on the transcriptome of MIA PaCa-2 human pancreatic cancer cells. PL treatment of pancreatic cancer cells resulted in differential expression of 683 mRNA transcripts with known protein functions, 351 of which were upregulated and 332 of which were downregulated compared to control-treated cells. Transcripts associated with oxidative stress, endoplasmic reticulum (ER) stress, and unfolded protein response pathways were significantly overexpressed with PL treatment. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to validate the RNA-seq results, which included upregulation of HO-1, IRE1α, cytochrome c, and ASNS. The results provide key insight into the mechanisms by which PL alters cancer cell physiology and identify that activation of oxidative stress and ER stress pathways is a critical avenue for PL anticancer effects.
    TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance
    Martha R. C. Bhattacharya, The Journal of Neuroscience - 2016
    Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway. SIGNIFICANCE STATEMENT Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system.
    Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi
    R Carballar-Lejarazú, Insect Molecular Biology - 2016
    DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.
    Effects of carbon nanotubes on intercellular communication and involvement of IL-1 genes
    Yke Jildouw Arnoldussen, Journal of Cell Communication and Signaling - 2016
    An increasing amount of products containing engineered nanoparticles is emerging. Among these particles are carbon nanotubes (CNTs) which are of interest for a wide range of industrial and biomedical applications. There have been raised concerns over the effects of CNTs on human health. Some types of CNTs are classified as group 2B carcinogens by the International Agency for Research on Cancer. CNTs may also induce pulmonary inflammatory and fibrotic effects. By utilizing CNTs of different lengths, we investigated the role of the proinflammatory cytokine, interleukin-1 (IL-1) on gap junctional intercellular communication (GJIC) by using IL-1 wild-type (IL1-WT) and IL-1 knock-out (IL1-KO) cells. GJIC decreased equally in both cell types after CNT exposure. Immunofluorescence staining showed Gja1 and Gjb2 in gap junctions and hemichannels for both cell types. Gjb1 and Gjb2 expression was low in IL1-KO cells, which was confirmed by protein analysis. Gja1 was upregulated with both CNTs, whereas Gjb1 was down-regulated by CNT-2 in IL1-WT cells. Connexin mRNA expression was regulated differently by the CNTs. CNT-1 affected Gja1 and Gjb2, whereas CNT-2 had an effect on Gjb1. CNTs negatively affect GJIC through gap junctions independently of the length of CNT and IL-1 status. Furthermore, connexin gene expression was affected by IL-1 at transcriptional and translational levels. As both CNTs used in this study are cytotoxic to the cells and reduce cell survival, we suggest that CNT-induced reduction in GJIC may be important for inhibiting transfer of cell survival signals between cells.
    PROMOTION OF WOUND HEALING - Northwestern University
    Amy Paller, US Patent - 2016
    SUMMARY OF THE INVENTION In some embodiments, the present invention provides methods of promoting wound healing comprising depleting gangliosides in a subject. In some embodiments, a ganglioside depletion agent (e.g., glucosylceramide synthase inhibitor) is administered to a subject to promote wound healing. In some embodiments, the present invention provides methods of promoting wound healing comprising administering one or more glucosylceramide synthase inhibitors to a subject with one or more cutaneous wounds. In some embodiments, the subject is diabetic (e.g., Type I diabetes, Type II diabetes, gestational diabetes, etc.). In some embodiments, one or more ganglioside precursors (e.g., GM3) are targeted to promote wound healing. In some embodiments, conversion of ganglioside precursors (e.g., GM3) into gangliosides is inhibited to promote wound healing. In some embodiments, the subject is not diabetic. In some embodiments, one or more cutaneous wounds comprise one or more of incisions, lacerations, abrasions, puncture wounds, and closed wounds (e.g., diabetic ulcers, such as a foot ulcer). In some embodiments, a glucosylceramide synthase inhibitor is selected from PDMP, D-threo-EtDO-P4, ((1R, 2R)-nonanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide-L-tartaric acid salt, AMP-DNM and analogues, homologues, and functional equivalents thereof. In some embodiments, a glucosylceramide synthase inhibitor is administered systemically. In some embodiments, a glucosylceramide synthase inhibitor is administered locally. In some embodiments, a glucosylceramide synthase inhibitor is administered topically. In some embodiments, administering a glucosylceramide synthase inhibitor accelerates the rate of wound repair (e.g., the wound heals twice as fast as without said inhibitor). In some embodiments, administering a glucosylceramide synthase inhibitor reduces the chance of said wound becoming infected. In some embodiments, a ganglioside depletion agent is administered systemically. In some embodiments, a ganglioside depletion agent is administered locally. In some embodiments, a ganglioside depletion agent is administered topically. In some embodiments, administering a ganglioside depletion agent accelerates the rate of wound repair (e.g., the wound heals at least 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× as fast as without said inhibitor). In some embodiments, administering a ganglioside depletion agent reduces the chance of said wound becoming infected. In some embodiments, a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides is administered systemically. In some embodiments, a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides is administered locally. In some embodiments, a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides is administered topically. In some embodiments, administering a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides accelerates the rate of wound repair (e.g., the wound heals twice as fast as without said inhibitor). In some embodiments, administering a composition that inhibits conversion of ganglioside precursors (e.g., GM3) into gangliosides reduces the chance of said wound becoming infected. In some embodiments, the present invention provides a composition for wound care comprising one or more ganglioside depletion agents (e.g., a glucosylceramide synthase inhibitors) and an application element. In some embodiments, the present invention provides a composition for wound care comprising one or more glucosylceramide synthase inhibitors and an application element. In some embodiments, a glucosylceramide synthase inhibitor is selected from PDMP, D-threo-EtDO-P4, ((1R, 2R)-nonanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide-L-tartaric acid salt, AMP-DNM and analogues, homologues, and functional equivalents thereof. In some embodiments, the application element is configured for topical application to a wound. In some embodiments, the application element comprises a liquid, cream, paste, salve, balm, or semi-solid. In some embodiments, the application element comprises a patch, wrap, or bandage. In some embodiments, a composition further comprises one or more additional wound care agents. In some embodiments, wound care agents are selected from antiseptic, antibiotic, local anesthetic, anti-inflammatory, pain reliever, etc.
    Compositions Useful for Treating Herpes Simplex Labialis and/or Herpes Esophagitis, and Methods Using Same - DREXEL UNIVERSITY
    Clifford, Jane E., US Patent - 2016
    1. A composition comprising an anti-herpetic agent and at least one inhibitor selected from the group consisting of an ATM inhibitor, a Chk2 inhibitor, and a salt, solvate or N-oxide thereof, wherein the composition treats or prevents a HSV-1 infection in a subject in need thereof, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 2. The composition of claim 1, wherein the ATM inhibitor and the Chk2 inhibitor are independently selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, small molecule, antagonist, aptamer, peptidomimetic, and any combinations thereof. 3. The composition of claim 2, wherein the ATM inhibitor is at least one small molecule selected from the group consisting of caffeine, wortmannin, chloroquine, CP-466722, KU-55933, KU-59403, KU-60019, and a salt, N-oxide or solvate thereof. 4. The composition of claim 2, wherein the Chk2 inhibitor is at least one small molecule selected from the group consisting of Chk2 inhibitor II, SC-203885, NSC-109555, and a salt, N-oxide or solvate thereof. 5. The composition of claim 1, wherein the anti-herpetic agent is at least one selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 6. A method of treating or preventing a HSV-1 infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-herpetic agent and an effective amount of at least one inhibitor selected from the group consisting of an ATM inhibitor and a Chk2 inhibitor, whereby the HSV-1 infection is treated or prevented in the subject, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 7. The method of claim 6, wherein the ATM inhibitor and the Chk2 inhibitor are independently selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, small molecule, antagonist, aptamer, peptidomimetic and any combinations thereof. 8. The method of claim 7, wherein the ATM inhibitor is at least one small molecule selected from the group consisting of caffeine, wortmannin, chloroquine, CP-466722, KU-55933, KU-59403, KU-60019, and a salt, N-oxide or solvate thereof. 9. The method of claim 7, wherein the Chk2 inhibitor is at least one small molecule selected from the group consisting of Chk2 inhibitor II, SC-203885, NSC-109555, and a salt, N-oxide or solvate thereof. 10. The method of claim 6, wherein the anti-herpetic agent is selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 11. The method of claim 6, wherein the at least one inhibitor and the anti-herpetic agent are co-administered to the subject. 12. The method of claim 6, wherein administration of the inhibitor to the subject reduces the amount of the anti-herpetic agent required to be administered to the subject to obtain the same therapeutic benefit obtained when the effective dose of the anti-herpetic agent in the absence of the inhibitor is administered to the subject. 13. The method of claim 6, wherein the subject experiences less frequent or less severe side effects of the anti-herpetic agent, as compared to when the effective dose of the anti-herpetic agent in the absence of the inhibitor is administered to the subject. 14. The method of claim 6, wherein development of resistance to the anti-herpetic agent is prevented or minimized in the subject, as compared to when the effective dose of the anti-herpetic agent in the absence of the inhibitor is administered to the subject. 15. A method of treating or preventing a HSV-1 infection in a subject in need thereof, wherein the infection is caused by a drug-resistant HSV-1 strain, the method comprising administering to the subject an effective amount of at least one inhibitor selected from the group consisting of an ATM inhibitor and a Chk2 inhibitor, wherein the subject is optionally further administered an effective amount of an anti-herpetic agent, wherein administration of the at least one inhibitor treats or prevents the HSV-1 infection in the subject, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 16. The method of claim 15, wherein the ATM inhibitor and the Chk2 inhibitor are independently selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, small molecule, antagonist, aptamer, peptidomimetic and any combinations thereof. 17. The method of claim 16, wherein the ATM inhibitor is at least one small molecule selected from the group consisting of caffeine, wortmannin, chloroquine, CP-466722, KU-55933, KU-59403, KU-60019, and a salt, N-oxide or solvate thereof. 18. The method of claim 16, wherein the Chk2 inhibitor is at least one small molecule selected from the group consisting of Chk2 inhibitor II, SC-203885, NSC-109555, and a salt, N-oxide or solvate thereof. 19. The method of claim 15, wherein the anti-herpetic agent is at least one selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 20. The method of claim 15, wherein the drug-resistant HSV-1 strain has a TK mutation. 21. The method of claim 15, wherein the strain is resistant to at least one selected from the group consisting of acyclovir, famciclovir, penciclovir, valacyclovir, acyclovir, trifluridine, penciclovir and valacyclovir. 22. A kit comprising at least one inhibitor selected from the group consisting of an ATM inhibitor and a Chk2 inhibitor, the kit further comprising an applicator; and an instructional material for the use of the kit, wherein the instruction material comprises instructions for treating, ameliorating or preventing a HSV-1 infection in a subject in need thereof, wherein the HSV-1 infection comprises at least one selected from the group consisting of herpes simplex labialis and herpes esophagitis. 23. The kit of claim 22, wherein the kit further comprises an anti-herpetic agent.
    IGSF10 mutations dysregulate gonadotropin-releasing hormone neuronal migration resulting in delayed puberty
    Sasha R Howard, EMBO Molecular Medicine - 2016
    Early or late pubertal onset affects up to 5% of adolescents and is associated with adverse health and psychosocial outcomes. Self-limited delayed puberty (DP) segregates predominantly in an autosomal dominant pattern, but the underlying genetic background is unknown. Using exome and candidate gene sequencing, we have identified rare mutations in IGSF10 in 6 unrelated families, which resulted in intracellular retention with failure in the secretion of mutant proteins. IGSF10 mRNA was strongly expressed in embryonic nasal mesenchyme, during gonadotropin-releasing hormone (GnRH) neuronal migration to the hypothalamus. IGSF10 knockdown caused a reduced migration of immature GnRH neurons in vitro, and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Additionally, loss-of-function mutations in IGSF10 were identified in hypothalamic amenorrhea patients. Our evidence strongly suggests that mutations in IGSF10 cause DP in humans, and points to a common genetic basis for conditions of functional hypogonadotropic hypogonadism (HH). While dysregulation of GnRH neuronal migration is known to cause permanent HH, this is the first time that this has been demonstrated as a causal mechanism in DP.
    Monocyte-Derived Macrophages Contribute to Spontaneous Long-Term Functional Recovery after Stroke in Mice
    Somsak Wattananit, The Journal of Neuroscience - 2016
    Stroke is a leading cause of disability and currently lacks effective therapy enabling long-term functional recovery. Ischemic brain injury causes local inflammation, which involves both activated resident microglia and infiltrating immune cells, including monocytes. Monocyte-derived macrophages (MDMs) exhibit a high degree of functional plasticity. Here, we determined the role of MDMs in long-term spontaneous functional recovery after middle cerebral artery occlusion in mice. Analyses by flow cytometry and immunocytochemistry revealed that monocytes home to the stroke-injured hemisphere., and that infiltration peaks 3 d after stroke. At day 7, half of the infiltrating MDMs exhibited a bias toward a proinflammatory phenotype and the other half toward an anti-inflammatory phenotype, but during the subsequent 2 weeks, MDMs with an anti-inflammatory phenotype dominated. Blocking monocyte recruitment using the anti-CCR2 antibody MC-21 during the first week after stroke abolished long-term behavioral recovery, as determined in corridor and staircase tests, and drastically decreased tissue expression of anti-inflammatory genes, including TGFβ, CD163, and Ym1. Our results show that spontaneously recruited monocytes to the injured brain early after the insult contribute to long-term functional recovery after stroke. SIGNIFICANCE STATEMENT For decades, any involvement of circulating immune cells in CNS repair was completely denied. Only over the past few years has involvement of monocyte-derived macrophages (MDMs) in CNS repair received appreciation. We show here, for the first time, that MDMs recruited to the injured brain early after ischemic stroke contribute to long-term spontaneous functional recovery through inflammation-resolving activity. Our data raise the possibility that inadequate recruitment of MDMs to the brain after stroke underlies the incomplete functional recovery seen in patients and that boosting homing of MDMs with an anti-inflammatory bias to the injured brain tissue may be a new therapeutic approach to promote long-term improvement after stroke.
    Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption
    Alexis Demonbreun, The American Journal of Pathology - 2016
    Dysferlin is a membrane-associated protein implicated in membrane resealing; loss of dysferlin leads to muscular dystrophy. We examined the same loss-of-function Dysf mutation in two different mouse strains, 129T2/SvEmsJ (Dysf129) and C57BL/6J (DysfB6). Although there are many genetic differences between these two strains, we focused on polymorphisms in Anxa6 because these variants were previously associated with modifying a pathologically distinct form of muscular dystrophy and increased the production of a truncated annexin A6 protein. Dysferlin deficiency in the C57BL/6J background was associated with increased Evan's Blue dye uptake into muscle and increased serum creatine kinase compared to the 129T2/SvEmsJ background. In the C57BL/6J background, dysferlin loss was associated with enhanced pathologic severity, characterized by decreased mean fiber cross-sectional area, increased internalized nuclei, and increased fibrosis, compared to that in Dysf129 mice. Macrophage infiltrate was also increased in DysfB6 muscle. High-resolution imaging of live myofibers demonstrated that fibers from DysfB6 mice displayed reduced translocation of full-length annexin A6 to the site of laser-induced sarcolemmal disruption compared to Dysf129 myofibers, and impaired translocation of annexin A6 associated with impaired resealing of the sarcolemma. These results provide one mechanism by which the C57BL/6J background intensifies dysferlinopathy, giving rise to a more severe form of muscular dystrophy in the DysfB6 mouse model through increased membrane leak and inflammation.
    LNCRHOXF1: a long noncoding RNA from the X-chromosome that suppresses viral response genes during development of the early human placenta
    Ian Penkala, Molecular and Cellular Biology - 2016
    Long noncoding RNAs (lncRNAs) can regulate gene expression in a cell-specific fashion during development. Here we identify a novel lncRNA from the X-chromosome that we named lncRHOXF1 and which is abundantly expressed in trophectoderm and primitive endoderm cells of human blastocyst-stage embryos. LncRHOXF1 is a spliced and polyadenylated lncRNA about 1 kb in length, found in both the nuclear and cytoplasmic compartments of in vitro differentiated human trophectoderm progenitor cells. Gain of function experiments in human embryonic stem cells, which normally lack lncRHOXF1 RNA, revealed that lncRHOXF1 reduced proliferation and favored cellular differentiation. LncRHOXF1 knockdown using siRNAs in human trophectoderm progenitors increased expression of viral response genes, including type I interferon. Sendai virus infection of human trophectoderm progenitor cells increased lncRHOXF1 RNA levels and siRNA-mediated disruption of lncRHOXF1 during infection reduced the expression of viral response genes leading to higher virus replication. Thus, lncRHOXF1 RNA is the first example of a lncRNA that regulates the host response to viral infections in human placental progenitor cells, and we propose that it functions as a repressor of the viral response during early human development.
    Bcl-2 Regulates Reactive Oxygen Species Signaling and a Redox-Sensitive Mitochondrial Proton Leak in Mouse Pancreatic β-Cells: Endocrinology: Vol 157, No 6
    Michal Aharoni-Simon, Endocrine Society - 2016
    In pancreatic β-cells, controlling the levels of reactive oxygen species (ROS) is critical to counter oxidative stress, dysfunction and death under nutrient excess. Moreover, the fine-tuning of ROS and redox balance is important in the regulation of normal β-cell physiology. We recently demonstrated that Bcl-2 and Bcl-xL, in addition to promoting survival, suppress β-cell glucose metabolism and insulin secretion. Here, we tested the hypothesis that the nonapoptotic roles of endogenous Bcl-2 extend to the regulation of β-cell ROS and redox balance. We exposed mouse islet cells and MIN6 cells to the Bcl-2/Bcl-xL antagonist Compound 6 and the Bcl-2-specific antagonist ABT-199 and evaluated ROS levels, Ca2+ responses, respiratory control, superoxide dismutase activity and cell death. Both acute glucose stimulation and the inhibition of endogenous Bcl-2 progressively increased peroxides and stimulated superoxide dismutase activity in mouse islets. Importantly, conditional β-cell knockout of Bcl-2 amplified glucose-induced formation of peroxides. Bcl-2 antagonism also induced a mitochondrial proton leak that was prevented by the antioxidant N-acetyl-L-cysteine and, therefore, secondary to redox changes. We further established that the proton leak was independent of uncoupling protein 2 but partly mediated by the mitochondrial permeability transition pore. Acutely, inhibitor-induced peroxides promoted Ca2+ influx, whereas under prolonged Bcl inhibition, the elevated ROS was required for induction of β-cell apoptosis. In conclusion, our data reveal that endogenous Bcl-2 modulates moment-to-moment ROS signaling and suppresses a redox-regulated mitochondrial proton leak in β-cells. These noncanonical roles of Bcl-2 may be important for β-cell function and survival under conditions of high metabolic demand. - See more at: http://press.endocrine.org/doi/abs/10.1210/en.2015-1964?journalCode=endo&#sthash.PLRoyXBy.dpuf
    Hyper-Variability in Circulating Insulin, High Fat Feeding Outcomes, and Effects of Reducing Ins2 Dosage in Male Ins1 -Null Mice in a Specific Pathogen-Free Facility
    Nicole M. Templeman, PLOS ONE - 2016
    Insulin is an essential hormone with key roles in energy homeostasis and body composition. Mice and rats, unlike other mammals, have two insulin genes: the rodent-specific Ins1 gene and the ancestral Ins2 gene. The relationships between insulin gene dosage and obesity has previously been explored in male and female Ins2 -/- mice with full or reduced Ins1 dosage, as well as in female Ins1 -/- mice with full or partial Ins2 dosage. We report herein unexpected hyper-variability in Ins1 -null male mice, with respect to their circulating insulin levels and to the physiological effects of modulating Ins2 gene dosage. Two large cohorts of Ins1 -/- : Ins2 +/- mice and their Ins1 -/- : Ins2 +/+ littermates were fed chow diet or high fat diet (HFD) from weaning, and housed in specific pathogen-free conditions. Cohort A and cohort B were studied one year apart. Contrary to female mice from the same litters, inactivating one Ins2 allele on the complete Ins1 -null background did not consistently cause a reduction of circulating insulin in male mice, on either diet. In cohort A, all HFD-fed males showed an equivalent degree of insulin hypersecretion and weight gain, regardless of Ins2 dosage. In cohort B the effects of HFD appeared generally diminished, and cohort B Ins1 -/- : Ins2 +/- males showed decreased insulin levels and body mass compared to Ins1 -/- : Ins2 +/+ littermates, on both diets. Although experimental conditions were consistent between cohorts, we found that HFD-fed Ins1 -/- : Ins2 +/- mice with lower insulin levels had increased corticosterone. Collectively, these observations highlight the phenotypic characteristics that change in association with differences in circulating insulin and Ins2 gene dosage, particularly in male mice.
    Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to Atlantic salmon (Salmo salar L.)
    Gyri T. Haugland, Aquaculture - 2016
    Cleaner fish such as lumpfish (Cyclopterus lumpus L.) and ballan wrasse (Labrus bergylta A.) are increasingly used to delouse farmed Atlantic salmon (Salmo salar L.). In 2014, > 20 million cleaner fish were placed into net-pens with farmed salmon in Norway. Amoebic gill disease (AGD), caused by the opportunistic, parasitic amoeba Paramoeba perurans, is emerging in salmon farming in Northern Europe. The amoeba displays low host specificity as it has been isolated from a range of fish species in addition to salmonids, such as wrasse and lumpfish cohabitating with farmed salmon. It is, however, not known to which degree lumpfish respond to P. perurans challenge, to which extent lumpfish may develop AGD, and if they can function as a vector for the spread of P. perurans to salmon. The present study shows that lumpfish can be infected with P. perurans under experimental conditions and develop AGD. However, lumpfish are more resistant and the development of pathology is slower compared to salmon. It is also shown that lumpfish can act as carriers and transmit parasitic amoebae to Atlantic salmon. Importantly, it is demonstrated that the gill lesion score system extensively used for evaluating AGD in Atlantic salmon is less suitable for lumpfish infected with P. perurans as the disease develops more slowly in lumpfish and because lumpfish may be non-symptomatic carriers. Statement of relevance 1) The last few years, lumpfish have been used as cleaner fish in order to delouse farmed Atlantic salmon. However, nothing is yet known about its susceptibility to the parasitic amoeba P. perurans. 2) Currently, nothing is known about amoebic gill disease (AGD) development in lumpfish and the possibility that amoeba may be transferred from lumpfish to salmon. 3) There is currently little information about the pathology (macroscopic and histological changes) in lumpfish. 4) We have found that the commonly used gill scoring system for salmon is not applicable for lumpfish, and we recommend that the lumpfish is screened, not scored before transfer to net-pens with salmon.
    Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation
    Eri Morimoto, Journal of Cellular Physiology - 2016
    Inhibition of Runx2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)-induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2dox cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2-driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT-qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co-treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox-driven alkaline phosphatase (ALP) activity in control ST2/Rx2dox cells, but not in cells in which Wif1 was silenced. Unlike its anti-mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S-phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2-driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2dox cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.
    Epigenetic silencing of miR-124 prevents spermine oxidase regulation: implications for Helicobacter pylori-induced gastric cancer
    T. Murray-Stewart, Oncogene - 2016
    Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3′-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.
    Maleah dissertation Final (3-29-16).pdf
    Angelia Maleah Holland, A dissertation submitted to the Graduate Faculty of Auburn University - 2016
    We investigated the effects of a low carbohydrate ketogenic diet ( KD),versus other diets, on adipose tissue, liver and serum biomarkers in exercise trained versus sedentary rodents . METHODS: Male Sprague -Dawley rats (~9 -10 weeks of age) remained sedentary (SED) or exercised daily with resistance loaded running wheels (EX) over 6 weeks. Rats were provided isocaloric amounts of KD (20.2% protein, 10.3% carbohydrate, 69.5% fat), Western diet (WD; 15.2% protein, 42.7 carbohydrate, 42.0% fat), or Standard Chow(SC; 24.0% protein, 58.0% carbohydrate, 18.0% fat); n=8 10 in each diet group for SED and EX rats.Upon euthanasia, body and select adipose tissue masses were recorded and preserved for analyses, and liver and serum were also removed and preserved for analyses. RESULTS: Body mass and feed efficiency was greater in WD and SC fed vs. KD fed rats (p<0.001). Diet (WD>KD=SC, p<0.05)and activity (SED>EX, p<0.05) effects existed for raw and relative (body mass adjusted) omental adipose tissue (OMAT)masses. OMAT adipocyte diameters were lowest in KD fed rats p<0.01). Activity effects (EX>SED, p<0.05 to p<0.001)existed for OMAT acetyl Co A carboxylase (ACC),phospho/pan p65 (Ser536), phosphopan-AMPK (Thr172), phosphor /pan-HSL (Ser563),and uncoupling protein 1 (UCP1) protein expression patterns. Raw inguinal/subcutaneous (SQ)masses were greater in WD vs. KD fed rats (p<0.01), and diet (WD>KD, p<0.05) and activity (SED>EX, p<0.01) effects existed for raw subscapular brown adipose tissue (BAT)masses. Diet effects existed for liver iii triglycerides (WD>KD=SC, p<0.001), and diet (WD=SC>KD, p<0.05) and activity effects (SED>EX, p=0.01) existed for liver phosphopan p65.Diet (WD=SC>KD, p<0.001) and activity (SED>EX, p<0.001) effects existed for serum insulin,and diet (KD>SC, p<0.01) and activity (SED>EX, p<0.01) effects existed for serum β hydroxybutyrate (BHB). In all rats, serum insulin was positively associated with body mass (r=0.54, p<0.001), feed efficiency (r=0.57, p<0.001), relative OMAT mass (r=0.57, p<0.001), and relative SQ mass (r=0.31, p<0.05). CONCLUSIONS: While KD fed rats weighed less and had lower adipose tissue masses compared to WD and/or SC-fed rats, exercise did not enhance body or fat mass maintenance and/or further alter adipose tissue, liver or serum biomarkers in KD-fed rats. Notwithstanding, the association data suggest lower insulin with KD and/or exercise may facilitate attenuation of adipose tissue mass accretion in rodents
    Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes
    Tor Paaske Utheim, PLOS ONE - 2016
    Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.
    Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases
    Shan Yu, The FASEB Journal - 2016
    Liver X receptor (LXR), a nuclear hormone receptor, is an essential regulator of immune responses. Activation of LXR-mediated transcription by synthetic agonists, such as T0901317 and GW3965, attenuates progression of inflammatory disease in animal models. However, the adverse effects of these conventional LXR agonists in elevating liver lipids have impeded exploitation of this intriguing mechanism for chronic therapy. Here, we explore the ability of a series of sterol-based LXR agonists to alleviate inflammatory conditions in mice without hepatotoxicity. We show that oral treatment with sterol-based LXR agonists in mice significantly reduces dextran sulfate sodium colitis-induced body weight loss, which is accompanied by reduced expression of inflammatory markers in the large intestine. The anti-inflammatory property of these agonists is recapitulated in vitro in mouse lamina propria mononuclear cells, human colonic epithelial cells, and human peripheral blood mononuclear cells. In addition, treatment with LXR agonists dramatically suppresses inflammatory cytokine expression in a model of traumatic brain injury. Importantly, in both disease models, the sterol-based agonists do not affect the liver, and the conventional agonist T0901317 results in significant liver lipid accumulation and injury. Overall, these results provide evidence for the development of sterol-based LXR agonists as novel therapeutics for chronic inflammatory diseases.—Yu, S., Li, S., Henke, A., Muse, E. D., Cheng, B., Welzel, G., Chatterjee, A. K., Wang, D., Roland, J., Glass, C. K., Tremblay, M. Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases.
    GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine
    David B. Finlay, PeerJ - 2016
    he orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.
    Retinoic acid receptor signaling preserves tendon stem cell characteristics and prevents spontaneous differentiation in vitro | Stem Cell Research & Therapy | Full Text
    Stuart Webb, Stem Cell Research & Therapy - 2016
    Previous studies have reported that adult mesenchymal stem cells (MSCs) tend to gradually lose their stem cell characteristics in vitro when placed outside their niche environment. They subsequently undergo spontaneous differentiation towards mesenchymal lineages after only a few passages. We observed a similar phenomenon with adult tendon stem cells (TSCs) where expression of key tendon genes such as Scleraxis (Scx), are being repressed with time in culture. We hypothesized that an environment able to restore or maintain Scleraxis expression could be of therapeutic interest for in vitro use and tendon cell-based therapies.
    Phenotypical and biochemical characterisation of resistance for parasitic weed (Orobanche foetida Poir.) in radiation-mutagenised mutants of chickpea
    Ines Brahmi, Pest Management Science - 2016
    BACKGROUND Some radiation-mutagenised chickpea mutants potentially resistant to the broomrape, Orobanche foetida Poir., were selected through field trials. The objectives of this work were to confirm resistance under artificial infestation, in pots and mini-rhizotron systems, and to determine the developmental stages of broomrape affected by resistance and the relevant resistance mechanisms induced by radiation mutagenesis. RESULTS Among 30 mutants tested for resistance to O. foetida, five shared strong resistance in both pot experiments and mini-rhizotron systems. Resistance was not complete, but the few individuals that escaped resistance displayed high disorders of shoot development. Results demonstrated a 2–3-fold decrease in stimulatory activity of root exudates towards broomrape seed germination in resistant mutants in comparison with non-irradiated control plants and susceptible mutants. Resistance was associated with an induction of broomrape necrosis early during infection. When infested, most of the resistant mutants shared enhanced levels of soluble phenolic contents, phenylalanine ammonia lyase activity, guaiacol peroxidase activity and polyphenol oxidase activity, in addition to glutathione and notably ascorbate peroxidase gene expression in roots. CONCLUSION Results confirmed enhanced resistance in chickpea radiation-mutagenised mutants, and demonstrated that resistance is based on alteration of root exudation, presumed cell-wall reinforcement and change in root oxidative status in response to infection. © 2016 Society of Chemical Industry
    A Closed Chondromimetic Environment within Magnetic-Responsive Liquified Capsules Encapsulating Stem Cells and Collagen II/TGF-β3 Microparticles
    Clara R. Correia, Advanced Healthcare Materials - 2016
    TGF-β3 is enzymatically immobilized by transglutaminase-2 action to poly(l-lactic acid) microparticles coated with collagen II. Microparticles are then encapsulated with stem cells inside liquified spherical compartments enfolded with a permselective shell through layer-by-layer adsorption. Magnetic nanoparticles are electrostatically bound to the multilayered shell, conferring magnetic-response ability. The goal of this study is to engineer a closed environment inside which encapsulated stem cells would undergo a self-regulated chondrogenesis. To test this hypothesis, capsules are cultured in chondrogenic differentiation medium without TGF-β3. Their biological outcome is compared with capsules encapsulating microparticles without TGF-β3 immobilization and cultured in normal chondrogenic differentiation medium containing soluble TGF-β3. Glycosaminoglycans quantification demosntrates that similar chondrogenesis levels are achieved. Moreover, collagen fibrils resembling the native extracellular matrix of cartilage can be observed. Importantly, the genetic evaluation of characteristic cartilage markers confirms the successful chondrogenesis, while hypertrophic markers are downregulated. In summary, the engineered capsules are able to provide a suitable and stable chondrogenesis environment for stem cells without the need of TGF-β3 supplementation. This kind of self-regulated capsules with softness, robustness, and magnetic responsive characteristics is expected to provide injectability and in situ fixation, which is of great advantage for minimal invasive strategies to regenerate cartilage.
    Gluten-free diet increases beta-cell volume and improves glucose tolerance in an animal model of type 2 diabetes
    Karsten Buschard, Diabetes/Metabolism Research and Reviews - 2016
    Background Gluten-free (GF) diet alleviates type 1 diabetes in animal models and possibly in humans. We recently showed that fatty acid-induced insulin secretion is enhanced by enzymatically digested gluten (gliadin) stimulation in INS-1E insulinoma cells. We therefore hypothesized that GF diet would induce beta-cell rest and ameliorate type 2 diabetes. Methods C57BL/6JBomTac (B6) mice were fed a high-fat (HF), gluten-free high-fat (GF–HF), standard (STD) or gluten-free (GF) diet for 42 weeks. Results Short-term (6–24 weeks) GF–HF versus HF feeding impaired glucose tolerance and increased fasting glucose. Long-term (36–42 weeks) GF–HF versus HF feeding improved glucose tolerance and decreased fasting leptin. Mice fed a GF–HF versus HF diet for 42 weeks showed higher volumes of beta cells, islets and pancreas. The beta-cell volume correlated with the islet- and pancreas volume as well as body weight. GF–HF versus HF diet did not influence toll-like receptor 4 (Tlr4), interleukin 1 (IL-1), interleukin 6 (IL-6) or tumour necrosis factor-alpha (TNF-alpha) mRNA expression in intestine. STD versus GF feeding did not affect any parameter studied. Conclusions Long-term feeding with GF–HF versus HF increases beta-cell volume and improves glucose tolerance in B6 mice. The mechanism may include beta-cell rest, but is unlikely to include TLR4 and proinflammatory cytokines in the intestine. Beta-cell volume correlates with pancreas volume and body weight, indicating that insulin secretion capacity controls pancreas volume. Thus, long-term GF diets may be beneficial for obese type 2 diabetes patients and trials should be performed. Copyright © 2016 John Wiley & Sons, Ltd.
    Impact of KRAS, BRAF, PIK3CA, TP53 status and intraindividual mutation heterogeneity on outcome after liver resection for colorectal cancer metastases
    Inger Marie Løes, International Journal of Cancer - 2016
    We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p < 0.001, 0.002 and 0.023, respectively). KRAS and BRAF mutations also predicted a reduced median disease-specific survival (DSS) (29 vs. 51 and 16 vs. 49 months; p <0.001 and 0.008, respectively). No effect of TP53 (60.4%) mutation status was observed. Postoperative, but not preoperative chemotherapy improved both TTR and DSS (p < 0.001 for both) with no interaction with gene mutation status. Among 94 patients harboring two or more metastatic deposits, 13 revealed mutation heterogeneity across metastatic deposits for at least one gene. Mutation heterogeneity predicted reduced median DSS compared to homogeneous mutations (18 vs. 37 months; p = 0.011 for all genes; 16 vs. 26 months; p < 0.001 analyzing BRAF or KRAS mutations separately). In multivariate analyses, KRAS or BRAF mutations consistently predicted poor TRR and DSS. Mutation heterogeneity robustly predicted DSS but not TTR, while postoperative chemotherapy improved both TTR and DSS. Our findings indicate that BRAF and KRAS mutations as well as mutation heterogeneity predict poor outcome in CRC patients subsequent to liver resections and might help guide treatment decisions.
    MicroRNA Expression during Bovine Oocyte Maturation and Fertilization
    Graham C. Gilchrist, International Journal of Molecular Sciences - 2016
    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.
    Glucagon-like peptide-1 and vitamin D: anti-inflammatory response in diabetic kidney disease in db/db mice and in cultured endothelial cells
    Yael Einbinder, Diabetes/Metabolism Research and Reviews - 2016
    Background Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that stimulates insulin secretion and may affect the inflammatory pathways involved in diabetes mellitus. Calcitriol, an active form of vitamin D, plays an important role in renal, endothelial and cardiovascular protection. We evaluated the anti-inflammatory and histologic effects of a GLP-1 analogue (liraglutide) and of calcitriol in a db/db mouse diabetes model and in endothelial cells exposed to a diabetes-like environment. Methods Diabetic db/db mice were treated with liraglutide and calcitriol for 14 weeks, after which the kidneys were perfused and removed for mRNA and protein analysis and histology. Endothelial cells were stimulated with advanced glycation end products (AGEs), glucose, liraglutide and calcitriol. Total RNA and protein were extracted and analysed for the expression of selected inflammatory markers. Results Typical histological changes, glomerular enlargement and mesangial expansion were seen in db/db mice compared with control mice. Glomerular hypertrophy was ameliorated with liraglutide, compared with db/db controls. Liraglutide up-regulated endothelial nitric oxide synthase protein expression compared with the db/db control group and down-regulated p65 protein expression. Calcitriol did not further improve the beneficial effect observed on protein expression. In endothelial cells, liraglutide treatment exhibited a dose-dependent ability to prevent an inflammatory response in the selected markers: thioredoxin-interacting protein, p65, IL6 and IL8. In most gene and protein expressions, addition of calcitriol did not enhance the effect of liraglutide. Conclusions The GLP-1 analogue liraglutide prevented the inflammatory response observed in endothelial cells exposed to a diabetes-like environment and in db/db mice at the level of protein expression and significantly ameliorated the glomerular hypertrophy seen in the diabetic control group. Copyright © 2016 John Wiley & Sons, Ltd.
    Acidified nitrite inhibits proliferation of Listeria monocytogenes — Transcriptional analysis of a preservation method
    Stefanie Müller-Herbst, International Journal of Food Microbiology - 2016
    Sodium nitrite (NaNO2) is added as a preservative during raw meat processing such as raw sausage production to inhibit growth of pathogenic bacteria. In the present study it was shown in challenge assays that the addition of sodium nitrite indeed inhibited growth and survival of Listeria monocytogenes in short-ripened spreadable raw sausages. Furthermore, in vitro growth analyses were performed, which took into account combinations of various parameters of the raw sausage ripening process like temperature, oxygen availability, pH, NaCl concentration, and absence or presence of NaNO2. Data based on 300 growth conditions revealed that the inhibitory effect of nitrite was most prominent in combination with acidification, a combination that is also achieved during short-ripened spreadable raw sausage production. At pH 6.0 and below, L. monocytogenes was unable to replicate in the presence of 200 mg/l NaNO2. During the adaptation of L. monocytogenes to acidified nitrite stress (pH 6.0, 200 mg/l NaNO2) in comparison to acid exposure only (pH 6.0, 0 mg/l NaNO2), a massive transcriptional adaptation was observed using microarray analyses. In total, 202 genes were up-regulated and 204 genes were down-regulated. In accordance with growth inhibition, a down-regulation of genes encoding for proteins which are involved in central cellular processes, like cell wall/membrane/envelope biogenesis, translation and ribosomal structure and biogenesis, transcription, and replication, recombination and repair, was observed. Among the up-regulated genes the most prominent group belonged to poorly characterized genes. A considerable fraction of the up-regulated genes has been shown previously to be up-regulated intracellularly in macrophages, after exposure to acid shock or to be part of the SigB regulon. These data indicate that the adaptation to acidified nitrite partly overlaps with the adaptation to stress conditions being present during host colonization.
    Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny
    José Antonio Mata-Sotresa, Elsevier - 2016
    In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24 h cycle in larvae reared under a 12 h light/12 h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60 days post-hatch. In each sampling day, larvae were sampled every 3 h during a complete 24 h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes.
    Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2–Dependent Bladder Cancer Cell Migration and Invasion
    Sounak Gupta, The American Journal of Pathology - 2016
    Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β–induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers.
    Fluid flow and Smad2 affects the response of vascular endothelial cells
    Linda Selene Tamez, UNIVERSITY OF CALGARY Thesis - 2016
    Human aortic endothelial cells (HAECs) have been observed to respond to fluid flow and shear stress by activating different signaling molecules both in vitro and in vivo. An important example of these flow activated molecules is Smad2. Smad2 is a signaling molecule and transcription factor that has shown to be indispensable for the maintenance of vascular integrity. The aim of this study was to understand the effect of shear stress and Smad2 knockdown on endothelial gene expression.HAEC were transfected with Smad2 siRNA, and exposed to steady laminar shear stress (10 dyne/cm 2). Our results showed that Smad2 siRNA and shear stress significantly up regulated genes involved in atherosclerosis, heart dysfunction, and angiogenes is. Furthermore, Smad2 siRNA had a negative impact on athero-protective genes under static conditions. This is the first reported Smad2 siRNA gene expression profile of endothelial cells. Our findings suggest that Smad2 may a have a protective role against cardiovascular diseases.
    Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles - 657534.pdf
    Mariona Esquerdo, PLOS One - 2016
    Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situhybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential sub-functionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.
    PLOS ONE: Pharmacological and Genetic Modulation of REV-ERB Activity and Expression Affects Orexigenic Gene Expression
    Ariadna Amador, Plos One - 2016
    The nuclear receptors REV-ERBα and REV-ERBβ are transcription factors that play pivotal roles in the regulation of the circadian rhythm and various metabolic processes. The circadian rhythm is an endogenous mechanism, which generates entrainable biological changes that follow a 24-hour period. It regulates a number of physiological processes, including sleep/wakeful cycles and feeding behaviors. We recently demonstrated that REV-ERB-specific small molecules affect sleep and anxiety. The orexinergic system also plays a significant role in mammalian physiology and behavior, including the regulation of sleep and food intake. Importantly, orexin genes are expressed in a circadian manner. Given these overlaps in function and circadian expression, we wanted to determine whether the REV-ERBs might regulate orexin. We found that acute in vivo modulation of REV-ERB activity, with the REV-ERB-specific synthetic ligand SR9009, affects the circadian expression of orexinergic genes in mice. Long term dosing with SR9009 also suppresses orexinergic gene expression in mice. Finally, REV-ERBβ-deficient mice present with increased orexinergic transcripts. These data suggest that the REV-ERBs may be involved in the repression of orexinergic gene expression.
    A simple, accurate and universal method for quantification of PCR
    Nicky Boulter, BMC Biotechnology - 2016
    Research into gene expression enables scientists to decipher the complex regulatory networks that control fundamental biological processes. Quantitative real-time PCR (qPCR) is a powerful and ubiquitous method for interrogation of gene expression. Accurate quantification is essential for correct interpretation of qPCR data. However, conventional relative and absolute quantification methodologies often give erroneous results or are laborious to perform.
    Molecular endocrine changes of Gh/Igf1 axis in gilthead sea bream (Sparus aurata L.) exposed to different environmental salinities during larvae to post-larvae stages
    Manuel Yúfera, Fish Physiology and Biochemistry - 2016
    The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.
    Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina
    Xu Wang, The Journal of Neuroscience - 2016
    Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.
    Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles
    Mariona Esquerdo, PLOS ONE - 2016
    Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.
    Inhibition of adhesion, migration and of α5β1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A
    Chiara Pelillo, Journal of Inorganic Biochemistry - 2016
    NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5β1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5β1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5β1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5β1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5β1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration.
    Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival
    Stéphanie St-Pierre, PLOS ONE - 2016
    It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.
    Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells
    Aleksandra M. Ochnik, Neoplasia - 2016
    Background Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. Materials and Methods To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)+ (MCF-7L) and ERα- (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. Results AIB1 positively regulated IGF-I-induced mRNA translation in both ERα+ and ERα- cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα+ and ERα- knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. Conclusion The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation.
    Seasonal variation in nifH abundance and expression of cyanobacterial communities associated with boreal feather mosses
    Denis Warshan, The ISME Journal - 2016
    Dinitrogen (N2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, ‘Stigonema cluster’ made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82–94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N2-fixation being greatly explained by variations in nifH expression of the ‘Stigonema cluster’. These results suggest that Stigonema is potentially the most influential N2-fixer in symbiosis with boreal forest feather mosses.
    Watnick, Randolph S, US Patent - 2016
    A method for evaluating a subject's responsiveness to treatment with a Psap peptide, the method comprising: determining a level of CD36 in a sample obtained from a subject having cancer, wherein an elevated level of CD36 in the sample compared to a control level indicates that the subject is responsive to or likely to be responsive to treatment with a Psap peptide. 2. The method of claim 1, wherein the level of CD36 in the sample is determined by performing an assay. 3. The method of claim 1, wherein the method further comprises: identifying the subject with an elevated level of CD36 in the sample compared to the control level as responsive to or likely to be responsive to treatment with a Psap peptide. 4. The method of claim 3, wherein the method further comprises: administering to the subject identified as responsive to or likely to be responsive to treatment with a Psap peptide an effective amount of a Psap peptide to treat the cancer. 5. A method for treating a subject with cancer, the method comprising: administering to a subject with cancer characterized by an elevated level of CD36 in a sample compared to a control level an effective amount of a Psap peptide to treat the cancer. 6. A method for treating a subject with cancer, the method comprising: (a) selecting a subject with cancer on the basis that the subject is known to have an elevated level of CD36 in a sample compared to a control level; and (b) administering an effective amount of a Psap peptide to the subject because the subject has an elevated level of CD36 in the sample compared to the control level. 7. The method of claim 1, wherein the control level is a level of CD36 from a non-cancerous cell or tissue obtained from the subject having the cancer. 8. The method of claim 1, wherein the control level is a level of CD36 in a cell or tissue obtained from a healthy subject or a population of healthy subjects. 9. The method of claim 1, wherein the control level is a predetermined level. 10. The method of claim 1, wherein the level of CD36 is a CD36 protein level. 11. The method of claim 1, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma. 12. The method of claim 1, wherein the Psap peptide comprises the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substitution variant thereof, wherein the amino acid substitution is: a) Tyrosine (Y) for Tryptophan (W); b) an amino acid substitution for Leucine (L) selected from Valine (V), Alanine (A) or Glycine (G), or a non-canonical amino acid of similar size, or a derivative thereof; c) Arginine (R) for Lysine (K); d) a D-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D) and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L); e) a D-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/or a D-isomer of Proline (P) for an L-isomer of Proline (P); or combinations thereof. 13. The method of claim 12, wherein the Psap peptide is 50 amino acids or fewer in length. 14. The method of claim 13, wherein the Psap peptide is 30 amino acids or fewer in length. 15. The method of claim 14, wherein the Psap peptide is 15 amino acids or fewer in length. 16. The method of claim 15, wherein the Psap peptide is 6 amino acids or fewer in length. 17. The method of claim 12, wherein the Psap peptide is a cyclic peptide. 18. The method of claim 12, wherein the non-canonical amino acid of similar size is methylvaline, methylleucine, or sarcosine. 19. 19-33. (canceled) 34. The method of claim 5, wherein the control level is a level of CD36 from a non-cancerous cell or tissue obtained from the subject having the cancer. 35. The method of claim 6, wherein the control level is a level of CD36 from a non-cancerous cell or tissue obtained from the subject having the cancer.
    Disruption of the sigS gene attenuates the local innate immune response to Staphylococcus aureus in a mouse mastitis model
    Vincent Peton, Veterinary Microbiology - 2016
    Staphylococcus aureus (S. aureus) is a major pathogen involved in ruminant mastitis and present worldwide. Clinical signs of S. aureus mastitis vary considerably and are largely dependent on strain-specific factors. A comparison of two S. aureus strains that reproducibly induced either severe (O11) or mild (O46) mastitis in ewes revealed that the transcriptional regulator sigS was mutated in O46 (Le Maréchal et al., 2011. PLoS One. 6 (11) e27354. doi:10.1371/journal.pone.0027354). In the present paper, we analysed the sigS sequence in 18 other S. aureus strains isolated from goat or ewe mastitis and found a 4-bp deletion similar to that of the O46 sigS gene in three strains associated with subclinical ewe mastitis. This sigS gene was disrupted in strain O11 (O11ΔsigS), so our aim was to investigate its involvement in the severity of infections in the context of mastitis. The wild type (wt) and mutant strains were then characterized in vitro to determine the involvement of sigS in the response S. aureus under various stress conditions, and assess its influence on the cytotoxicity of the pathogen, its invasive capacity and biofilm formation. The strains were compared in vivo in an experimental mouse mastitis model in which clinical signs and cytokine production were evaluated at 24 h post-infection. While no significant differences in the effect on bacterial growth between O11 and O11ΔsigS were observed either in vitro or in vivo, a significantly weaker in vivo production of interleukin (IL)-1α, IL-1β, and Tumor Necrosis Factor (TNF)-α was measured in the mammary glands infected with the mutant strain, suggesting that infection with O11ΔsigS induced an attenuated local innate immune response. These results suggest an impact of sigS disruption on S. aureus pathogenesis in a ruminant mastitis context. This disruption is probably involved in, and may partly explain, the milder symptoms previously observed in S. aureus O46-induced mastitis in ewes.
    microRNA 338-3p exhibits tumor suppressor role and its down-regulation is associated with adverse clinical outcome in prostate cancer patients
    Ashraf Bakkar, Molecular Biology Reports - 2016
    MicroRNAs (miRNAs) are small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. Several miRNAs have been implicated in regulating prostate cancer (PCa) progression. Deregulations of miRNA regulatory networks have been reported in ERG positive PCa, which accounts for ~50 % of PCa and have been suggested to affect tumor aggressiveness. The function of miR338-3p, its prognostic significance, and its association with ERG positive PCa has not been fully investigated. Using microarray expression profiling, we identified miRNA338-3p as among the top deregulated miRNAs associated with ERG status in PCa. We investigated miR338-3p function using in vitro and in vivo experimental models and its expression was assessed and validated in clinical samples and a public cohort of localized and metastatic prostate cancer. miR338-3p was significantly down-regulated with disease progression from benign prostate tissue to primary and metastatic lesions. In localized disease, patients with lower miR338-3p expression levels showed increased association to biochemical recurrence and several adverse pathological parameters compared to patients with higher miRNA338-3p tissue expression levels. Using in vitro PCa cell models, overexpression of miR338-3p resulted in a decrease in cell invasion and expression of chemokine signalling genes CXCL12, CXCR4, and CXCR7. In vivo, orthotropic implantation of PC3 cells stably expressing miR338-3p was associated with a significant decrease in tumor weights compared to control cells. miR338-3p has anti-proliferative and anti-invasive properties. It affects CXCR4 axis, and its down-regulation is associated with adverse clinical outcomes in PCa patients.
    microRNA 338-3p exhibits tumor suppressor role and its down-regulation is associated with adverse clinical outcome in prostate cancer patients
    Mohammed Alshalalfa, Molecular Biology Reports - 2016
    MicroRNAs (miRNAs) are small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. Several miRNAs have been implicated in regulating prostate cancer (PCa) progression. Deregulations of miRNA regulatory networks have been reported in ERG positive PCa, which accounts for ~50 % of PCa and have been suggested to affect tumor aggressiveness. The function of miR338-3p, its prognostic significance, and its association with ERG positive PCa has not been fully investigated. Using microarray expression profiling, we identified miRNA338-3p as among the top deregulated miRNAs associated with ERG status in PCa. We investigated miR338-3p function using in vitro and in vivo experimental models and its expression was assessed and validated in clinical samples and a public cohort of localized and metastatic prostate cancer. miR338-3p was significantly down-regulated with disease progression from benign prostate tissue to primary and metastatic lesions. In localized disease, patients with lower miR338-3p expression levels showed increased association to biochemical recurrence and several adverse pathological parameters compared to patients with higher miRNA338-3p tissue expression levels. Using in vitro PCa cell models, overexpression of miR338-3p resulted in a decrease in cell invasion and expression of chemokine signalling genes CXCL12, CXCR4, and CXCR7. In vivo, orthotropic implantation of PC3 cells stably expressing miR338-3p was associated with a significant decrease in tumor weights compared to control cells. miR338-3p has anti-proliferative and anti-invasive properties. It affects CXCR4 axis, and its down-regulation is associated with adverse clinical outcomes in PCa patients.
    Inhibition of O-GlcNAc transferase activity reprograms prostate
    Harri M. Itkonen, Oncotarget - 2016
    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific.
    Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes : Scientific Reports
    M. Schaeck, Scientific Reports - 2016
    The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3′/5′ integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.
    A Neurobiological Pathway That Mediates Stress-Induced Drug Use - viewcontent.cgi
    Sarit Aviel-Ronen, Scientific Report - 2016
    Aims: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), β-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. Methods: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, β-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. Results: AMACR and β-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and β-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. Conclusions: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of β-catenin.
    B.M Habiyambere, British Journal of Medicine & Medical Research - 2016
    Objectives: The aim for this study was to investigate the effects of chronic restraint stress on the expression of mucins that comprises the integral component of the gastrointestinal barrier. Design and Methods: A completely randomized design was used for the study. Sixteen ICR male mice were randomly divided into treated and control animals. Restraint stress was applied to the treated mice for 21 days. Body weight and feed consumed were regularly recorde d. After 21 days mice were euthanized and blood and gastrointestinal tissues were collected. Serum corticosterone levels were determined. Intestinal sections were fixed, paraffin embedded, sectioned, stained with alcian blue periodic acid Schiff stain to visualize goblet cells. Expression of mucosa mucins was determined using qPCR and western blotting.Results: As expected stressed mice showed higher corticosterone levels than the control mice (5082±1975 vs 1566±1236 pg/ml, P= 0.002). At the end of the study the body weight was lower in the stressed group (32±2 vs 36.0±3 g) even though the cumulative food consumed in both groups was not different. Stressed mice had lower goblet cell count in the duodenum, jejunum, ileum and colon (8±2, 7±1, 9±1, and 25±8 vs14±1, 14±1, 11±2 and 40±9, respectively), and shorter villi in the Original Research Article Habiyambere and Onyango ; BJMMR, 13(6): 1-14, 2016; Article no.BJMMR.237642 duodenum and jejunum (316±82 and 256±73 vs 440±73 and 357±35 μm, respectively). Expressions of mRNA for Muc1 and Muc4 in the colon of stressed animals were upregulated (1.5 and 2.2 fold increase, and P values of 0.05 and 0.01, respectively). This mRNA upregulation was accompanied by increased expression of Muc1 and 4 proteins in the colon.Conclusion: These findings suggest that chronic stress affects the intestinal barrier by reducing the number of the mucin-producing goblet cells, shortening the villi and upregulating Muc1 and Muc4 in the colon.
    Mesenchymal stromal cells from the foreskin: Tissue isolation, cell characterization and immunobiological properties
    Mehdi Najar, Cytotherapy - 2016
    Background aims Because of their self-renewal capacity, multilineage potential and immunomodulatory properties, MSCs are an attractive tool for cell-based immunotherapy strategies. Foreskin, considered as a biological waste material, has been shown to be a reservoir of therapeutic cells. Methods MSCs were isolated from different foreskin samples, maintained under in vitro culture and defined according to the International Society for Cellular Therapy (ISCT) criteria. We subsequently determined their main cell characteristics as well as their immunobiological properties. The following parameters were determined: (i) morphology and phenotype, (ii) proliferative and clonogenic potentials, (iii) tri-lineage differentiation ability, (iv) immunological profile, (v) immunomodulatory properties and (vi) protein and messenger RNA expression/secretion profile of immunoregulatory cytokines/factors as well as the pattern of toll-like receptors (TLRs). By using a pro-inflammatory cytokine cocktail, we also evaluated the influence of an inflammatory environment on their biology. Results With a typical fibroblast-like morphology and an ISCT-compliant phenotype, foreskin-MSCs (FSK-MSCs) were highly proliferative and had a great clonogenic potential. They displayed multilineage capacities and interesting immunomodulatory properties. Of importance, FSK-MSCs were not immunogenetic and were further able to inhibit T-cell proliferation. We showed that several immunoregulatory cytokines and factors might be potentially involved in FSK-MSC immunomodulation with particular attention to hepatocyte growth factor and interleukin-11. Moreover, FSK-MSCs expressed several TLRs and were sensitive to the inflammatory environment by properly adjusting their profile and fate. Conclusions Foreskin represents a new alternative source for MSCs that is compliant with ISCT criteria. Their unique immunobiological properties allow consideration of FSK-MSCs as a valuable tolerogenic product for cell-based immunotherapy.
    Characterization of aromatic aminotransferases from Ephedra sinica Stapf
    Korey Kilpatrick, Amino Acids - 2016
    Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
    GAPDH Binding to TNF-α mRNA Contributes to Posttranscriptional Repression in Monocytes: A Novel Mechanism of Communication between Inflammation and Metabolism
    Patrick Millet, The Journal of Immunology - 2016
    Expression of the inflammatory cytokine TNF is tightly controlled. During endotoxin tolerance, transcription of TNF mRNA is repressed, although not entirely eliminated. Production of TNF cytokine, however, is further controlled by posttranscriptional regulation. In this study, we detail a mechanism of posttranscriptional repression of TNF mRNA by GAPDH binding to the TNF 3′ untranslated region. Using RNA immunoprecipitation, we demonstrate that GAPDH–TNF mRNA binding increases when THP-1 monocytes are in a low glycolysis state, and that this binding can be reversed by knocking down GAPDH expression or by increasing glycolysis. We show that reducing glycolysis decreases TNF mRNA association with polysomes. We demonstrate that GAPDH–TNF mRNA binding results in posttranscriptional repression of TNF and that the TNF mRNA 3′ untranslated region is sufficient for repression. Finally, after exploring this model in THP-1 cells, we demonstrate this mechanism affects TNF expression in primary human monocytes and macrophages. We conclude that GAPDH–TNF mRNA binding regulates expression of TNF based on cellular metabolic state. We think this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock.
    Unraveling the Tissue-Specific Gene Signatures of Gilthead Sea Bream (Sparus aurata L.) after Hyper- and Hypo-Osmotic Challenges
    Juan Antonio Martos-Sitcha, PLoS ONE - 2016
    A custom microarray was used for the transcriptomic profiling of liver, gills and hypothalamus in response to hypo- (38‰ → 5‰) or hyper- (38‰ → 55‰) osmotic challenges (7 days after salinity transfer) in gilthead sea bream (Sparus aurata) juveniles. The total number of differentially expressed genes was 777. Among them, 341 and 310 were differentially expressed in liver after hypo- and hyper-osmotic challenges, respectively. The magnitude of changes was lower in gills and hypothalamus with around 131 and 160 responsive genes in at least one osmotic stress condition, respectively. Regardless of tissue, a number of genes were equally regulated in either hypo- and hyper-osmotic challenges: 127 out of 524 in liver, 11 out of 131 in gills and 19 out of 160 in hypothalamus. In liver and gills, functional analysis of differentially expressed genes recognized two major clusters of overlapping canonical pathways that were mostly related to “Energy Metabolism” and “Oxidative Stress”. The later cluster was represented in all the analyzed tissues, including the hypothalamus, where differentially expressed genes related to “Cell and tissue architecture” were also over-represented. Overall the response for “Energy Metabolism” was the up-regulation, whereas for oxidative stress-related genes the type of response was highly dependent of tissue. These results support common and different osmoregulatory responses in the three analyzed tissues, helping to load new allostatic conditions or even to return to basal levels after hypo- or hyper-osmotic challenges according to the different physiological role of each tissue.
    Estrogen receptor β regulates endometriotic cell survival through serum and glucocorticoid–regulated kinase activation
    Diana Monsivais, Fertility and Sterility - 2016
    Objective To determine the expression and biological roles of serum and glucocorticoid–regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. Design Case-control. Setting University research setting. Patient(s) Premenopausal women. Intervention(s) Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. Main Outcome Measure(s) Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) β. Result(s) Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERβ-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERβ based on small interfering RNA knockdown and chromatin immunoprecipitation of ERβ followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. Conclusion(s) ERβ leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.
    Methods and Compounds for the Inhibition of Cellular Proliferation
    Gerhard Wagner, US Patent - 2015
    Methods for inhibiting translation, treating a cellular proliferative disorder, and inhibiting proliferation of cells using the compounds disclosed herein are provided.
    Effect of genetic background and postinfectious stress on visceral sensitivity in Citrobacter rodentium-infected mice
    S. U. Mondelaers, Neurogastroenterology & Motility - 2016
    Background Infectious gastroenteritis is a major risk factor to develop postinfectious irritable bowel syndrome (PI-IBS). It remains unknown why only a subgroup of infected individuals develops PI-IBS. We hypothesize that immunogenetic predisposition is an important risk factor. Hence, we studied the effect of Citrobacter rodentium infection on visceral sensitivity in Th1-predominant C57BL/6 and Th2-predominant Balb/c mice. Methods Eight-week-old mice were gavaged with C. rodentium, followed by 1 h of water avoidance stress (WAS) at 5 weeks PI. At 10, 14 days, and 5 weeks PI, samples were assessed for histology and inflammatory gene expression by RT-qPCR. Visceral sensitivity was evaluated by visceromotor response recordings (VMR) to colorectal distension. Key Results Citrobacter rodentium evoked a comparable colonic inflammatory response at 14 days PI characterized by increased crypt length and upregulation of Th1/Th17 cytokine mRNA levels (puncorrected < 0.05) in both C57BL/6 and Balb/c mice. At 5 weeks PI, inflammatory gene mRNA levels returned to baseline in both strains. The VMR was maximal at 14 days PI in C57BL/6 (150 ± 47%; p = 0.02) and Balb/c mice (243 ± 52%; p = 0.03). At 3 weeks PI, the VMR remained increased in Balb/c (176 ± 23%; p = 0.02), but returned to baseline in C57BL/6 mice. At 5 weeks PI, WAS could not re-introduce visceral hypersensitivity (VHS). Conclusions & Inferences Citrobacter rodentium infection induces transient VHS in C57BL/6 and Balb/c mice, which persisted 1 week longer in Balb/c mice. Although other strain-related differences may contribute, a Th2 background may represent a risk factor for prolonged PI-VHS. As PI-VHS is transient, other factors are crucial for persistent VHS development as observed in PI-IBS.
    Targeting NF-κB in glioblastoma: A therapeutic approach
    Dinorah Friedmann-Morvinski, Science Advances - 2016
    Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas, which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2), expression of a IκBαM super repressor, or using a NEMO (NF-κB essential modifier)–binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1, one of the NF-κB target genes significantly up-regulated in GBM, was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM. Inhibition of the transcription factor NF-κB or its target genes should be considered for the treatment of patients with glioblastoma multiforme. Inhibition of the transcription factor NF-κB or its target genes should be considered for the treatment of patients with glioblastoma multiforme.
    The auxin response factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes
    Naden Krogan, New Phytologist - 2016
    * The regulatory effect auxin has on its own transport is critical in numerous self-organizing plant patterning processes. However, our understanding of the molecular mechanisms linking auxin signal transduction and auxin transport is still fragmentary, and important regulatory genes remain to be identified. * To track a key link between auxin signaling and auxin transport in development, we established an Arabidopsis thaliana genetic background in which fundamental patterning processes in both shoot and root were essentially abolished and the expression of PIN FORMED (PIN) auxin efflux facilitators was dramatically reduced. * In this background, we demonstrate that activating a steroid-inducible variant of the auxin response factor (ARF) MONOPTEROS (MP) is sufficient to restore patterning and PIN gene expression. Further, we show that MP binds to distinct promoter elements of multiple genetically defined PIN genes. * Our work identifies a direct regulatory link between central, well-characterized genes involved in auxin signal transduction and auxin transport. The steroid-inducible MP system directly demonstrates the importance of this molecular link in multiple patterning events in embryos, shoots and roots, and provides novel options for interrogating the properties of self-regulated auxin-based patterning in planta.
    Soybean Meal and Soy Protein Concentrate in Early Diet Elicit Different Nutritional Programming Effects on Juvenile Zebrafish
    Erick Perera, Zebrafish - 2015
    There is now strong evidence that early nutrition plays an important role in shaping later physiology. We assessed here whether soy protein concentrate (SPC) or soybean meal (SBM) in early diet would modify zebrafish responses to these products in later life. We fed zebrafish larvae with SPC-, SBM-, or a control-diet for the first 3 days of feeding and then grew all larvae on the control diet up to juveniles. Finally, we assessed the expression in juveniles of genes involved in inflammation/immunity, the breakdown of extracellular matrix, luminal digestion, and intestinal nutrient absorption/trafficking. First feeding SBM had wider, stronger, and more persistent effects on gene expression with respect to SPC. Juveniles fed with SPC at first feeding were more prone to inflammation after refeeding with SPC than fish that never experienced SPC before. Conversely, zebrafish that faced SBM at first feeding were later less responsive to refeeding with SBM through inflammation and had higher expression of markers of peptide absorption and fatty acid transport. Results indicate that some features of inflammation/remodeling, presumably at the intestine, and nutrient absorption/transport in fish can be programmed by early nutrition. These findings sustain the rationale of using zebrafish for depicting molecular mechanisms involved in nutritional programming.
    A genome-wide association study identifies four novel susceptibility loci underlying inguinal hernia
    Eric Jorgenson, Nature Communications - 2016
    Inguinal hernia repair is one of the most commonly performed operations in the world, yet little is known about the genetic mechanisms that predispose individuals to develop inguinal hernias. We perform a genome-wide association analysis of surgically confirmed inguinal hernias in 72,805 subjects (5,295 cases and 67,510 controls) and confirm top associations in an independent cohort of 92,444 subjects with self-reported hernia repair surgeries (9,701 cases and 82,743 controls). We identify four novel inguinal hernia susceptibility loci in the regions of EFEMP1, WT1, EBF2 and ADAMTS6. Moreover, we observe expression of all four genes in mouse connective tissue and network analyses show an important role for two of these genes (EFEMP1 and WT1) in connective tissue maintenance/homoeostasis. Our findings provide insight into the aetiology of hernia development and highlight genetic pathways for studies of hernia development and its treatment.
    Caspase-3 cleaved p65 fragment dampens NF-κB-mediated anti-apoptotic transcription by interfering with the p65/RPS3 interaction - 56420a8508aec448fa621bec.pdf
    Eric M. Wier, Federation of European Biochemical Societies. - 2015
    Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF- j B)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF- j B transactiva- tion. Here we show that the p65 1-97 fragment generated by Caspase-3 cleavage interferes with ribo- somal protein S3 (RPS3), an NF- j B ‘‘specifier ” subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF- j B-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF- j B-conferred anti-apoptotic transcription by the frag- ment from partial p65 cleavage by activated Caspase-3.
    In-Vitro Adhesion and Invasion Properties of Salmonella Typhimurium Competing with Bacteriophage in Epithelial Cells and Chicken Macrophages
    H.Y Lee, Revista Brasileira de Ciência Avícola - 2015
    This study was designed to assess the role of bacteriophage P22 in the adhesion, invasion, intracellular survival of, and cellular immune response to Salmonella Typhimurium in intestinal epithelial INT-407 and chicken macrophage-like HD11 cells. The ability of S. Typhimurium to adhere, invade, and survive to INT-407 and HD11cells was evaluated under Salmonella infection alone (control), phage treatment followed by Salmonella infection (PS), Salmonella infection followed by phage treatment (SP), and a combination treatment with Salmonella and phage (S+P). The number of S. Typhimurium associated on INT-407 cells was reduced from 4.2 to 2.7 log cfu/cm2 by phage treatment (SP). The number of intracellular S. Typhimurium within INT-407 cells was significantly reduced to below the detection limit (0.7 log cfu/cm2) compared with the control (3.4 log cfu/cm2). S. Typhimurium remained inside HD11 cells at 49% and 17% levels in the absence and presence of phages, respectively, at 24 h post-infection (hpi). The expression levels of IFN-g, IL-10, IL-1b, IL-6, IL-8, iNOS, and IL-12 increased in HD11 cells regardless the absence and presence of phages, while those of IL-16, TLR2-1, TLR3, and TLR7 were decreased at 0 and 24 hpi. This study sheds new light on our understanding of the role of phages in Salmonella adhesion, invasion, survival, and cellular immune responses.Key words: Adhesion; Bacteriophage; Invasion; Salmonella; Toll-like receptor
    Insulin Receptor Substrate 1: The Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation
    Nathan Law, Journal of Biological Chemistry - 2015
    The ubiquitous phosphatidylinositol-3 kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells (GCs) as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)- dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, GCs secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser789. Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through AKT and forkhead box O1 (FOXO1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.
    20-HETE Activates the Transcription of Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding
    Victor Garcia, Journal of Pharmacology and Experimental Therapeutics - 2015
    Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that in human microvascular endothelial cells (HMVECs), 20-HETE increases angiotensin converting enzyme (ACE) mRNA, protein, and ACE activity via an EGFR-tyrosine kinase-MAPK-IKKβ-mediated signaling pathway. Here we show that similar to EGF, 20-HETE (10nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of NF-kB activation prevented the 4.58-fold (±0.78; p<0.05) 20-HETE-mediated induction of ACE. 20-HETE increased NF-kB binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (±0.18; p<0.05) and 2.53-fold (± 0.24; p<0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two and one putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. ChIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs. vehicle; p<0.05). This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.
    Visualizing estrogen receptor-α-expressing neurons using a new ERα-ZsGreen reporter mouse line
    Kenji Saito, Metabolism - 2016
    Background A variety of biological functions of estrogens, including regulation of energy metabolism, are mediated by neurons expressing estrogen receptor-α (ERα) in the brain. However, complex intracellular processes in these ERα-expressing neurons are difficult to unravel, due to the lack of strategy to visualize ERα-expressing neurons, especially in unfixed brain tissues. Results and conclusions Here we generated a novel ERα-ZsGreen reporter mouse line in which expression of a green fluorescent reporter protein, ZsGreen, is driven by a 241 kb ERα gene promoter. We validated that ZsGreen is highly colocalized with endogenous ERα in the brain. Native ZsGreen signals were visualized in unfixed brain tissue, and were used to assist single cell collection and electrophysiological recordings. Finally, we demonstrated that this ERα-ZsGreen mouse allele can be used in combination with other genetic reporter alleles to allow experiments in highly selective neural populations.
    Discrete Responses to Limitation for Iron and Manganese in Agrobacterium tumefaciens: Influence on Attachment and Biofilm Formation
    Jason E. Heindl, Journal of Bacteriology - 2015
    Transition metals such as iron and manganese are crucial trace nutrients for the growth of most bacteria, functioning as catalytic cofactors for many essential enzymes. Dedicated uptake and regulatory systems have evolved to ensure their acquisition for growth, while preventing toxicity.Transcriptomic analysis of the iron- and manganese-responsive regulons of Agrobacterium tumefaciens revealed that there are discrete regulatory networks that respond to changes in iron and manganese levels. Complementing earlier studies, the iron-responsive gene network is quite large, and includes many aspects of iron-dependent metabolism and the iron-sparing response. In contrast, the manganese-responsive network is restricted to a limited number of genes, many of which can be linked to transport and utilization of the transition metal. Several of the target genes predicted to drive manganese uptake are required for growth under manganese-limited conditions, and an A. tumefaciens mutant with a manganese transport deficiency is attenuated for plant virulence. Iron and manganese limitation independently inhibit biofilm formation by A. tumefaciens, and several candidate genes that could impact biofilm formation were identified in each regulon. The biofilm inhibitory effects of iron and manganese do not rely on recognized metal-responsive transcriptional regulators, suggesting alternate mechanisms influencing biofilm formation. However, under low manganese the dcpA operon is upregulated, a system that controls levels of the cyclic-diguanosine monophosphate second messenger. Mutation of this regulatory pathway dampens the effect of manganese limitation. IMPORTANCE Responses to changes in transition metal levels, such as those of manganese and iron, are important for normal metabolism and growth in bacteria. Our study uses global gene expression profiling to understand the response of the plant pathogen Agrobacterium tumefaciens to changes of transition metal availability. Among the properties that are affected by both iron and manganese levels are those required for normal surface attachment and biofilm formation, but the requirement for each of these transition metals is mechanistically independent from the other.
    Nerve Growth Factor is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs
    Krock Emerson, Journal of Biological Chemistry - 2015
    Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity, but more recently were found to be activated by endogenous alarmins such as fragmented extra-cellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1β (control) treatment increased NGF, BDNF and IL-1β gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1β treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2 and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-κB, and to a lesser extent p38, but not ERK1/2 activity blocked TLR2-driven NGF upregulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-κB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-κB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.
    Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5
    Kahlilia C. Morris-Blanco, Scientific Reports - 2016
    not listed
    Antisense RNA-Mediated Epigenetic Regulation of Brain-Derived Neurotrophic Factor. - viewcontent.cgi
    Roya Pedram Fatemi, University of Miami Scholarly Repository DISSERTATION - 2015
    Long noncoding RNAs (lncRNAs) regulate chromatin remodeling through their interactions with epigenetic enzymes during development and disease. The inhibition of the natural antisense transcript of Brain-derived neurotrophic factor (BDNF-AS), results in BDNF promoter de-repression and transcriptional upregulation, both in vitro and in vivo. Recently, we showed that BDNF-AS interacts with the histone methyltransferase enhancer of zeste homolog 2 (EZH2) to suppress BDNF mRNA and protein expression. BDNF is an important neurotrophin that is required for neural development and maintenance of the nervous system. Dysregulation of BDNF occurs in a number of neurological disorders, including: Alzheimer’s Disease, Parkinson’s Disease, Rett syndrome, and amyotrophic lateral sclerosis. Previous attempts to upregulate BDNF by administering the recombinant form in various parts of the central nervous system have failed, mostly due to the challenge of delivering BDNF to the correct cells and neural networks. Our approach to upregulating BDNF by modulating its interaction with an epigenetic enzyme is a highly specific target with potential therapeutic value. To achieve this, we developed a novel pharmacological assay to characterize the interaction between long noncoding RNAs and their epigenetic targets using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. With this assay, we are able to quantify lncRNA-protein interactions rapidly for the purpose of high throughput screening, enabling drug discovery efforts for this novel class of drug targets. In this work, we present our assay development and screening findings, including the identification of potential small molecule modulators of lncRNA-protein interactions. Furthermore, we describe the application of this lncRNA-protein interaction assay to detect RNA requirements for EZH2 recruitment, a much debated and important question that lingers in the field. From our work, it is evident that BDNF-AS has several regions of RNA that are required for EZH2 recruitment, potentially due to the importance of this transcript in regulating BDNF. This work describes exploratory drug discovery for a novel class of drug targets as well as applications to understand the basic biochemistry
    Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts
    S. M. Hosseini, BMC Developmental Biology - 2015
    This study describes the generation and analysis of the transcriptional profile of bovine inner cell mass (ICM) and trophectoderm (TE), obtained from in vivo developed embryos by using a bovine-embryo specific array (EmbryoGENE) containing 37,238 probes.
    Macrophage and adipocyte IGF1 maintain adipose tissue homeostasis during metabolic stresses
    Hye Rim Chang, Obesity - 2016
    Objective Insulin-like growth factor-1 (IGF1) regulates differentiation and growth of tissues and reduces stress and injury. IGF1 also in a tissue-specific manner modulates the differentiation and lipid storage capacity of adipocytes in vitro, but its roles in adipose tissue development and response to stress are not known. Methods To study IGF1 in vivo, the cellular sources of adipose tissue Igf1 expression were identified and mice were generated with targeted deletion in adipocytes and macrophages. The effects of adipocyte and macrophage deficiency of IGF1 on adipose tissue development and the response to chronic (high-fat feeding) and acute (cold challenge) stress were studied. Results The expression of Igf1 by adipose tissue was derived from multiple cell types including adipocytes and macrophages. In lean animals, adipocytes were the primary source of IGF1, but in obesity expression by adipocytes was reduced and by macrophages increased, so as to maintain overall adipose tissue Igf1 expression. Genetic deletion studies revealed that adipocyte-derived IGF1 regulated perigonadal but not subcutaneous adipose tissue mass during high-fat feeding and the development of obesity. Conversely, macrophage-derived IGF1 acutely modulated perigonadal adipose tissue mass during thermogenic challenges. Conclusions Local IGF1 is not required in lean adipose tissue development but is required to maintain homeostasis during both chronic and acute metabolic stresses.
    Conserved and unique features of the homeologous maize Aux/IAA proteins ROOTLESS WITH UNDETECTABLE MERISTEM 1 and RUM1-like 1
    Yanxiang Zhang, Journal of Experimental Botany - 2015
    The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1) is a key regulator of lateral and seminal root formation. An ancient maize genome duplication resulted in the emergence of its homeolog rum1-like1 (rul1), which displays 92% amino acid sequence identity with RUM1. Both, RUL1 and RUM1 exhibit the canonical four domain structure of Aux/IAA proteins. Moreover, both are localized to the nucleus, are instable and have similar short half-lives of ~23min. Moreover, RUL1 and RUM1 can be stabilized by specific mutations in the five amino acid degron sequence of domain II. In addition, proteins encoded by both genes interact in vivo with auxin response factors (ARFs) such as ZmARF25 and ZmARF34 in protoplasts. Although it was demonstrated that RUL1 and RUM1 can homo and heterodimerize in vivo, rul1 expression is independent of rum1. Moreover, on average rul1 expression is ~84-fold higher than rum1 in the 12 tested tissues and developmental stages, although the relative expression levels in different root tissues are very similar. While RUM1 and RUL1 display conserved biochemical properties, yeast-two-hybrid in combination with BiFC experiments identified a RUM1-associated protein 1 (RAP1) that specifically interacts with RUM1 but not with RUL1. This suggests that RUM1 and RUL1 are at least in part interwoven into different molecular networks.
    FMR6 may play a role in the pathogenesis of fragile X-associated premature ovarian insufficiency
    Shai E. Elizur, Gynecological Endocrinology - 2015
    The aim of this study was to evaluate whether long noncoding RNA accumulation play a role in the pathophysiology of fragile X-associated premature ovarian insufficiency (FXPOI). The study population consisted of 22 consecutive fragile X mental retardation 1 (FMR1) premutation carriers (CGGn 55–199 repeats) undergoing in vitro fertilization and pre-implantation genetic diagnosis (IVF–PGD) treatment. The control group consists of 11 patients, with <55 CGG repeats, undergoing IVF–ICSI for male factor infertility, matched by age, treated in the same period. After oocyte retrieval, granulosa cells from follicular fluid were washed and stored at −80 °C. RNA was transcribed to generate cDNA and the RNA levels were measured using RT–PCR. Transcripts levels in granulosa cells of long noncoding RNA’s FMR4 and FMR6 were measured. In FMR1 premutation carriers there was a significant nonlinear association between the number of CGG repeats and the levels of FMR6 (p = 0.03), but not FMR4. The highest level of FMR6 was seen in women with mid-size CGG repeats (80–120). In addition, a significant negative linear correlation was observed between the number of oocytes retrieved and the RNA levels in granulosa cells of FMR6 (r = −0.53, p = 0.01) but not FMR4. Our study supports previous findings suggesting RNA toxic gain-of-function as one of the possible pathophysiologic mechanisms underlying FXPOI.
    An Immunofluorescence-assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood
    Kazunori Hoshino, Journal of Circulating Biomarkers - 2015
    Circulating tumour cells (CTCs) are important indicators of metastatic cancer and may provide critical information for individualized treatment. As CTCs are usually very rare, the techniques to obtain information from very small numbers of cells are crucial. Here, we propose a method to perform a single cell quantitative reverse transcription polymerase chain reaction (qPCR) analysis of rare tumour cells. We utilized a microfluidic immunomagnetic assay to separate cancer cells from blood. A combination of detailed immunofluorescence and laser microdissection enabled the precise selection of individual cells. Cancer cells that were spiked into blood were successfully separated and picked up for a single cell PCR analysis. The breast cancer cell lines MCF7, SKBR3 and MDAMB231 were tested with 10 different genes. The result of the single cell analysis matched the results from a few thousand cells. Some markers (e.g., ER, HER2) that are commonly used for cancer identification showed relatively large deviations in expression levels. However, others (e.g., GRB7) showed deviations that are small enough to supplement single cell disease profiling.
    Detection of emergent strains of West Nile virus with a blood screening assay
    Helen M. Faddy, Transfusion - 2015
    BACKGROUND West Nile virus (WNV) is a threat to transfusion safety. WNV Kunjin strain (WNVKUN) is endemic across parts of Australia; however, human infection is believed to be infrequent and is often associated with relatively minor symptoms. A virulent strain, closely related to WNVKUN (termed WNVNSW2011) was recently identified as the etiologic agent of encephalitis in Australian horses. The aim of this project was to investigate whether a commercially available WNV blood screening assay can detect different strains of WNVKUN, including the virulent WNVNSW2011, in human blood donor samples. STUDY DESIGN AND METHODS Plasma samples were spiked with four different strains of WNVKUN, as well as a prototype WNV strain, at high, medium, and low viral loads. Spiking was confirmed with real-time reverse transcription–polymerase chain reaction (RT-PCR), before testing with the Procleix WNV transcription-mediated amplification (TMA) blood screening assay (Grifols). RESULTS All WNV strains used were detectable by RT-PCR after being spiked into plasma. Additionally, all viral spiked samples were reactive by WNV TMA. CONCLUSION We experimentally demonstrate that a commercially available WNV blood screening assay can detect different strains of WNVKUN. Given that WNV can be transfusion transmissible, it is essential to confirm that emergent strains are detectable by existing blood screening methods.
    Blood-Borne Activity-Dependent Neuroprotective Protein (ADNP) is Correlated with Premorbid Intelligence, Clinical Stage, and Alzheimer’s Disease Biomarkers
    Malishkevich, Anna, Journal of Alzheimer's Disease - 2015
    Biomarkers for Alzheimer’s disease (AD) are vital for disease detection in the clinical setting. Discovered in our laboratory, activity-dependent neuroprotective protein (ADNP) is essential for brain formation and linked to cognitive functions. Here, we revealed that blood borne expression of ADNP and its paralog ADNP2 is correlated with premorbid intelligence, AD pathology, and clinical stage. Age adjustment showed significant associations between: 1) higher premorbid intelligence and greater serum ADNP, and 2) greater cortical amyloid and lower ADNP and ADNP2 mRNAs. Significant increases in ADNP mRNA levels were observed in patients ranging from mild cognitive impairment (MCI) to AD dementia. ADNP2 transcripts showed high correlation with ADNP transcripts, especially in AD dementia lymphocytes. ADNP plasma/serum and lymphocyte mRNA levels discriminated well between cognitively normal elderly, MCI, and AD dementia participants. Measuring ADNP blood-borne levels could bring us a step closer to effectively screening and tracking AD.
    High-content screening identifies a role for Na+ channels in insulin production
    Marta Szabat, Royal Society Open Science - 2015
    Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production.
    Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle
    Camilla Brolin, Molecular Therapy — Nucleic Acids - 2015
    Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue.
    Activation of Autophagy by Unfolded Proteins during Endoplasmic Reticulum Stress
    Xiaochen Yang, The Plant Journal - 2015
    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum and is caused by conditions such as heat or endoplasmic reticulum stress agents, including tunicamycin (TM) and dithiothreitol (DTT). Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in an INOSITOL-REQUIRING ENZYME 1b (IRE1b)-dependent manner and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, overexpression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker green fluorescent protein-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. This article is protected by copyright. All rights reserved.
    Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells
    Lisbeth Ramírez-Carvajala, Antiviral Research - 2015
    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the “master regulator” of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.
    Zinc enhances the number of regulatory T cells in allergen-stimulated cells from atopic subjects
    Eva Rosenkranz, European Journal of Nutrition - 2015
    Purpose The trace element zinc is essential for immune function and its regulation. Since zinc deficiency and allergic hyperresponsive reactions are often accompanied, the influence of zinc on allergen-induced cell growth, CD4+ regulatory T (Treg) cell numbers and cytokine expression during allergic immune reactions was investigated. Methods Peripheral blood mononuclear cells (PBMCs) from non-atopic and atopic subjects were treated with timothy grass allergen pre-incubated with or without zinc. Proliferation was determined by analyzing the incorporation of 3H-thymidine. Intracellular zinc and Foxp3 levels and cell surface antigens were measured by FACS, cytokine expression by ELISA and real-time PCR. Results Incubation with 50 μM zinc sulfate (Zn50) enhances cytosolic zinc concentrations in CD3+ T cells. The data also reveal that the combination of Zn50 plus allergen significantly reduces PBMC proliferation of atopic subjects. Additionally, Zn50 plus allergen enhances Th1 cytokine responses shown by increased interferon (IFN)-γ/interleukin (IL)-10 ratios as well as enhanced tumor necrosis factor-α release. In response to allergen, zinc increases Treg cells and upregulates the mRNA expression of cytotoxic T-lymphocyte antigen-4 in atopic subjects. Interestingly, Zn50 alone leads to an increase of CD4+CD25high(hi)+ cells in atopic and non-atopic subjects. Conclusions Zinc may regulate unwanted hyperresponsive immune reactions by suppressing proliferation through a significant shift from IL-10 to the Th1 cytokine IFN-γ, and enhanced regulatory T cell numbers. Therefore, zinc supplementation may be a promising tool for the therapy of allergies, without negatively affecting the immune system.
    Active FOXO1 is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming
    Caroline H. Diep, Molecular Cancer Research - 2015
    Progesterone promotes differentiation coupled to proliferation and pro-survival in the breast, but inhibits estrogen-driven growth in the reproductive tract and ovaries. Herein, it is demonstrated, using progesterone receptor (PR) isoform-specific ovarian cancer model systems, that PR-A and PR-B promote distinct gene expression profiles that differ from PR-driven genes in breast cancer cells. In ovarian cancer models, PR-A primarily regulates genes independently of progestin, while PR-B is the dominant ligand-dependent isoform. Notably, FOXO1 and the PR/FOXO1 target-gene p21 (CDKN1A) are repressed by PR-A, but induced by PR-B. In the presence of progestin, PR-B, but not PR-A, robustly induced cellular senescence via FOXO1-dependent induction of p21 and p15 (CDKN2B). Chromatin immunoprecipitation (ChIP) assays performed on PR-isoform specific cells demonstrated that while each isoform is recruited to the same PRE-containing region of the p21 promoter in response to progestin, only PR-B elicits active chromatin marks. Overexpression of constitutively active FOXO1 in PR-A-expressing cells conferred robust ligand-dependent upregulation of the PR-B target genes GZMA, IGFBP1, and p21, and induced cellular senescence. In the presence of endogenous active FOXO1, PR-A was phosphorylated on Ser294 and transactivated PR-B at PR-B target genes; these events were blocked by the FOXO1 inhibitor (AS1842856). PR isoform-specific regulation of the FOXO1/p21 axis recapitulated in human primary ovarian tumor explants treated with progestin; loss of progestin sensitivity correlated with high AKT activity. Implications: This study indicates FOXO1 as a critical component for progesterone signaling to promote cellular senescence and reveals a novel mechanism for transcription factor control of hormone sensitivity.
    Migration of CD8+ T Cells into the Central Nervous System Gives Rise to Highly Potent Anti-HIV CD4dimCD8bright T Cells in a Wnt Signaling–Dependent Manner
    Maureen H. Richards, The Journal of Immunology - 2015
    The role of CD8+ T cells in HIV control in the brain and the consequences of such control are unclear. Approximately 3% of peripheral CD8+ T cells dimly express CD4 on their surface. This population is known as CD4dimCD8bright T cells. We evaluated the role of CD4dimCD8bright and CD8 single positive T cells in HIV-infected brain using NOD/SCID/IL-2rcγ−/− mice reconstituted with human PBMCs (NSG-huPBMC). All three T cell populations (CD4 single positive, CD8 single positive, and CD4dimCD8bright) were found in NSG-huPBMC mouse brain within 2 wk of infection. Wnts secreted from astrocytes induced CD4dimCD8bright T cells by 2-fold in vitro. Injection of highly purified CD8 single positive T cells into mouse brain induced CD4dimCD8bright T cells by 10-fold, which were proliferative and exhibited a terminally differentiated effector memory phenotype. Brain CD4dimCD8bright T cells from HIV-infected mice exhibited anti-HIV–specific responses, as demonstrated by induction of CD107ab post exposure to HIV peptide–loaded targets. Further, higher frequency of CD4dimCD8bright T cells (R = −0.62; p ≤ 0.001), but not CD8 single positive T cells (R = −0.24; p ≤ 0.27), negatively correlated with HIV gag mRNA transcripts in HIV-infected NSG-huPBMC brain. Together, these studies indicate that single positive CD8+ T cells entering the CNS during HIV infection can give rise to CD4dimCD8bright T cells, likely through a Wnt signaling–dependent manner, and that these cells are associated with potent anti-HIV control in the CNS. Thus, CD4dimCD8bright T cells are capable of HIV control in the CNS and may offer protection against HIV-associated neurocognitive disorders.
    Anti-inflammatory Effects of Poly-l-lysine in Intestinal Mucosal System Mediated by Calcium-Sensing Receptor Activation
    Yoshinori Mine, Journal of Agricultural and Food Chemistry - 2015
    Calcium-sensing receptor (CaSR) is involved in maintaining cellular homeostasis and promoting recovery of damaged intestinal epithelial cells (IECs). Poly-l-lysine (PL) is a basic polypeptide identified for its role in the activation of CaSR through allosteric binding. The primary goal of the current study was to identify the modulatory effect of PL on intestinal inflammation and to determine whether these effects were mediated by CaSR activation. We used human intestinal epithelial cell lines, Caco-2 and HT-29, to assess PL anti-inflammatory activities in vitro. We found that PL reduced the IL-8 secretion from tumor necrosis factor (TNF)-α-treated human intestinal epithelial cell lines. On the other hand, the gene expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1? was inhibited by PL supplementation. We subsequently evaluated the anti-inflammatory activity of PL in vivo using a DSS-induced mouse colitis model. PL supplementation was shown to prevent dextran sulfate sodium salt (DSS)-induced loss of weight, colitic symptoms, and shortening of colon length but maintained colonic morphology. The pro-inflammatory cytokine expression in the mouse colon, including TNF-α, IL-6, INF-?, IL-17, and IL-1?, was significantly up-regulated by DSS treatment, but was inhibited upon PL administration. As shown by the results from both in vitro and in vivo studies, the reduction of inflammatory cytokine production caused by PL was reversed by NPS-2143 pretreatment. In the present study, we provide evidence that PL exerts anti-inflammatory effects on the gut system, which is primarily mediated by allosteric ligand activation of CaSR.
    Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance : Nature : Nature Publishing Group
    Kari R. Fischer, Nature - 2015
    The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.
    CDX2 downregulation is associated with poor differentiation and MMR deficiency in colon cancer
    J. Olsen, Experimental and Molecular Pathology - 2015
    Background Homeobox genes are often deregulated in cancer and can have both oncogenic and tumor-suppressing potential. The Caudal-related homeobox transcription factor 2 (CDX2) is an intestine-specific transcription factor. CDX2 has been implicated in differentiation, proliferation, cell adhesion, and migration. In this study, we investigated CDX2 mRNA and protein expression in relation to the clinicopathological characteristics of colon cancer, including mismatch repair status and recurrence risk. Methods Tumor samples were obtained from colon cancer patients. Biopsies from tumor tissue and normal adjacent tissue were fixed in liquid nitrogen for RNA extraction or in formalin and paraffin embedded (FFPE) for immunohistochemical staining. CDX2 mRNA expression was evaluated by RT-qPCR. FFPE sections were stained for MLH1, MSH2, MSH6, PMS2, and CDX2. Results A total of 191 patient samples were included in the study and analyzed by immunohistochemistry. Of these samples, 97 were further evaluated by RT-qPCR. There was no significant difference in CDX2 mRNA expression between tumor and normal tissues. CDX2 mRNA expression was significantly lower in right-sided tumors (p < 0.05), poorly differentiated tumors (p < 0.05), and MMR-deficient tumors (p < 0.05). Similarly, CDX2 protein expression was more often low or absent in right-sided tumors (p < 0.01), poorly differentiated tumors (p < 0.001), and MMR-deficient tumors (p < 0.001). Low CDX2 protein or mRNA expression was not associated with recurrence risk. Conclusion We found that CDX2 downregulation is associated with MMR deficiency, right-sided tumors, and poor differentiation at both the mRNA and protein level. Whether CDX2 plays an active role in tumor progression in MSI/MMR-deficient tumors remains to be elucidated.
    Induction of lytic cycle sensitizes Epstein-Barr virus infected B cells to NK cell killing that is counteracted by virus-mediated NK cell evasion mechanisms in late lytic cycle
    Luke R. Williams, Journal of Virology - 2015
    Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV infected cells undergoing lytic replication in-vitro and studies in both humans, and mice with reconstituted human immune systems have shown NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV infected cells undergoing lytic replication, and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as yet unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, whilst cells entering lytic cycle become sensitized to NK cell killing, we observed that cells in late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV infected B cells undergoing lytic replication is dependent upon the phase of lytic cycle. Induction of lytic cycle is associated with acquired sensitization to NK cell killing, while progress through late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, implicating important roles for the BZLF1 immediate-early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.
    Chronic exposure to low doses of lipopolysaccharide and high‐fat feeding increases body mass without affecting glucose tolerance in female rats
    Anete Dudele, Physiological Reports - 2015
    Obesity‐related inflammation may have a causal role in the development of diabetes and insulin resistance, and studies using animal models of chronic experimental endotoxemia have shown the link. However, many studies use only males, and much less is known about the role of obesity‐related inflammation in females. Therefore, we addressed how experimentally induced chronic inflammation affects body mass, energy intake, and glucose metabolism in female rats. Adult female Sprague Dawley rats were instrumented with slow release pellets that delivered a constant daily dose of 53 or 207 μg of lipopolysaccharide (LPS) per rat for 60 days. Control rats were instrumented with vehicle pellets. Due to inflammatory nature of high‐fat diet (HFD) half of the rats received HFD (60% of calories from lard), while the other half remained on control diet to detect possible interactions between two modes of induced inflammation. Our results showed that chronic LPS administration increased female rat body mass and calorie intake in a dose‐dependent manner, and that HFD further exacerbated these effects. Despite these effects, no effects of LPS and HFD were evident on female rat glucose metabolism. Only LPS elevated expression of inflammatory markers in the hypothalamus. To conclude, female rats respond to experimentally induced chronic inflammation by increasing body mass, but do not develop glucose intolerance in the given period of time.
    Expansion of inflammatory innate lymphoid cells in patients with common variable immune deficiency
    Montserrat Cols, Journal of Allergy and Clinical Immunology - 2015
    Background Common variable immunodeficiency (CVID) is an antibody deficiency treated with immunoglobulin; however, patients can have noninfectious inflammatory conditions that lead to heightened morbidity and mortality. Objectives Modular analyses of RNA transcripts in whole blood previously identified an upregulation of many interferon-responsive genes. In this study we sought the cell populations leading to this signature. Methods Lymphoid cells were measured in peripheral blood of 55 patients with CVID (31 with and 24 without inflammatory/autoimmune complications) by using mass cytometry and flow cytometry. Surface markers, cytokines, and transcriptional characteristics of sorted innate lymphoid cells (ILCs) were defined by using quantitative PCR. Gastrointestinal and lung biopsy specimens of subjects with inflammatory disease were stained to seek ILCs in tissues. Results The linage-negative, CD127+, CD161+ lymphoid population containing T-box transcription factor, retinoic acid–related orphan receptor (ROR) γt, IFN-γ, IL-17A, and IL-22, all hallmarks of type 3 innate lymphoid cells, were expanded in the blood of patients with CVID with inflammatory conditions (mean, 3.7% of PBMCs). ILCs contained detectable amounts of the transcription factors inhibitor of DNA binding 2, T-box transcription factor, and RORγt and increased mRNA transcripts for IL-23 receptor (IL-23R) and IL-26, demonstrating inflammatory potential. In gastrointestinal and lung biopsy tissues of patients with CVID, numerous IFN-γ+RORγt+CD3− cells were identified, suggesting a role in these mucosal inflammatory states. Conclusions An expansion of this highly inflammatory ILC population is a characteristic of patients with CVID with inflammatory disease; ILCs and the interferon signature are markers for the uncontrolled inflammatory state in these patients.
    Comparative effects of whey protein versus l-leucine on skeletal muscle protein synthesis and markers of ribosome biogenesis following resistance exercise - Online First - Springer
    C. Brooks Mobley, Amino Acids - 2015
    We compared immediate post-exercise whey protein (WP, 500 mg) versus l-leucine (LEU, 54 mg) feedings on skeletal muscle protein synthesis (MPS) mechanisms and ribosome biogenesis markers 3 h following unilateral plantarflexor resistance exercise in male, Wistar rats (~250 g). Additionally, in vitro experiments were performed on differentiated C2C12 myotubes to compare nutrient (i.e., WP, LEU) and ‘exercise-like’ treatments (i.e., caffeine, hydrogen peroxide, and AICAR) on ribosome biogenesis markers. LEU and WP significantly increased phosphorylated-rpS6 (Ser235/236) in the exercised (EX) leg 2.4-fold (P < 0.01) and 2.7-fold (P < 0.001) compared to the non-EX leg, respectively, whereas vehicle-fed control (CTL) did not (+65 %, P > 0.05). Compared to the non-EX leg, MPS levels increased 32 % and 52 % in the EX leg of CTL (P < 0.01) and WP rats (P < 0.001), respectively, but not in LEU rats (+15 %, P > 0.05). Several genes associated with ribosome biogenesis robustly increased in the EX versus non-EX legs of all treatments; specifically, c-Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA, Npm1 mRNA, Fb1 mRNA, and Xpo-5 mRNA. However, only LEU significantly increased 45S pre-rRNA levels in the EX leg (63 %, P < 0.001). In vitro findings confirmed that ‘exercise-like’ treatments similarly altered markers of ribosome biogenesis, but only LEU increased 47S pre-rRNA levels (P < 0.01). Collectively, our data suggests that resistance exercise, as well as ‘exercise-like’ signals in vitro, acutely increase the expression of genes associated with ribosome biogenesis independent of nutrient provision. Moreover, while EX with or without WP appears superior for enhancing translational efficiency (i.e., increasing MPS per unit of RNA), LEU administration (or co-administration) may further enhance ribosome biogenesis over prolonged periods with resistance exercise.
    A novel stem cell culture model of recurrent glioblastoma
    Maleeha A. Qazi, Journal of Neuro-Oncology - 2015
    Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with average disease relapse at 9 months and median survival rarely extending beyond 15 months. Brain tumor stem cells (BTSCs) have been implicated in not only initiating GBM but also conferring resistance to therapy. However, it is not clear whether the BTSC population that initiates tumor growth is also responsible for GBM recurrence. In this study, we have developed a novel in vitro treatment model to profile the evolution of primary treatment-naïve GBM BTSCs through chemoradiotherapy. We report that our in vitro model enriched for a CD15+/CD133− BTSC population, mirroring the phenotype of BTSCs in recurrent GBM. We also show that in vitro treatment increased stem cell gene expression as well as self-renewal capacity of primary GBMs. In addition, the chemoradiotherapy-refractory gene signature obtained from gene expression profiling identified a hyper-aggressive subtype of glioma. The delivery of in vitro chemoradiotherapy to primary GBM BTSCs models several aspects of recurrent GBM biology, and could be used as a discovery and drug-screening platform to uncover new biological drivers and therapeutic targets in GBM.
    Interactive effects of chronic waterborne copper and cadmium exposure on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas)
    Melissa K. Driessnacka, Elsevier - 2016
    The present study was carried out to examine the interactive effects of chronic waterborne copper (Cu) and cadmium (Cd) on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas). Trios (1 male: 2 female; n = 5) of fish were exposed for 21 days to: (i) control (no added Cu or Cd), (ii) waterborne Cu (75 μg/L), (iii) waterborne Cd (5 μg/L), and (iv) Cu and Cd mixture (75 and 5 μg/L, respectively). Reproductive output (cumulative egg production) was significantly reduced by Cu but not by Cd. Interestingly however, no spawning occurred in fish exposed to the mixture of waterborne Cu and Cd. In general, both Cu and Cd accumulation in target tissues (gill, liver, gonad and carcass) increased significantly in fish exposed to Cu and Cd mixture, and no interaction between Cu and Cd accumulation was observed in any tissues, except in the liver where Cu accumulation was significantly reduced by Cd. The expression of female hepatic estrogen receptor genes (ER-α and ER-β) was most significantly elevated in fish exposed to Cu and Cd mixture, whereas vitellogenin gene expression was reduced maximally in the same exposure. Similarly, the hepatic expression of the metallothionein gene was most significantly upregulated in fish exposed to Cu and Cd mixture. Moreover, the circulating estradiol level in females was significantly decreased only during the co-exposure of waterborne Cu and Cd. Overall, the present study indicates that the interaction of chronic waterborne Cu and Cd exposure may elicit greater than additive effect on reproductive output in fish.
    Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD)
    161, Clinical Immunology - 2015
    Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.
    Caspase-3 cleaved p65 fragment dampens NF-κB-mediated anti-apoptotic transcription by interfering with the p65/RPS3 interaction
    Eric M. Wier, FEBS Letters - 2015
    Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF-κB)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF-κB transactivation. Here we show that the p651-97 fragment generated by Caspase-3 cleavage interferes with ribosomal protein S3 (RPS3), an NF-κB “specifier” subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF-κB-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF-κB-conferred anti-apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase-3.
    Transcriptome profiling of wheat glumes in wild emmer, hulled landraces and modern cultivars
    Hongda Zou, BMC Genomics - 2015
    Wheat domestication is considered as one of the most important events in the development of human civilization. Wheat spikelets have undergone significant changes during evolution under domestication, resulting in soft glumes and larger kernels that are released easily upon threshing. Our main goal was to explore changes in transcriptome expression in glumes that accompanied wheat evolution under domestication.
    Acute Psychological Stress Modulates the Expression of Enzymes Involved in the Kynurenine Pathway throughout Corticolimbic Circuits in Adult Male Rats
    Haley A. Vecchiarelli, Neural Plasticity - 2015
    Tryptophan is an essential dietary amino acid that is necessary for protein synthesis, but also serves as the precursor for serotonin. However, in addition to these biological functions, tryptophan also serves as a precursor for the kynurenine pathway, which has neurotoxic (quinolinic acid) and neuroprotective (kynurenic acid) metabolites. Glucocorticoid hormones and inflammatory mediators, both of which are increased by stress, have been shown to bias tryptophan along the kynurenine pathway and away from serotonin synthesis; however, to date, there is no published data regarding the effects of stress on enzymes regulating the kynurenine pathway in a regional manner throughout the brain. Herein, we examined the effects of an acute psychological stress (120 min restraint) on gene expression patterns of enzymes along the kynurenine pathway over a protracted time-course (1–24 h post-stress termination) within the amygdala, hippocampus, hypothalamus, and medial prefrontal cortex. Time-dependent changes in differential enzymes along the kynurenine metabolism pathway, particularly those involved in the production of quinolinic acid, were found within the amygdala, hypothalamus, and medial prefrontal cortex, with no changes seen in the hippocampus. These regional differences acutely may provide mechanistic insight into processes that become dysregulated chronically in stress-associated disorders.
    Nahmias Yaakov, United States Patent Application 20150268224 - 2015
    Systems, kits and methods for non-invasive, long-term, real-time monitoring of one or more physiological parameters of a cell, including but not limited to oxygen uptake, are provided.
    The effect of B-cell depletion in the Theiler's model of multiple sclerosis
    Francesca Gilli, Journal of the Neurological Sciences - 2015
    B cell depletion (BCD) is being considered as a treatment for multiple sclerosis (MS), but there are many uncertainties surrounding the use of this therapy, such as its potential effect in individuals with concurrent viral infections. We sought to discover what effect BCD, induced by an anti-CD20 monoclonal antibody, would have on Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Mice were injected with the anti-CD20 monoclonal antibody 5D2, 14 days before or 14 days after infection with TMEV. Efficacy of depletion of B cells was assessed by flow cytometry of CD19+ cells. Mouse disability was measured by Rotarod, viral load was measured by real time PCR for TMEV RNA. Binding and neutralizing antibody levels were determined in sera and CSF by ELISA, and in CNS by real time PCR for IgG RNA. Inflammation, microglial activation, axonal damage and demyelination were assessed using immunohistochemistry. 5D2-induced BCD was confirmed by demonstration of nearly absent CD19+ cells in the blood and lymphoid tissue. Systemic and CNS antibody responses were suppressed during 5D2 treatment. Higher viral loads were detected in 5D2-treated mice than in controls, and the viral levels correlated negatively with IgG production in the brain. Overall, 5D2 caused worsening of the early encephalitis and faster progression of disability, as well as exacerbation of the pathology of TMEV-IDD at the end stage of the disease. These data indicate that BCD in humans might worsen CNS viral infections and might not improve disability accrual in MS.
    Conductive interpenetrating networks of polypyrrole and polycaprolactone encourage electrophysiological development of cardiac cells
    Benjamin S. Spearman, Acta Biomaterialia - 2015
    Conductive and electroactive polymers have the potential to enhance engineered cardiac tissue function. In this study, an interpenetrating network of the electrically-conductive polymer polypyrrole (PPy) was grown within a matrix of flexible polycaprolactone (PCL) and evaluated as a platform for directing the formation of functional cardiac cell sheets. PCL films were either treated with sodium hydroxide to render them more hydrophilic and enhance cell adhesion or rendered electroactive with PPy grown via chemical polymerization yielding PPy–PCL that had a resistivity of 1.0 ± 0.4 kΩ cm, which is similar to native cardiac tissue. Both PCL and PPy–PCL films supported cardiomyocyte attachment; increasing the duration of PCL pre-treatment with NaOH resulted in higher numbers of adherent cardiomyocytes per unit area, generating cell densities which were more similar to those on PPy–PCL films (1568 ± 126 cells mm−2, 2880 ± 439 cells mm−2, 3623 ± 456 cells mm−2 for PCL with 0, 24, 48 h of NaOH pretreatment, respectively; 2434 ± 166 cells mm−2 for PPy–PCL). When cardiomyocytes were cultured on the electrically-conductive PPy–PCL, more cells were observed to have peripheral localization of the gap junction protein connexin-43 (Cx43) as compared to cells on NaOH-treated PCL (60.3 ± 4.3% vs. 46.6 ± 5.7%). Cx43 gene expression remained unchanged between materials. Importantly, the velocity of calcium wave propagation was faster and calcium transient duration was shorter for cardiomyocyte monolayers on PPy–PCL (1612 ± 143 μm/s, 910 ± 63 ms) relative to cells on PCL (1129 ± 247 μm/s, 1130 ± 20 ms). In summary, PPy–PCL has demonstrated suitability as an electrically-conductive substrate for culture of cardiomyocytes, yielding enhanced functional properties; results encourage further development of conductive substrates for use in differentiation of stem cell-derived cardiomyocytes and cardiac tissue engineering applications. Statement of Significance Current conductive materials for use in cardiac regeneration are limited by cytotoxicity or cost in implementation. In this manuscript, we demonstrate for the first time the application of a biocompatible, conductive polypyrrole–polycaprolactone film as a platform for culturing cardiomyocytes for cardiac regeneration. This study shows that the novel conductive film is capable of enhancing cell–cell communication through the formation of connexin-43, leading to higher velocities for calcium wave propagation and reduced calcium transient durations among cultured cardiomyocyte monolayers. Furthermore, it was demonstrated that chemical modification of polycaprolactone through alkaline-mediated hydrolysis increased overall cardiomyocyte adhesion. The results of this study provide insight into how cardiomyocytes interact with conductive substrates and will inform future research efforts to enhance the functional properties of cardiomyocytes, which is critical for their use in pharmaceutical testing and cell therapy.
    Ammonium-induced architectural and anatomical changes with altered suberin and lignin levels significantly change water and solute permeabilities of rice (Oryza sativa L.) roots - Online First - Springer
    Kosala Ranathunge, Planta - 2015
    Main conclusion Non-optimal ammonium levels significantly alter root architecture, anatomy and root permeabilities for water and nutrient ions. Higher ammonium levels induced strong apoplastic barriers whereas it was opposite for lower levels. Application of nitrogen fertilizer increases crop productivity. However, non-optimal applications can have negative effects on plant growth and development. In this study, we investigated how different levels of ammonium (NH4 +) [low (30 or 100 μM) or optimum (300 μM) or high (1000 or 3000 μM)] affect physio-chemical properties of 1-month-old, hydroponically grown rice roots. Different NH4 + treatments markedly altered the root architecture and anatomy. Plants grown in low NH4 + had the longest roots with a weak deposition of suberised and lignified apoplastic barriers, and it was opposite for plants grown in high NH4 +. The relative expression levels of selected suberin and lignin biosynthesis candidate genes, determined using qRT-PCR, were lowest in the roots from low NH4 +, whereas, they were highest for those grown in high NH4 +. This was reflected by the suberin and lignin contents, and was significantly lower in roots from low NH4 + resulting in greater hydraulic conductivity (Lp r) and solute permeability (P sr) than roots from optimum NH4 +. In contrast, roots grown at high NH4 + had markedly greater suberin and lignin contents, which were reflected by strong barriers. These barriers significantly decreased the P sr of roots but failed to reduce the Lp r below those of roots grown in optimum NH4 +, which can be explained in terms of the physical properties of the molecules used and the size of pores in the apoplast. It is concluded that, in rice, non-optimal NH4 + levels differentially affected root properties including Lp r and P sr to successfully adapt to the changing root environment.
    Correlating quantitative real-time PCR to rapid diagnostic test and RNA transcript expression in isolated gametocytemia and asexual parasitemia of Plasmodium falciparum malaria
    Rachel Lau, Tropical Diseases, Travel Medicine and Vaccines - 2015
    Background At present, only microscopic examination of stained thick and thin blood smears for malaria can differentiate clinically relevant asexual parasitemia from clinically irrelevant isolated gametocytemia. Microscopy is time consuming, labour intensive, and requires significant technical expertise to perform. Simple and rapid tests that can distinguish asexual from isolated sexual parasitemia are needed. Methods To determine if parasitemia and cycle threshold (CT) values on Plasmodium genus and P. falciparum-specific quantitative polymerase chain reaction (qPCR) assays correlate to positivity of rapid diagnostic test (RDT), and 18S rRNA gene copy number, we analyzed blood samples from Ontario patients with isolated P. falciparum gametocytemia or asexual stages. RNA transcripts were evaluated to determine whether there is correlation of expression to different life cycle stages of P. falciparum. Results 45 specimens containing isolated P. falciparum gametocytes, and 40 specimens containing isolated asexual stages by microscopy were identified and analyzed. By RDT, 40 of 45 (88.9 %) isolated gametocytemia specimens and 40 of 40 (100 %) asexual-stage specimens were positive for Plasmodium falciparum-specific histidine rich protein-2 (HRP-2). Fourteen of 45 (31.1 %) isolated gametocytemia specimens, and 36 of 40 (90 %) asexual-stage specimens were positive for Plasmodium genus aldolase T2 band. Positivity of the aldolase T2 band was associated with lower mean Plasmodium genus and P. falciparum-specific CT values, and to higher mean 18S rRNA gene copy by qPCR for both isolated gametocytemia and asexual-stage specimens. There was also a negative correlation of asexual parasitemia to both CT values, and positive correlation to 18S rRNA gene copy number. Analysis of asexual stage-specific erythrocyte binding antigen (eba-175) transcripts on 25 isolated gametocytemia and 20 asexual-stage specimens gave a positive predictive value of 62.5 % and negative predictive value of 100 % for asexual parasitemia. Thus, an absence of eba-175 transcripts excluded the presence of asexual (clinically relevant) parasitemia. Conclusions Positivity of the aldolase T2 band of BinaxNow RDT correlated to higher parasite load in both isolated gametocytemia and asexual-stage specimens. Asexual stage-specific eba-175 RNA transcript expression provided reasonable negative predictive value for exclusion of asexual parasitemia in clinical samples, but was present in both isolated gametocytemia and asexual stage specimens.
    Shemi, Amotz, United States Patent Application 20150238517 - 2015
    Treatment of cancer by regional and prolonged release of one or more nucleotide-based agents is provided.
    The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and act as potential endocrine disrupters in chicken LMH cells
    Solomon Asnake, Toxicology in Vitro - 2015
    Increased exposure of birds to endocrine disrupting compounds has resulted in developmental and reproductive dysfunctions. We have recently identified the flame retardants, allyl-2,4,6-tribromophenyl ether (TBP-AE), 2-3-dibromopropyl-2,4,6-tribromophenyl ether (TBP-DBPE) and the TBP-DBPE metabolite 2-bromoallyl-2,4,6-tribromophenyl ether (TBP-BAE) as antagonists to both the human androgen receptor (AR) and the zebrafish AR. In the present study, we aimed at determining whether these compounds also interact with the chicken AR. In silico modeling studies showed that TBP-AE, TBP-BAE and TBP-DBPE were able to dock into to the chicken AR ligand-binding pocket. In vitro transfection assays revealed that all three brominated compounds acted as chicken AR antagonists, inhibiting testosterone induced AR activation. In addition, qRT-PCR studies confirmed that they act as AR antagonists and demonstrated that they also alter gene expression patterns of apoptotic, anti-apoptotic, drug metabolizing and amino acid transporter genes. These studies, using chicken LMH cells, suggest that TBP-AE, TBP-BAE and TBP-DBPE are potential endocrine disrupters in chicken.
    Kidney-Specific Reduction of Oxidative Phosphorylation Genes Derived from Spontaneously Hypertensive Rat
    Jason A. Collett, PLoS ONE - 2015
    Mitochondrial (Mt) dysfunction contributes to the pathophysiology of renal function and promotes cardiovascular disease such as hypertension. We hypothesize that renal Mt-genes derived from female spontaneously hypertensive rats (SHR) that exhibit hypertension have reduced expression specific to kidney cortex. After breeding a female Okamoto-Aoki SHR (SAP = 188mmHg) with Brown Norway (BN) males (SAP = 100 and 104 mmHg), hypertensive female progeny were backcrossed with founder BN for 5 consecutive generations in order to maintain the SHR mitochondrial genome in offspring that contain over increasing BN nuclear genome. Mt-protein coding genes (13 total) and nuclear transcription factors mediating Mt-gene transcription were evaluated in kidney, heart and liver of normotensive (NT: n = 20) vs. hypertensive (HT: n = 20) BN/SHR-mtSHR using quantitative real-time PCR. Kidney cortex, but not liver or heart Mt-gene expression was decreased ~2–5 fold in 12 of 13 protein encoding genes of HT BN/SHR-mtSHR. Kidney cortex but not liver mRNA expression of the nuclear transcription factors Tfam, NRF1, NRF2 and Pgc1α were also decreased in HT BN/SHR-mtSHR. Kidney cortical tissue of HT BN/SHR-mtSHR exhibited lower cytochrome oxidase histochemical staining, indicating a reduction in renal oxidative phosphorylation but not in liver or heart. These results support the hypothesis that renal cortex of rats with SHR mitochondrial genome has specifically altered renal expression of genes encoding mitochondrial proteins. This kidney-specific coordinated reduction of mitochondrial and nuclear oxidative metabolism genes may be associated with heritable hypertension in SHR.
    Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa
    Dorit Cohen, PNAS - 2015
    The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP–specific phosphodies- terases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to pro- duce 5`-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG). However, to de- grade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)—a3ʹ→5ʹexonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteo- bacteria—as the primary enzyme responsible for pGpG degradation inPseudomonas aeruginosacells. We found that aP. aeruginosaΔ ornmutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce bio- film. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corre- sponding with this activity,Δorncells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL- dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP inΔorncells. Thus, we propose that Orn pro- vides homeostatic control of intracellular pGpG under native physio- logical conditions and that this activity is fundamental to c-di-GMP signal transduction.
    Tissue- and age-dependent expression of the bovine DEFB103 gene and protein - Online First - Springer
    Ali Mirabzadeh-Ardakani, Cell and Tissue Research - 2015
    Beta-defensin 103 (DEFB103) shares little homology with 8 other members of the bovine beta-defensin family and in other species DEFB103 protein has diverse functions, including antimicrobial activity, a chemoattractant for dendritic cells, enhancing epithelial wound repair and regulating hair colour. Expression of the bovine DEFB103 gene was surveyed in 27 tissues and transcript was most abundant in tissues with stratified squamous epithelium. Oral cavity epithelial tissues and nictitating membrane consistently expressed high levels of DEFB103 gene transcript. An age-dependent decrease (P < 0.05) in DEFB103 gene expression was only observed for buccal epithelium when comparing healthy 10- to 14-day-old and 10- to 12-month-old calves. A bovine herpesvirus-1 respiratory infection did, however, significantly (P < 0.05) up-regulate DEFB103 gene expression in the buccal epithelium of 6- to 8-month-old calves. Finally, DEFB103 transcript was low in lymph nodes draining the skin and at the limit of detection in other internal organs such as lung, intestine and kidney. Affinity-purified rabbit antisera to bovine DEFB103 was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells prior to birth. Beta-defensins have been identified as regulators of dendritic cell (DC) chemokine responses and we observed a close association between DCs and epithelial cells expressing DEFB103 in both the fetus and newborn calf. In conclusion, bovine DEFB103 gene expression is most abundant in stratified squamous epithelium with DEFB103 protein localised to basal epithelial cells. These observations are consistent with proposed roles for DEFB103 in DC recruitment and repair of stratified squamous epithelium.
    Functional Activation of the Flagellar Type III Secretion Export Apparatus
    Andrew M. Phillips, PLoS Genet - 2015
    Author Summary Bacteria build needle-like injectsomes to secrete toxins into host cells and build propeller-like flagella to swim through their environment using a molecular machine called the type III secretion system (T3SS). Both the injectisome and the flagellum are large self-assembling complexes and regulation of the T3SS ensures that proteins are secreted sequentially for proper structure and function. Here we report genetic and cytological data that the SwrB protein of Bacillus subtilis helps the base of the flagellum adopt a completed conformation which in turn activates the enclosed T3SS to export proteins for the next stage of flagellar assembly. Thus SwrB presents a novel mechanism to supervise an early structural checkpoint regulating machine assembly. Targeting functional regulators like SwrB could inhibit T3SS-based strategies of pathogens.
    Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell–dependent manner through cysteinyl leukotriene receptor 1 and E-prostanoid receptor 3
    Vinay Kondeti, Journal of Allergy and Clinical Immunology - 2015
    Background Although arachidonic acid metabolites, cysteinyl leukotrienes (cys-LTs; leukotriene [LT] C4, LTD4, and LTE4), and prostaglandin (PG) E2 are generated at the site of inflammation, it is not known whether crosstalk exists between these 2 classes of inflammatory mediators. Objective We sought to determine the role of LTD4-PGE2 crosstalk in inducing vascular inflammation in vivo, identify effector cells, and ascertain specific receptors and pathways involved in vitro. Methods Vascular (ear) inflammation was assessed by injecting agonists into mouse ears, followed by measuring ear thickness and histology, calcium influx with Fura-2, phosphorylation and expression of signaling molecules by means of immunoblotting, PGD2 and macrophage inflammatory protein 1β generation by using ELISA, and expression of transcripts by using RT-PCR. Candidate receptors and signaling molecules were identified by using antagonists and inhibitors and confirmed by using small interfering RNA. Results LTD4 plus PGE2 potentiated vascular permeability and edema, gearing the system toward proinflammation in wild-type mice but not in KitW-sh mice. Furthermore, LTD4 plus PGE2, through cysteinyl leukotriene receptor 1 (CysLT1R) and E-prostanoid receptor (EP) 3, enhanced extracellular signal-regulated kinase (Erk) and c-fos phosphorylation, inflammatory gene expression, macrophage inflammatory protein 1β secretion, COX-2 upregulation, and PGD2 generation in mast cells. Additionally, we uncovered that this synergism is mediated through Gi, protein kinase G, and Erk signaling. LTD4 plus PGE2–potentiated effects are partially sensitive to CysLT1R or EP3 antagonists but completely abolished by simultaneous treatment both in vitro and in vivo. Conclusions Our results unravel a unique LTD4-PGE2 interaction affecting mast cells through CysLT1R and EP3 involving Gi, protein kinase G, and Erk and contributing to vascular inflammation in vivo. Furthermore, current results also suggest an advantage of targeting both CysLT1R and EP3 in attenuating inflammation.
    Serotonin promotes acinar de-differentiation following pancreatitis-induced regeneration in the adult pancreas
    Enrica Saponara, The Journal of Pathology - 2015
    The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help develop therapeutic interventions. Serotonin (5-hydroxytryptamine, 5-HT) has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, namely inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar de-differentiation and cell cycle progression during the regenerative phase were investigated over the course of two weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect clonal regeneration of mature acinar cells. Serotonin was however required for acinar de-differentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes, delayed formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in the progenitor-based regeneration, as it promoted acinar cell de-differentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas activated by zymogen secretion. Our findings indicate that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting de-differentiation of acinar cells.
    DNA methylation signature of interleukin 1 receptor type II in asthma
    Valérie Gagné-Ouellet, Clinical Epigenetics - 2015
    Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
    The effect of energy balance on the transcriptome of bovine granulosa cells at 60 days post-partum
    Annie Girard, Theriogenology - 2015
    Dairy cows expend great amounts of energy during the lactation peak to cope with milk production. A state of negative energy balance was suggested as a cause for the suboptimal fertility observed during this period, via an interaction with ovarian function. The objective of this study was to identify the impact of negative energy balance (NEB) on gene expression in granulosa cells of dairy cows at 60 days post-partum, and to suggest a potential treatment to improve ovarian function. Dairy cows at 60 days post-partum from ten typical medium-sized farms were synchronized using a single injection of prostaglandin (PG). Dominant follicles were collected 42 h later by transvaginal aspiration. Blood concentrations of beta-hydroxybutyrate (BHB) on the day of aspiration were used to classify animals into two groups: severe NEB (high BHB, n=12) and mild NEB (low BHB, n=12). The transcriptomes of granulosa cells from both groups were contrasted using microarrays and the differently expressed genes were analysed using Ingenuity Pathway Analysis to identify affected functions and potential upstream regulators. Genes linked with cellular organization (KRT4 and PPL), proliferation (TACSTD2) and fatty acids metabolism (VNN2) were downregulated in granulosa cells from animals with severe NEB. Several genes linked to Decitabine, a hypomethylating agent, and with beta-estradiol, were downregulated in the severe NEB group. Numerous genes linked to vitamins A and D were also downregulated in this group of cows, suggesting a potential deficiency of these vitamins in dairy cows during the post-partum period. This study supports the idea that energy balance has an impact on follicular dynamics which could be detrimental to resumption of fertility after calving.
    dentification and characterization of a novel family of cysteine-rich peptides
    Marco Gerdol, Genome Biology and Evolution - 2015
    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis . These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre- propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, i.e. gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.
    Expression of barley SUSIBA2 transcription factor yields high-starch low-methane rice
    J. Su, Nature - 2015
    Atmospheric methane is the second most important greenhouse gas after carbon dioxide, and is responsible for about 20% of the global warming effect since pre-industrial times. Rice paddies are the largest anthropogenic methane source and produce 7–17% of atmospheric methane. Warm waterlogged soil and exuded nutrients from rice roots provide ideal conditions for methanogenesis in paddies with annual methane emissions of 25–100-million tonnes. This scenario will be exacerbated by an expansion in rice cultivation needed to meet the escalating demand for food in the coming decades. There is an urgent need to establish sustainable technologies for increasing rice production while reducing methane fluxes from rice paddies. However, ongoing efforts for methane mitigation in rice paddies are mainly based on farming practices and measures that are difficult to implement. Despite proposed strategies to increase rice productivity and reduce methane emissions, no high-starch low-methane-emission rice has been developed. Here we show that the addition of a single transcription factor gene, barley SUSIBA2 (refs 7, 8), conferred a shift of carbon flux to SUSIBA2 rice, favouring the allocation of photosynthates to aboveground biomass over allocation to roots. The altered allocation resulted in an increased biomass and starch content in the seeds and stems, and suppressed methanogenesis, possibly through a reduction in root exudates. Three-year field trials in China demonstrated that the cultivation of SUSIBA2 rice was associated with a significant reduction in methane emissions and a decrease in rhizospheric methanogen levels. SUSIBA2 rice offers a sustainable means of providing increased starch content for food production while reducing greenhouse gas emissions from rice cultivation. Approaches to increase rice productivity and reduce methane emissions as seen in SUSIBA2 rice may be particularly beneficial in a future climate with rising temperatures resulting in increased methane emissions from paddies.
    Integrated genomic and functional analyses of histone demethylases identify oncogenic KDM2A isoform in breast cancer
    Hui Liu, Molecular Carcinogenesis - 2015
    Histone lysine demethylases (KDMs) comprise a large class of enzymes that catalyze site-specific demethylation of lysine residues on histones and other proteins. They play critical roles in controlling transcription, chromatin architecture, and cellular differentiation. However, the genomic landscape and clinical significance of KDMs in breast cancer remain poorly characterized. Here, we conducted a meta-analysis of 24 KDMs in breast cancer and identified associations among recurrent copy number alterations, gene expression, breast cancer subtypes, and clinical outcome. Two KDMs, KDM2A and KDM5B, had the highest frequency of genetic amplification and overexpression. Furthermore, among the 24 KDM genes, KDM2A had the highest correlation between copy number and mRNA expression, and high mRNA levels of KDM2A were significantly associated with shorter survival of breast cancer patients. KDM2A has two isoforms: the long isoform is comprised of a JmjC domain, CXXC-zinc finger, PHD zinc finger, F-box, and the AMN1 protein domain; whereas the short isoform of KDM2A lacks the N-terminal JmjC domain but contains all other motifs. Detailed characterization of KDM2A in breast cancer revealed that the short isoform of KDM2A is more abundant than the long isoform at DNA, mRNA, and protein levels in a subset of breast cancers. Furthermore, our data indicate that the short isoform of KDM2A has oncogenic potential and functions as an oncogenic isoform in a subset of breast cancers. Taken together, our findings suggest that amplification and overexpression of the KDM2A short isoform is critical in breast cancer progression. © 2015 Wiley Periodicals, Inc.
    Klotho expression in cervical cancer: differential expression in adenocarcinoma and squamous cell carcinoma
    Sarit Aviel-Ronen, Journal of Clinical Pathology - 2015
    Aims Klotho is a trans-membrane protein that serves as a tumour suppressor in a wide array of malignancies. Recent data suggest it as an epigenetically silenced tumour suppressor in cervical cancer. Yet, the expression pattern of klotho in cervical cancer has not been determined. We aimed to study the expression of klotho in squamous cell carcinomas (SQCC) and adenocarcinoma (ADC) of the cervix. Methods Klotho expression was analysed by immunohistochemistry in 44 SQCC samples, 38 ADC samples and the adjacent normal tissue. For each sample, percentage of positive stained cells, staining intensity and a combined staining score were recorded. Staining was validated by measuring klotho mRNA levels, using quantitative RT-PCR, in 18 of the samples. Results Klotho expression was high in all endocervical and exocervical normal tissues adjacent to tumour. No expression of klotho was noted in 7 out of 38 (18.4%) ADC samples and in 2 out of 44 (4.5%) SQCC samples. Staining intensity, number of positively stained cells and combined intensity score were all lower in tumours compared with normal adjacent tissues in ADC and SQCC. Klotho mRNA levels highly correlated with immunohistochemical (IHC) staining (p=0.008). Conclusions We found reduced klotho expression in cervical carcinoma, especially in ADC, compared with normal adjacent tissue. Our results support the role of klotho as a potential tumour suppressor in cervical cancer. Further studies are required in order to establish the therapeutic role of klotho in cervical carcinoma and identify patients who may benefit from it.
    Impact of reduced ATGL-mediated adipocyte lipolysis
    Gabriele Schoiswohl, Endocrine Society - 2015
    Emerging evidence suggests that impaired regulation of adipocyte lipolysis contributes to the pro-inflammatoryimmunecell infiltration of metabolic tissues in obesity, a process that is proposed to contribute to the development and exacerbation of insulin resistance. To test this hypothesis in vivo, we generated mice with adipocyte-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme catalyzing triacylglycerol hydrolysis. In contrast to prior models, adiponectin (Adipoq)-driven Cre expression was used for targeted ATGL deletion. The resulting adipocytespecific ATGL knockout (AAKO) mice were then characterized for metabolic and immune phenotypes. Lean and diet-induced obese AAKO mice had reduced adipocyte lipolysis, serum lipids, systemic lipid oxidation, and expression of PPAR target genes in adipose tissue and liver. These changes did not increase overall body weight or fat mass in AAKO mice by 24 weeks of age, in part due to reduced expression of genes involved in lipid uptake, synthesis, and adipogenesis. Systemic glucose and insulin tolerance were improved in AAKO mice, primarily due to enhanced hepatic insulin signaling, which was accompanied by marked reduction in diet-induced hepatic steatosis as well as hepatic immune cell infiltration and activation. In contrast, although adipocyte ATGL deletion reduced adipose tissue immune cell infiltration in response to an acute lipolytic stimulus, it was not sufficient to ameliorate, and may even exacerbate, chronic inflammatory changes that occur in adipose tissue in response to diet-induced obesity.
    Xanthohumol impairs glucose uptake by a human first trimester extravillous trophoblast cell line (HTR-8/SVneo cells) and impacts the process of placentation
    Ana Correia-Branco, Oxford Journals - 2015
    In this study we aimed to investigate modulation of glucose uptake by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line by a series of compounds and to study its consequences upon cell proliferation, viability and migration. We observed that uptake of 3H-deoxy-D-glucose (3H-DG; 10 nM) was time-dependent, saturable, inhibited by cytochalasin B (50 and 100 µM), phloretin (0.5 mM) and phloridzin (1 mM), insulin-insensitive and sodium-independent. In the short-term (30 min), neither 5-HT (100-1000 µM), melatonin (10 nM) nor the drugs of abuse ethanol (100 mM), nicotine (100 µM), cocaine (25 µM), amphetamine (10-25 µM) and 3,4-methylenedioxy-N-methamphetamine (MDMA, 10 µM) affected 3H-DG uptake, while dexamethasone (100-1000 µM), fluoxetine (100-300 µM), quercetin, epigallocatechin-3-gallate (30-1000 µM), xanthohumol (XH) and resveratrol (1-500 µM) decreased it. XH was the most potent inhibitor (IC50= 3.55 (1.37-9.20) µM) of 3H-DG uptake, behaving as a non-competitive inhibitor of 3H-DG uptake, both after short- and long-term (24h) treatment. The effect of XH (5 µM; 24h) upon 3H-DG uptake involved mammalian target of rapamycin, tyrosine kinases and c-Jun N-terminal kinases intracellular pathways. Moreover, XH appeared to decrease cellular uptake of lactate due to inhibition of the monocarboxylate transporter 1 (MCT1). Additionally, XH (24h; 5 µM) decreased cell viability, proliferation, culture growth and migration. The effects of XH upon cell viability and culture growth, but not the antimigratory effect, were mimicked by low extracellular glucose conditions and reversed by high extracellular glucose conditions. We thus suggest that XH, by inhibiting glucose cellular uptake and impairing HTR-8/SVneo cell viability and proliferation, may have a deleterious impact in the process of placentation.
    Methods and Compositions for Treating Prostate Cancer
    , United States Patent Application - 2015
    Treatment of prostate cancer by regional and prolonged release of one or more nucleotide-based RNAi agents is provided.
    Ex vivo evaluation of acellular and cellular collagen-glycosaminoglycan flowable matrices
    Tom Hodgkinson, Biomedical Materials - 2015
    Collagen-glycosaminoglycan flowable matrices (CGFM) are increasingly finding utility in a diversifying number of cutaneous surgical procedures. Cellular in-growth and vascularisation of CGFM remain rate-limiting steps, increasing cost and decreasing efficacy. Through in vitro and ex vivo culture methods, this study investigated the improvement of injectable CGFM by the incorporation of hyaluronan (HA) and viable human cells (primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs)). Ex vivo investigations included the development and evaluation of a human cutaneous wound healing model for the comparison of dermal substitutes. Cells mixed into the Integra Flowable Wound Matrix (IFWM), a commercially available CGFM, were confirmed to be viable and proliferative through MTT assays (p  <  0.05). PHDFs proliferated with greater rapidity than BM-MSCs up to 1 week in culture (p  <  0.05), with PHDF proliferation further enhanced by HA supplementation (p  <  0.05). After scaffold mixing, gene expression was not significantly altered (qRT-PCR). PHDF and BM-MSC incorporation into ex vivo wound models significantly increased re-epithelialisation rate, with maximal effects observed for BM-MSC supplemented IFWM. HA supplementation to PHDF populated IFWM increased re-epithelialisation but had no significant effect on BM-MSC populated IFWM. In conclusion, when combined with PHDF, HA increased re-epithelialisation in IFWM. BM-MSC incorporation significantly improved re-epithelialisation in ex vivo models over acellular and PHDF populated scaffolds. Viable cell incorporation into IFWM has potential to significantly benefit wound healing in chronic and acute cutaneous injuries by allowing a point-of-care matrix to be formed from autologous or allogenic cells and bioactive molecules.
    The Dual EGFR/HER2 Inhibitor Lapatinib Synergistically Enhances the Antitumor Activity of the Histone Deacetylase Inhibitor Panobinostat in Colorectal Cancer Models
    Melissa LaBonte, Cancer Research - 2011
    As key molecules that drive progression and chemoresistance in gastrointestinal cancers- epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L, lapatinib range 7.6-25.8 \{micro\}mol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks- caspase-8 activation- and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-\{kappa\}B1- IRAK1- and CCND1. Panobinostat treatment induced downregulation of EGFR- HER2- and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model- the combination showed greater antitumor activity than either agent alone- with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment. Cancer Res, 71(10), 3635-48. (C)2011 AACR.
    RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation
    Tanja Rosel, The EMBO Journal - 2011
    The U1 snRNP particle contains three U1-specific proteins- including U1C- and is involved in 5' splice-site recognition during spliceosome assembly. Here- RNA-Seq analysis of a zebrafish U1C null mutant identifies a gene-specific role for U1C in regulating alternative splicing.- Precise 5′ splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish- hi1371- to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable- but U1C-deficient U1 snRNPs. Surprisingly- genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity- as well as the functional conservation of the effects observed. In addition- sequence motif analysis of the U1C-dependent 5′ splice sites uncovered an association with downstream intronic U-rich elements. In sum- our findings provide evidence for a new role of a general snRNP protein- U1C- as a mediator of alternative splicing regulation.
    Genome Expansion and Differential Expression of Amino Acid Transporters at the Aphid/Buchnera Symbiotic Interface
    Daniel Price, Molecular Biology and Evolution - 2011
    In insects- some of the most ecologically important symbioses are nutritional symbioses that provide hosts with novel traits and thereby facilitate exploitation of otherwise inaccessible niches. One such symbiosis is the ancient obligate intracellular symbiosis of aphids with the \{gamma\}-proteobacteria- Buchnera aphidicola. Although the nutritional basis of the aphid/Buchnera symbiosis is well understood- the processes and structures that mediate the intimate interactions of symbiotic partners remain uncharacterized. Here- using a de novo approach- we characterize the complement of 40 amino acid polyamine organocation (APC) superfamily member amino acid transporters (AATs) encoded in the genome of the pea aphid- Acyrthosiphon pisum. We find that the A. pisum APC superfamily is characterized by extensive gene duplications such that A. pisum has more APC superfamily transporters than other fully sequenced insects- including a ten paralog aphid-specific expansion of the APC transporter slimfast. Detailed expression analysis of 17 transporters selected on the basis of their phylogenetic relationship to five AATs identified in an earlier bacteriocyte expressed sequence tag study distinguished a subset of eight transporters that have been recruited for amino acid transport in bacteriocyte cells at the symbiotic interface. These eight transporters include transporters that are highly expressed and/or highly enriched in bacteriocytes and intriguingly- the four AATs that show bacteriocyte-enriched expression are all members of gene family expansions- whereas three of the four that are highly expressed but not enriched in bacteriocytes retain one-to-one orthology with transporters in other genomes. Finally- analysis of evolutionary rates within the large A. pisum slimfast expansion demonstrated increased rates of molecular evolution coinciding with two major shifts in expression: 1) a loss of gut expression and possibly a gain of bacteriocyte expression and 2) loss of expression in all surveyed tissues in asexual females. Taken together- our characterization of nutrient AATs at the aphid/Buchnera symbiotic interface provides the first examination of the processes and structures operating at the interface of an obligate intracellular insect nutritional symbiosis- offering unique insight into the types of genomic change that likely facilitated evolutionary maintenance of the symbiosis.
    Differential regulation of Igf1 and Igf2 mRNA levels in tilapia hepatocytes: effects of insulin and cortisol on GH sensitivity
    Andrew Pierce, Journal of Endocrinology - 2011
    Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals- liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life- whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly- and by increasing hepatocyte sensitivity to GH. We examined the regulation of igf1 and igf2 mRNA levels by GH- insulin- and cortisol- and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia- a cichlid teleost. GH increased mRNA levels of both igf1 and igf2 in a concentration-related and biphasic manner over the physiological range- with a greater effect on igf2 mRNA level. Insulin increased basal igf2 mRNA level- and strongly increased GH-stimulated igf2 mRNA level- but slightly reduced basal igf1 mRNA level and did not affect GH-stimulated igf1 mRNA level. Cortisol inhibited GH stimulation of igf1- but increased GH stimulation of igf2 mRNA level. The synergistic effect of insulin and GH on igf2 mRNA level was confirmed in vivo. These results indicate that insulin and cortisol differentially modulate the response of igf1 and igf2 mRNA to GH in tilapia hepatocytes- and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.
    Endotoxin Tolerance Represents a Distinctive State of Alternative Polarization (M2) in Human Mononuclear Cells
    Olga Pena, The Journal of Immunology - 2011
    Classical (M1) and alternative (M2) polarization of mononuclear cells (MNCs) such as monocyte and macrophages is known to occur in response to challenges within a microenvironment- like the encounter of a pathogen. LPS- also known as endotoxin- is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in MNCs known as endotoxin tolerance- defined as the reduced capacity of a cell to respond to LPS activation after an initial exposure to this stimulus. Using systems biology approaches in PBMCs- monocytes- and monocyte-derived macrophages involving microarrays and advanced bioinformatic analysis- we determined that gene responses during endotoxin tolerance were similar to those found during M2 polarization- featuring gene and protein expression critical for the development of key M2 MNC functions- including reduced production of proinflammatory mediators- expression of genes involved in phagocytosis- as well as tissue remodeling. Moreover- expression of different metallothionein gene isoforms- known for their role in the control of oxidative stress and in immunomodulation- were also found to be consistently upregulated during endotoxin tolerance. These results demonstrate that after an initial inflammatory stimulus- human MNCs undergo an M2 polarization probably to control hyperinflammation and heal the affected tissue.
    Endometriosis expresses a molecular pattern consistent with decreased retinoid uptake- metabolism and action
    Mary Pavone, Human Reproduction - 2011
    BACKGROUNDRetinoic acid (RA) regulates key biological processes- including differentiation- apoptosis and cell survival. RA mediates induction of 17 beta-hydroxysteroid dehydrogenase type 2 mRNA- catalyzing the conversion of estradiol to estrone- in endometrium but not endometriosis because of a defect in endometriotic stromal cells. This defect may involve both the uptake and metabolism of RA. In this study- we analyze the expression of genes involved in RA signaling in normal endometrium and endometriosis. METHODSTissue and stromal cells from ovarian endometriomas and eutopic endometrium from disease-free women were collected. Real-time reverse transcription-polymerase chain reaction was used to measure mRNA levels. Western blotting was used to evaluate protein expression. RESULTSWe found that endometriotic tissue and stromal cells demonstrated significantly decreased mRNA expression of the major genes involved in RA signaling- including STRA6- CRBP1- ALDH1A2- CRABP2 and FABP5. We found increased levels of CYP26B1- responsible for RA metabolism. Nuclear extracts showed that RAR- RXR and PPAR\{beta\}/\{delta\} were underexpressed in both tissues and stromal cells from endometriotic tissue. Differences in protein levels were confirmed by western blotting. CONCLUSIONSEndometriosis is characterized by a gene expression pattern suggesting a decrease in uptake and metabolism of RA. Because RA is integral in regulating key biological processes involved in cell survival- this alteration could partially explain the resistance to apoptosis found in endometriosis.
    Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the gammac cytokines and TGF-beta1
    Yuk Park, Blood - 2011
    Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor- also expressed on subsets of T cells- whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein- thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-\{beta\}1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis- indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-\{beta\}1 has an opposite and dominant effect to IL-2. TGF-\{beta\}1 treatment decreases DAP10- as its presence inhibits the association of RNA polymerase II with the DAP10 promoter- but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and- as a consequence- NKG2D protein as well. Finally- we show that other \{gamma\}c cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.
    Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms
    Julien Ochala, Journal of Physiology - 2011
    Non-technical summary Wasting and severely impaired function of skeletal muscle is frequently observed in critically ill intensive care unit (ICU) patients- with negative consequences for recovery and quality of life. An experimental rat ICU model has been used to study the mechanisms underlying this unique wasting condition in neuromuscularly blocked and mechanically ventilated animals at durations varying between 6 h and 2 weeks. The complete mechanical silencing' of skeletal muscle (removal of both weight bearing and activation) resulted in a specific myopathy frequently observed in ICU patients and characterized by a preferential loss of the motor protein myosin. A highly complex and coordinated protein synthesis and degradation system was observed in the time-resolved analyses. It is suggested the mechanical silencing' of skeletal muscle is a dominating factor triggering the specific myopathy associated with the ICU intervention- and strongly supporting the importance of interventions counteracting the complete unloading in ICU patients. Abstract The muscle wasting and impaired muscle function in critically ill intensive care unit (ICU) patients delay recovery from the primary disease- and have debilitating consequences that can persist for years after hospital discharge. It is likely that- in addition to pernicious effects of the primary disease- the basic life support procedures of long-term ICU treatment contribute directly to the progressive impairment of muscle function. This study aims at improving our understanding of the mechanisms underlying muscle wasting in ICU patients by using a unique experimental rat ICU model where animals are mechanically ventilated- sedated and pharmacologically paralysed for duration varying between 6 h and 14 days. Results show that the ICU intervention induces a phenotype resembling the severe muscle wasting and paralysis associated with the acute quadriplegic myopathy (AQM) observed in ICU patients- i.e. a preferential loss of myosin- transcriptional down-regulation of myosin synthesis- muscle atrophy and a dramatic decrease in muscle fibre force generation capacity. Detailed analyses of protein degradation pathways show that the ubiquitin proteasome pathway is highly involved in this process. A sequential change in localisation of muscle-specific RING finger proteins 1/2 (MuRF1/2) observed during the experimental period is suggested to play an instrumental role in both transcriptional regulation and protein degradation. We propose that- for those critically ill patients who develop AQM- complete mechanical silencing- due to pharmacological paralysis or sedation- is a critical factor underlying the preferential loss of the molecular motor protein myosin that leads to impaired muscle function or persisting paralysis.
    Nek10 Mediates G2/M Cell Cycle Arrest and MEK Autoactivation in Response to UV Irradiation
    Larissa Moniz, Molecular and Cellular Biology - 2011
    Appropriate cell cycle checkpoint control is essential for the maintenance of cell and organismal homeostasis. Members of the Nek (NIMA-related kinase) family of serine/threonine protein kinases have been implicated in the regulation of various aspects of the cell cycle. We explored the cellular functions of Nek10- a novel member of the Nek family- and demonstrate a role for Nek10 in the cellular UV response. Nek10 was required for the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling upon UV irradiation but not in response to mitogens- such as epidermal growth factor stimulation. Nek10 physically associated with Raf-1 and MEK1 in a Raf-1-dependent manner- and the formation of this complex was necessary for Nek10-mediated MEK1 activation. Nek10 did not affect the kinase activity of Raf-1 but instead promoted the autophosphorylation-dependent activation of MEK1. The appropriate maintenance of the G2/M checkpoint following UV irradiation required Nek10 expression and ERK1/2 activation. Taken together- our results uncover a role for Nek10 in the cellular response to UV irradiation.
    Effective fiber hypertrophy in satellite cell-depleted skeletal muscle
    John McCarthy, Development (Cambridge- England) - 2011
    An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue- a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of {\textgreater}90\% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy- the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload- satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers- resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers- long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force- Ca2+ sensitivity- rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response- both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion- indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.
    Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18a?
    Ken Matsui, Journal of proteome research - 2011
    The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis we identified Myo18aalpha as a novel binding partner of ezrin. Myo18aalpha is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18aalpha varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly- BCR stimulation increased the association between ezrin and Myo18aalpha- and induced co-segregation of Myo18aalpha with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18aalpha/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.
    DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration
    Hiroki Kaneko, Nature - 2011
    Geographic atrophy (GA)- an untreatable advanced form of age-related macular degeneration- results from retinal pigmented epithelium (RPE) cell death. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA- and that conditional ablation of Dicer1- but not seven other miRNA-processing enzymes- induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA- and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA- and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation- show that Alu RNA can directly cause human pathology- and identify new targets for a major cause of blindness.
    Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus
    Franz Kainz, Development - 2011
    Arthropods and vertebrates display a segmental body organisation along all or part of the anterior-posterior axis. Whether this reflects a shared- ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates- segments are formed sequentially by a segmentation clock' of oscillating gene expression involving Notch pathway components. Recent studies in spiders and basal insects have suggested that segmentation in these arthropods also involves Notch-based signalling. These observations have been interpreted as evidence for a shared- ancestral gene network for insect- arthropod and bilaterian segmentation. However- because this pathway can play multiple roles in development- elucidating the specific requirements for Notch signalling is important for understanding the ancestry of segmentation. Here we show that Delta- a ligand of the Notch pathway- is not required for segment formation in the cricket Gryllus bimaculatus- which retains ancestral characteristics of arthropod embryogenesis. Segment patterning genes are expressed before Delta in abdominal segments- and Delta expression does not oscillate in the pre-segmental region or in formed segments. Instead- Delta is required for neuroectoderm and mesectoderm formation, embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus- what initially appear to be segmentation phenotypes' can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods- and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.
    Progesterone and Mifepristone Regulate L-Type Amino Acid Transporter 2 and 4F2 Heavy Chain Expression in Uterine Leiomyoma Cells
    Xia Luo, Journal of Clinical Endocrinology & Metabolism - 2009
    Context: Progesterone and its receptor (PR) play key roles in uterine leiomyoma growth. Previously- using chromatin immunoprecipitation-based cloning- we uncovered L-type amino acid transporter 2 (LAT2) as a novel PR target gene. LAT2 forms heterodimeric complexes with 4F2 heavy chain (4F2hc)- a single transmembrane domain protein essential for LAT2 to exert its function in the plasma membrane. Until now- little is known about the roles of LAT2/4F2hc in the regulation of the growth of human uterine leiomyoma. Objective: The aim of the study is to investigate the regulation of LAT2 and 4F2hc by progesterone and the antiprogestin mifepristone and their functions in primary human uterine leiomyoma smooth muscle (LSM) cells and tissues from 39 premenopausal women. Results: In primary LSM cells- progesterone significantly induced LAT2 mRNA levels- and this was blocked by cotreatment with mifepristone. Progesterone did not alter 4F2hc mRNA levels- whereas mifepristone significantly induced 4F2hc mRNA expression. Small interfering RNA knockdown of LAT2 or 4F2hc markedly increased LSM cell proliferation. LAT2- PR-B- and PR-A levels were significantly higher in freshly isolated LSM cells vs. adjacent myometrial cells. In vivo- mRNA levels of LAT2 and PR but not 4F2hc were significantly higher in leiomyoma tissues compared with matched myometrial tissues. Conclusion: We present evidence that progesterone and its antagonist mifepristone regulate the amino acid transporter system LAT2/4F2hc in leiomyoma tissues and cells. Our findings suggest that products of the LAT2/4F2hc genes may play important roles in leiomyoma cell proliferation. We speculate that critical ratios of LAT2 to 4F2hc regulate leiomyoma growth.
    prtH2- Not prtH- Is the Ubiquitous Cell Wall Proteinase Gene in Lactobacillus helveticus
    M. Genay, Applied and Environmental Microbiology - 2009
    Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus- two CEPs with low percentages of amino acid identity have been described- i.e.- PrtH and PrtH2. However- the distribution of the genes that encode CEPs still remains unclear- rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of L. helveticus isolated from various biotopes- both in terms of the presence or absence of these genes and in terms of nucleotide sequence- and studied their transcription in dairy matrices. After defining three sets of primers for both the prtH and prtH2 genes- we studied the distribution of the genes by using PCR and Southern blotting experiments. The prtH2 gene was ubiquitous in the 29 strains of L. helveticus studied- whereas only 18 of them also exhibited the prtH gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally- expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains- ITG LH77 and CNRZ32- which possess one and two CEP-encoding genes- respectively. Both genes were shown to be expressed by L. helveticus at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening.
    Derivation of induced pluripotent stem cells from pig somatic cells
    Toshihiko Ezashi, Proceedings of the National Academy of Sciences U.S.A. - 2009
    For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here- we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes- hOCT4- hSOX2- hKLF4- and hc-MYC- the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after \{approx\}22 days- providing an overall reprogramming efficiency of \{approx\}0.1\%. The cells expressed porcine OCT4- NANOG- and SOX2 and had high telomerase activity- but also continued to express the 4 human transgenes. Unlike human ESC- the porcine iPSC (piPSC) were positive for SSEA-1- but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line- ID6- had a normal karyotype- a cell doubling time of \{approx\}17 h- and has been maintained through {\textgreater}220 doublings. The ID6 line formed embryoid bodies- expressing genes representing all 3 germ layers when cultured under differentiating conditions- and teratomas containing tissues of ectoderm- mesoderm- and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.
    Gene expression and muscle fiber function in a porcine ICU model
    Varuna Banduseela, Physiological Genomics - 2009
    Skeletal muscle wasting and impaired muscle function in response to mechanical ventilation and immobilization in intensive care unit (ICU) patients are clinically challenging partly due to 1) the poorly understood intricate cellular and molecular networks and 2) the unavailability of an animal model mimicking this condition. By employing a unique porcine model mimicking the conditions in the ICU with long-term mechanical ventilation and immobilization- we have analyzed the expression profile of skeletal muscle biopsies taken at three time points during a 5-day period. Among the differentially regulated transcripts- extracellular matrix- energy metabolism- sarcomeric and LIM protein mRNA levels were downregulated- while ubiquitin proteasome system- cathepsins- oxidative stress responsive genes and heat shock proteins (HSP) mRNAs were upregulated. Despite 5 days of immobilization and mechanical ventilation single muscle fiber cross-sectional areas as well as the maximum force generating capacity at the single muscle fiber level were preserved. It is proposed that HSP induction in skeletal muscle is an inherent- primary- but temporary protective mechanism against protein degradation. To our knowledge- this is the first study that isolates the effect of immobilization and mechanical ventilation in an ICU condition from various other cofactors.
    Association of Human Cytochrome P450 1A1 (CYP1A1) and Sulfotransferase 1A1 (SULT1A1) Polymorphisms with Differential Metabolism and Cytotoxicity of Aminoflavone
    Qiang Zheng, Molecular Cancer Therapeutics - 2010
    Aminoflavone (AF)- a clinically investigational novel anticancer agent- requires sequential metabolic activation by CYP1A1 and SULT1A1 to exert its antitumor activities. The purpose of this study was to determine the functional significance of common polymorphisms of human CYP1A1 and SULT1A1 on the metabolism and cytotoxicity of AF. To this end- Chinese Hamster V79 cells were genetically engineered to stably express human CYP1A1*1 (wild-type)- CYP1A1*2C (I462V)- or CYP1A1*4 (T461N) and coexpress human CYP1A1*1 with human SULT1A1*1 (wild-type)- SULT1A1*2 (R213H)- or SULT1A1*3 (M223V). The metabolism and cytotoxicity of AF were evaluated in these cellular models. All common variants of CYP1A1 and SULT1A1 were actively involved in the metabolic activation of AF- but with a varying degree of activity. Whereas CYP1A1 I462V variant exhibited a superior activity (mainly caused by a significantly higher Vmax) for hydroxylations of AF- expression of different CYP1A1 variants did not confer cell differential sensitivity to AF. The cells coexpressing CYP1A1*1 with SULT1A1*1- SULT1A1*2- or SULT1A1*3 displayed SULT1A1 allele-specific sensitivity to AF: SULT1A1*3 exhibited the highest sensitivity (IC50- 0.01 \{micro\}mol/L)- followed by SULT1A1*1 (IC50- 0.5 \{micro\}mol/L)- and SULT1A1*2 showed the lowest sensitivity (IC50- 4.4 \{micro\}mol/L). These data suggest that the presence of low-activity SULT1A1*2 may predict poor response to AF- whereas the presence of high-activity CYP1A1/SULT1A1 alleles- especially combination of CYP1A1*2C and SULT1A1*3 or SULT1A1*1- may be beneficial to patients receiving AF. The present study provides a foundation for future clinical investigations of potential genetic biomarkers that may enable selection of patients for the greatest potential benefit from AF treatment. Mol Cancer Ther, 9(10), 2803-13. (C)2010 AACR.
    Expression and Localization of Angiogenic Growth Factors in Developing Porcine Mesonephric Glomeruli
    De Ward, Journal of Histochemistry & Cytochemistry - 2010
    The development and growth of renal glomeruli is regulated by specific angiogenic growth factors- including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis- but it remains to be elucidated during the development of the embryonic mesonephros- which can function as an interesting model for glomerular development and senescence. In this study- the presence of the angiogenic growth factors was studied in developing porcine mesonephroi- using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition- mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli- which may be explained by the continuous growth of the glomeruli- as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli- whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis- suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations. (J Histochem Cytochem 58:1045-1056- 2010)
    Broad-Spectrum Suppression of Innate Immunity Is Required for Colonization of Arabidopsis Roots by the Fungus Piriformospora indica
    Sophie Jacobs, Plant Physiology - 2011
    Piriformospora indica is a root-colonizing basidiomycete that confers a wide range of beneficial traits to its host. The fungus shows a biotrophic growth phase in Arabidopsis (Arabidopsis thaliana) roots followed by a cell death-associated colonization phase- a colonization strategy that- to our knowledge- has not yet been reported for this plant. P. indica has evolved an extraordinary capacity for plant root colonization. Its broad host spectrum encompasses gymnosperms and monocotyledonous as well as dicotyledonous angiosperms- which suggests that it has an effective mechanism(s) for bypassing or suppressing host immunity. The results of our work argue that P. indica is confronted with a functional root immune system. Moreover- the fungus does not evade detection but rather suppresses immunity triggered by various microbe-associated molecular patterns. This ability to suppress host immunity is compromised in the jasmonate mutants jasmonate insensitive1-1 and jasmonate resistant1-1. A quintuple-DELLA mutant displaying constitutive gibberellin (GA) responses and the GA biosynthesis mutant ga1-6 (for GA requiring 1) showed higher and lower degrees of colonization- respectively- in the cell death-associated stage- suggesting that P. indica recruits GA signaling to help establish proapoptotic root cell colonization. Our study demonstrates that mutualists- like pathogens- are confronted with an effective innate immune system in roots and that colonization success essentially depends on the evolution of strategies for immunosuppression.
    T-Lymphocyte Responses to Intestinally Absorbed Antigens Can Contribute to Adipose Tissue Inflammation and Glucose Intolerance during High Fat Feeding
    Yuehui Wang, PLoS ONE - 2010
    Background Obesity is associated with inflammation of visceral adipose tissues- which increases the risk for insulin resistance. Animal models suggest that T-lymphocyte infiltration is an important early step- although it is unclear why these cells are attracted. We have recently demonstrated that dietary triglycerides- major components of high fat diets- promote intestinal absorption of a protein antigen (ovalbumin- “OVA�). The antigen was partly transported on chylomicrons- which are prominently cleared in adipose tissues. We hypothesized that intestinally absorbed gut antigens may cause T-lymphocyte associated inflammation in adipose tissue. Methodology/Principal Findings Triglyceride absorption promoted intestinal absorption of OVA into adipose tissue- in a chylomicron-dependent manner. Absorption tended to be higher in mesenteric than subcutaneous adipose tissue- and was lowest in gonadal tissue. OVA immunoreactivity was detected in stromal vascular cells- including endothelial cells. In OVA-sensitized mice- OVA feeding caused marked accumulation of CD3+ and osteopontin+ cells in mesenteric adipose tissue. The accumulating T-lymphocytes were mainly CD4+. As expected- high-fat (60\% kCal) diets promoted mesenteric adipose tissue inflammation compared to low-fat diets (10\% Kcal)- as reflected by increased expression of osteopontin and interferon-gamma. Immune responses to dietary OVA further increased diet-induced osteopontin and interferon-gamma expression in mesenteric adipose. Inflammatory gene expression in subcutaneous tissue did not respond significantly to OVA or dietary fat content. Lastly- whereas OVA responses did not significantly affect bodyweight or adiposity- they significantly impaired glucose tolerance. Conclusions/Significance Our results suggest that loss or lack of immunological tolerance to intestinally absorbed T-lymphocyte antigens can contribute to mesenteric adipose tissue inflammation and defective glucose metabolism during high-fat dieting.
    Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation
    Sumithra Urs, The FASEB Journal - 2010
    Development of bone and adipose tissue are linked processes arising from a common progenitor cell- but having an inverse relationship in disease conditions such as osteoporosis. Cellular differentiation of both tissues relies on growth factor cues- and we focus this study on Sprouty1 (Spry1)- an inhibitor of growth factor signaling. We tested whether Spry1 can modify the development of fat cells through its activity in regulating growth factors known to be important for adipogenesis. We utilized conditional expression and genetic-null mouse models of Spry1 in adipocytes using the fatty acid binding promoter (aP2). Conditional deletion of Spry1 results in 10\% increased body fat and decreased bone mass. This phenotype was rescued on Spry1 expression- which results in decreased body fat and increased bone mass. Ex vivo bone marrow experiments indicate Spry1 in bone marrow and adipose progenitor cells favors differentiation of osteoblasts at the expense of adipocytes by suppressing CEBP-Beta and PPARgamma while up regulating TAZ. Age and gender-matched littermates expressing only Cre recombinase were used as controls. Spry1 is a critical regulator of adipocyte differentiation and mesenchymal stem cell (MSC) lineage allocation- potentially acting through regulation of CEBP-Beta and TAZ.—Urs- S.- Venkatesh- D.- Tang- Y.- Henderson- T.- Yang- X.- Friesel- R. E.- Rosen- C. J.- Liaw- L. Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation.
    Identification and Characterization of Noncoding Small RNAs in Streptococcus pneumoniae Serotype 2 Strain D39
    Ho-Ching Tsui, Journal of Bacteriology - 2010
    We report a search for small RNAs (sRNAs) in the low-GC- Gram-positive human pathogen Streptococcus pneumoniae. Based on bioinformatic analyses by Livny et al. (J. Livny- A. Brencic- S. Lory- and M. K. Waldor- Nucleic Acids Res. 34:3484-3493- 2006)- we tested 40 candidates by Northern blotting and confirmed the expression of nine new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (A. Halfmann- M. Kovacs- R. Hakenbeck- and R. Bruckner- Mol. Microbiol. 66:110-126- 2007) that are positively controlled by the CiaR response regulator. We characterized 3 of these 14 sRNAs: Spd-sr17 (144 nucleotides [nt], decreased in stationary phase)- Spd-sr37 (80 nt, strongly expressed in all growth phases)- and CcnA (93 nt, induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures- whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes- indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth- certain stress responses- global transcription- or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 ΔciaR mutants- but attempts to link CcnA to -E to comC as a target were inconclusive in ciaR+ strains. These results show that S. pneumoniae- which lacks known RNA chaperones- expresses numerous sRNAs- but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.
    Stat3 Directly Controls the Expression of Tbx5- Nkx2.5- and GATA4 and Is Essential for Cardiomyocyte Differentiation of P19CL6 Cells
    Marylynn Snyder, Journal of Biological Chemistry - 2010
    The transcription factor Stat3 (signal transducer and activator of transcription 3) mediates many physiological processes- including embryogenesis- stem cell self-renewal- and postnatal survival. In response to gp130 receptor activation- Stat3 becomes phosphorylated by the receptor-associated Janus kinase- forms dimers- and enters the nucleus where it binds to Stat3 target genes and regulates their expression. In this report- we demonstrate that Stat3 binds directly to the promoters and regulates the expression of three genes that are essential for cardiac differentiation: Tbx5- Nkx2.5- and GATA4. We further demonstrate that Tbx5- Nkx2.5- and GATA4 expression is dependent on Stat3 in response to ligand treatment and during ligand-independent differentiation of P19CL6 cells into cardiomyocytes. Finally- we show that Stat3 is necessary for the differentiation of P19CL6 cells into beating cardiomyocytes. All together- these results demonstrate that Stat3 is required for the differentiation of cardiomyocytes through direct transcriptional regulation of Tbx5- Nkx2.5- and GATA4.
    STAT3 Signaling in CD4+ T Cells Is Critical for the Pathogenesis of Chronic Sclerodermatous Graft-Versus-Host Disease in a Murine Model
    Vedran Radojcic, The Journal of Immunology - 2010
    Donor CD4+ T cells are thought to be essential for inducing delayed host tissue injury in chronic graft-versus-host disease (GVHD). However- the relative contributions of distinct effector CD4+ T cell subpopulations and the molecular pathways influencing their generation are not known. We investigated the role of the STAT3 pathway in a murine model of chronic sclerodermatous GVHD. This pathway integrates multiple signaling events during the differentiation of naive CD4+ T cells and impacts their homeostasis. We report that chimeras receiving an allograft containing STAT3-ablated donor CD4+ T cells do not develop classic clinical and pathological manifestations of alloimmune tissue injury. Analysis of chimeras showed that abrogation of STAT3 signaling reduced the in vivo expansion of donor-derived CD4+ T cells and their accumulation in GVHD target tissues without abolishing antihost alloreactivity. STAT3 ablation did not significantly affect Th1 differentiation while enhancing CD4+CD25+Foxp3+ T cell reconstitution through thymus-dependent and -independent pathways. Transient depletion of CD25+ T cells in chimeras receiving STAT3-deficient T cells resulted in delayed development of alloimmune gut and liver injury. This delayed de novo GVHD was associated with the emergence of donor hematopoietic stem cell-derived Th1 and Th17 cells. These results suggest that STAT3 signaling in graft CD4+ T cells links the alloimmune tissue injury of donor graft T cells and the emergence of donor hematopoietic stem cell-derived pathogenic effector cells and that both populations contribute- albeit in different ways- to the genesis of chronic GVHD after allogenic bone marrow transplantation in a murine model.
    Gene position in a long operon governs motility development in Bacillus subtilis
    Loralyn Cozy, Molecular microbiology - 2010
    Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor σD. Here we show that chaining cells were OFF for σD-dependent gene expression because σD levels fell below a threshold- and σD activity was inhibited by the anti-sigma factor FlgM. The probability that σD exceeded the threshold was governed by the position of the sigD genes. The proportion of ON cells increased when sigD was artificially moved forward in the 27kb fla/che operon. In addition- we identified a new σD-dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally- we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension.
    Prolactin restores branchial mitochondrion-rich cells expressing Na+/Cl- cotransporter in hypophysectomized Mozambique tilapia
    Jason Breves, American Journal of Physiology - Regulatory- Integrative and Comparative Physiology - 2010
    Hypophysectomy and hormone replacement therapy were conducted to investigate the regulation of branchial mitochondrion-rich cell (MRC) recruitment and hormone receptor expression in euryhaline tilapia (Oreochromis mossambicus). Gene expression and immunolocalization of Na+/Cl- cotransporter (NCC) and Na+/K+/2Cl- cotransporter (NKCC) were used as markers for freshwater (FW)- and seawater (SW)-type MRCs- respectively. In FW fish- hypophysectomy resulted in a significant drop in plasma osmolality- an effect associated with a marked reduction of NCC gene expression and the disappearance of MRCs with apical-NCC immunoreactivity. In contrast- hypophysectomy in SW fish did not impact plasma osmolality- NKCC- or Na+- K+-ATPasealpha1 gene expression- or the recruitment of MRCs with basolateral-NKCC. Hypophysectomized fish in SW exhibited reduced mRNA levels of prolactin (PRL) receptor 1 and growth hormone (GH) receptor in the gill, GH receptor expression was also reduced following hypophysectomy in FW. PRL replacement therapy restored NCC gene expression and the appearance of MRCs with apical NCC in both FW and SW, there was no interaction of PRL with cortisol. In FW- cortisol modestly stimulated NKCC mRNA levels- while no effect of GH was evident. In SW- no clear effects of hormone replacement on gene expression of NKCC- Na+- K+-ATPasealpha1- or hormone receptors were detected. Taken together- the essential nature of PRL to survival of Mozambique tilapia in FW is derived- at least in part- from its ability to stimulate the recruitment of MRCs that express NCC- while recruitment of SW-type MRCs does not require pituitary mediation in this euryhaline tilapia.
    Episodes of Prolactin Gene Expression in GH3 Cells Are Dependent on Selective Promoter Binding of Multiple Circadian Elements
    Sudeep Bose, Endocrinology - 2010
    Prolactin (PRL) gene expression in mammotropes occurs in pulses- but the mechanism(s) underlying this dynamic process remains obscure. Recent findings from our laboratory of an E-box in the rat PRL promoter (E-box133) that can interact with the circadian factors- circadian locomoter output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1- and was necessary for pulse activity raised the intriguing possibility that the circadian system may be central to this oscillatory process. In this study- we used serum-shocked GH3 cells- established previously to synchronize PRL pulses between cells in culture- to reveal that pulses of PRL mRNA are linked temporally to the expression of bmal1- cry1- per1- and per3 mRNA in these cells. Moreover- we found that each of these circadian factors binds to the rat PRL promoter by chromatin immunoprecipitation analysis. Using EMSA analysis- we observed that two sites present in the proximal promoter region- E-box133 and E-box10- bind circadian factors differentially (E-box133 interacted with BMAL1- cryptochrome-1- period (PER)-1- and PER3 but not PER2 and E-box10 bound BMAL1- cryptochrome-1- PER2- PER3 but not PER1). More importantly- down-regulation of any factor binding E-box133 significantly reduced PRL mRNA levels during pulse periods. Our results demonstrate clearly that certain circadian elements binding to the E-box133 site are required for episodes of PRL mRNA expression in serum-shocked GH3 cultures. Moreover- our findings of binding-related differences between functionally distinct E-boxes demonstrate not only that E-boxes can bind different components but suggest that the number and type of circadian elements that bind to an E-box is central in dictating its function.
    Induction and rescue of Nod2-dependent Th1-driven granulomatous inflammation of the ileum
    Amlan Biswas, Proceedings of the National Academy of Sciences U.S.A. - 2010
    Mutations in the NOD2 gene are strong genetic risk factors for ileal Crohn's disease. However- the mechanism by which these mutations predispose to intestinal inflammation remains a subject of controversy. We report that Nod2-deficient mice inoculated with Helicobacter hepaticus- an opportunistic pathogenic bacterium- developed granulomatous inflammation of the ileum- characterized by an increased expression of Th1-related genes and inflammatory cytokines. The Peyer's patches and mesenteric lymph nodes were markedly enlarged with expansion of IFN-\{gamma\}-producing CD4 and CD8 T cells. Rip2-deficient mice exhibited a similar phenotype- suggesting that Nod2 function likely depends on the Rip2 kinase in this model. Transferring wild-type bone marrow cells into irradiated Nod2-deficient mice did not rescue the phenotype. However- restoring crypt antimicrobial function of Nod2-deficient mice by transgenic expression of \{alpha\}-defensin in Paneth cells rescued the Th1 inflammatory phenotype. Therefore- through the regulation of intestinal microbes- Nod2 function in nonhematopoietic cells of the small intestinal crypts is critical for protecting mice from a Th1-driven granulomatous inflammation in the ileum. The model may provide insight into Nod2 function relevant to inflammation of ileal Crohn's disease.
    Altered Retinoid Uptake and Action Contributes to Cell Survival in Endometriosis
    Mary Pavone, Journal of Clinical Endocrinology & Metabolism - 2010
    Context: Retinoic acid (RA) controls multiple biological processes via exerting opposing effects on cell survival. Retinol uptake into cells is controlled by stimulated by RA 6 (STRA6). RA is then produced from retinol in the cytosol. Partitioning of RA between the nuclear receptors RA receptor \{alpha\} and peroxisome-proliferator-activated receptor \{beta\}/\{delta\} is regulated by cytosol-to-nuclear shuttling proteins cellular RA binding protein 2 (CRABP2) and fatty acid binding protein 5 (FABP5)- which induce apoptosis or enhance survival- respectively. The roles of these mechanisms in endometrium or endometriosis remain unknown. Objective: The aim was to determine the regulation of retinoid uptake and RA action in primary stromal cells from endometrium (n = 10) or endometriosis (n = 10). Results: Progesterone receptor was necessary for high STRA6 and CRABP2 expression in endometrial stromal cells. STRA6- which was responsible for labeled retinoid uptake- was strikingly lower in endometriotic cells compared to endometrial cells. CRABP2 knockdown in endometrial cells increased survival- and FABP5 knockdown in endometriotic cells decreased survival without altering the expression of downstream nuclear retinoic acid receptor \{alpha\} and peroxisome-proliferator-activated receptor \{beta\}/\{delta\}. Conclusions: In endometrial stromal cells- progesterone receptor up-regulates expression of STRA6 and CRABP2- which control retinol uptake and growth-suppressor actions of RA. In endometriotic stromal cells- decreased expression of these genes leads to decreased retinol uptake and dominant FABP5-mediated prosurvival activity.
    Follistatin-like 1 regulates renal IL-1beta expression in cisplatin nephrotoxicity
    Leif Oxburgh, Renal Physiology - 2010
    Follistatin-like 1 (FSTL1) is a secreted protein with homology to both Follistatin and the SPARC/BM40 family of matricellular proteins. In this study- we sought to determine the expression patterns of Fstl1 and its cognate receptor Dip2a in the adult- and to assess the consequences of Fstl1 inactivation on development and homeostasis of the kidney. We find that FSTL1 circulates at high levels in both the human and the mouse and that it is also locally expressed in the loop of Henle in the kidney. To begin to understand the in vivo functions of Fstl1- we generated a mouse mutant using a genetrap approach. The hypomorphic Fstl1 genetrap strain displays a strong reduction in FSTL1 expression at the protein level- but it does not show overt developmental defects. FSTL1 has previously been implicated in diverse disease processes as a regulator of inflammatory cytokine expression- and we therefore evaluated the response of the genetrap strain to cisplatin-mediated acute kidney injury- a disease model with highly cytokine-dependent pathology. We find that although TNF-\{alpha\} and Il6 levels are unchanged relative to wild-type- renal Il-1\{beta\} expression is increased in genetrap mice following cisplatin treatment. Furthermore- histopatological analysis- expression of the tissue injury marker Havcr1- and measurement of serum creatinine demonstrate that reduction of Fstl1 expression sensitizes the kidney to acute cisplatin nephrotoxicity- suggesting a role for FSTL1-mediated Il-1\{beta\} suppression in protection of the kidney from acute nephrotoxic injury.
    Differential Contribution to Neuroendocrine Tumorigenesis of Parallel Egfr Signaling in Cancer Cells and Pericytes
    Olivier Nolan-Stevaux, Genes & Cancer - 2010
    Factors associated with tumor sensitivity to epidermal growth factor receptor (EGFR) inhibitors in the context of wild-type EGFR remain elusive. This study investigates the mechanistic basis of responsiveness to EGFR inhibitors in the RIP1-Tag2 (RT2) mouse model of pancreatic neuroendocrine tumorigenesis (PNET). Upon treatment of RT2 mice with EGFR inhibitors- PNET tumors harboring wild-type- nonamplified alleles of Egfr grow at a markedly reduced rate and display a significant increase in tumor cell apoptosis- as well as reduced neovascularization. The authors identify Tgf- and Hb-egf as key limiting mediators of separable pathological functions of Egfr in neuroendocrine tumor progression: Tgf- mutant tumors present with an elevated apoptotic index- whereas Hb-egf mutant lesions exhibit decreased angiogenic switching and neovascularization. This study not only associates Tgf- and Hb-egf expression with wild-type Egfr oncogenicity but also ascribes the proangiogenic activity of Egfr in this tumor model to a novel mesenchymal Hb-egf/Egfr signaling axis- whereby endothelial and pericyte-derived Hb-egf activates Egfr specifically in tumor-associated perivascular cells- leading to increased pericyte coverage of the tumor endothelium and enhanced angiogenesis.
    Murine leukemia virus glycosylated Gag (gPr80gag) facilitates interferon-sensitive virus release through lipid rafts
    Takayuki Nitta, Proceedings of the National Academy of Sciences U.S.A. - 2010
    Murine leukemia viruses encode a unique form of Gag polyprotein- gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65gag- the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses- its mechanism of action has been unclear- although it facilitates virus production at a late assembly or release step. Here- it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular- gPr80gag-facilitated release occurs through lipid rafts- because gPr80gag-negative M-MuLV has a lower cholesterol content- is less sensitive to inhibition of release by the cholesterol-depleting agent M\{beta\}CD- and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.
    NLR family member NLRC5 is a transcriptional regulator of MHC class I genes
    Torsten Meissner, Proceedings of the National Academy of Sciences - 2010
    MHC class I plays a critical role in the immune defense against viruses and tumors by presenting antigens to CD8 T cells. An NLR protein- class II transactivator (CIITA)- is a key regulator of MHC class II gene expression that associates and cooperates with transcription factors in the MHC class II promoter. Although CIITA also transactivates MHC class I gene promoters- loss of CIITA in humans and mice results in the severe reduction of only MHC class II expression- suggesting that additional mechanisms regulate the expression of MHC class I. Here- we identify another member of the NLR protein family- NLRC5- as a transcriptional regulator of MHC class I genes. Similar to CIITA- NLRC5 is an IFN-gamma-inducible nuclear protein- and the expression of NLRC5 resulted in enhanced MHC class I expression in lymphoid as well as epithelial cell lines. Using chromatin immunoprecipitation and reporter gene assays- we show that NLRC5 associates with and activates the promoters of MHC class I genes. Furthermore- we show that the IFN-gamma-induced up-regulation of MHC class I requires NLRC5- because knockdown of NLRC5 specifically impaired the expression of MHC class I. In addition to MHC class I genes- NLRC5 also induced the expression of Beta2-microglobulin- transporter associated with antigen processing- and large multifunctional protease- which are essential for MHC class I antigen presentation. Our results suggest that NLRC5 is a transcriptional regulator- orchestrating the concerted expression of critical components in the MHC class I pathway.
    Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7-12-dimethylbenz[a]anthracene
    Lisa Becks, BMC Cancer - 2010
    Background Activation of nuclear factor erythroid 2-related factor (Nrf2)- which belongs to the basic leucine zipper transcription factor family- is a strategy for cancer chemopreventive phytochemicals. It is an important regulator of genes induced by oxidative stress- such as glutathione S-transferases- heme oxygenase-1 and peroxiredoxin 1- by activating the antioxidant response element (ARE). We hypothesized that (1) the citrus coumarin auraptene may suppress premalignant mammary lesions via activation of Nrf2/ARE- and (2) that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis. Methods Premalignant lesions and mammary carcinomas were induced by medroxyprogesterone acetate and 7-12-dimethylbenz[a]anthracene treatment. The 10-week pre-malignant study was performed in which 8 groups of 10 each female wild-type (WT) and KO mice were fed either control diet or diets containing auraptene (500 ppm). A carcinogenesis study was also conducted in KO vs. WT mice (n = 30-34). Comparisons between groups were evaluated using ANOVA and Kaplan-Meier Survival statistics- and the Mann-Whitney U-test. Results All mice treated with carcinogen exhibited premalignant lesions but there were no differences by genotype or diet. In the KO mice- there was a dramatic increase in mammary carcinoma growth rate- size- and weight. Although there was no difference in overall survival- the KO mice had significantly lower mammary tumor-free survival. Also- in the KO mammary carcinomas- the active forms of NF-kB and Beta-catenin were increased {\textasciitilde}2-fold whereas no differences in oxidized proteins were observed. Many other tumors were observed- including lymphomas. Interestingly- the incidences of lung adenomas in the KO mice were significantly higher than in the WT mice. Conclusions We report- for the first time- that there was no apparent difference in the formation of premalignant lesions- but rather- the KO mice exhibited rapid- aggressive mammary carcinoma progression.
    Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development
    Shunsuke Ichi, Journal of Biological Chemistry - 2010
    The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp-/-) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp-/- embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138- miR-148a- miR-185- and miR-339-5p. In our model- folate reversed these epigenetic marks in folate-rescued Sp-/- embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5- the proliferative stage- and E12.5- when neural differentiation begins. In Sp-/- embryos compared with WT- levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5- and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp-/- embryos. Thus- one of the mechanisms by which folate may rescue the Sp-/- phenotype is by increasing the expression of KDM6B- which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.
    Live Attenuated Shigella dysenteriae Type 1 Vaccine Strains Overexpressing Shiga Toxin B Subunit
    Tao Wu, Infection and Immunity - 2011
    Shigella dysenteriae serotype 1 (S. dysenteriae 1) is unique among the Shigella species and serotypes in the expression of Shiga toxin which contributes to more severe disease sequelae and the ability to cause explosive outbreaks and pandemics. S. dysenteriae 1 shares characteristics with other Shigella species- including the capability of causing clinical illness with a very low inoculum (10 to 100 CFU) and resistance to multiple antibiotics- underscoring the need for efficacious vaccines and therapeutics. Following the demonstration of the successful attenuating capacity of deletion mutations in the guaBA operon in S. flexneri 2a vaccine strains in clinical studies- we developed a series of S. dysenteriae 1 vaccine candidates containing the fundamental attenuating mutation in guaBA. All strains are devoid of Shiga toxin activity by specific deletion of the gene encoding the StxA subunit- which encodes enzymatic activity. The StxB subunit was overexpressed in several derivatives by either plasmid-based constructs or chromosomal manipulation to include a strong promoter. All strains are attenuated for growth in vitro in the HeLa cell assay and for plaque formation and were safe in the Sereny test and immunogenic in the guinea pigs. Each strain induced robust serum and mucosal anti-S. dysenteriae 1 lipopolysaccharide (LPS) responses and protected against wild-type challenge. Two strains engineered to overexpress StxB induced high titers of Shiga toxin neutralizing antibodies. These candidates demonstrate the potential for a live attenuated vaccine to protect against disease caused by S. dysenteriae 1 and potentially to protect against the toxic effects of other Shiga toxin 1-expressing pathogens.
    Characterization of the mononuclear phagocyte system in zebrafish
    Valerie Wittamer, Blood - 2011
    The evolutionarily conserved immune system of the zebrafish (Danio rerio)- in combination with its genetic tractability- position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish- namely macrophages and dendritic cells (DCs)- but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene- we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs- macrophages- DCs- and B lymphocytes. Examination of mhc2dab:GFP, cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFPhi, cd45:DsRedhi cells were exclusively mature monocytes/macrophages and DCs- as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs- but were most abundant in the intestine and spleen- where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally- mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction- enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.
    IL-1 Blockade Attenuates Islet Amyloid Polypeptide-Induced Proinflammatory Cytokine Release and Pancreatic Islet Graft Dysfunction
    Clara Westwell-Roper, The Journal of Immunology - 2011
    Islets from patients with type 2 diabetes exhibit \{beta\} cell dysfunction- amyloid deposition- macrophage infiltration- and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP)- the main component of islet amyloid- might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP- but not nonamyloidogenic rodent islet amyloid polypeptide- caused release of CCL2 and CXCL1 by islets and induced secretion of TNF-- IL-1- IL-1\{beta\}- CCL2- CCL3- CXCL1- CXCL2- and CXCL10 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF- secretion was markedly diminished in MyD88-- but not TLR2- or TLR4-deficient macrophages- and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction- islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50\% more macrophages than wild-type grafts- an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88- but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to \{beta\} cell dysfunction in type 2 diabetes and islet transplantation.
    Sterol Regulatory Element-Binding Protein 2 Couples HIV-1 Transcription to Cholesterol Homeostasis and T Cell Activation
    Harry Taylor, Journal of Virology - 2011
    Cholesterol plays an essential role in the life cycle of several enveloped viruses. Many of these viruses manipulate host cholesterol metabolism to facilitate their replication. HIV-1 infection of CD4+ T cells activates the sterol regulatory element-binding protein 2 (SREBP2) transcriptional program- which includes genes involved in cholesterol homeostasis. However- the role of SREBP2-dependent transcription in HIV-1 biology has not been fully examined. Here- we identify TFII-I- a gene critical for HIV-1 transcription in activated T cells- as a novel SREBP2 target gene. We found TFII-I expression increased after HIV-1 infection or activation of human primary CD4+ T cells. We show that inhibition of SREBP2 activity reduced TFII-I induction in response to these stimuli. More importantly- small interfering RNA (siRNA)-mediated gene silencing of either SREBP2 or TFII-I significantly reduced HIV-1 production in CD4+ T cells. We also found that TFII-I potentiates Tat-dependent viral gene expression- consistent with a role at the level of HIV-1 transcription. Collectively- our results demonstrate for the first time that HIV-1 transcription in T cells is linked to cholesterol homeostasis through control of TFII-I expression by SREBP2.
    Musashi expression in Beta-cells coordinates insulin expression- apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes
    M Szabat, Cell Death & Disease - 2011
    Diabetes is associated with the death and dysfunction of insulin-producing pancreatic Beta-cells. In other systems- Musashi genes regulate cell fate via Notch signaling- which we recently showed regulates Beta-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms- as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of Beta-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1- while it decreased Ins1 and Ins2 expression- revealing a molecular link between ER stress and Beta-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1- stimulate proliferation- inhibit apoptosis and reduce insulin expression- whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together- these results demonstrate overlapping- but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and Beta-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory Beta-cell proliferation and the loss of Beta-cell gene expression seen in specific phases of the progression to type 2 diabetes.
    The role of interleukin-12 on modulating myeloid-derived suppressor cells- increasing overall survival and reducing metastasis
    Catherine Steding, Immunology - 2011
    Myeloid-derived suppressor cells (MDSC) are important to the tumour microenvironment as they actively suppress the immune system and promote tumour progression and metastasis. These cells block T-cell activation in the tumour microenvironment- preventing anti-tumour immune activity. The ability of a treatment to alter the suppressive function of these cells and promote an immune response is essential to enhancing overall therapeutic efficacy. Interleukin-12 (IL-12) has the potential not only to promote anti-tumour immune responses but also to block the activity of cells capable of immune suppression. This paper identifies a novel role for IL-12 as a modulator of MDSC activity- with implications for IL-12 as a therapeutic agent. Treatment with IL-12 was found to alter the suppressive function of MDSC by fundamentally altering the cells. Interleukin-12-treated MDSC exhibited up-regulation of surface markers indicative of mature cells as well as decreases in nitric oxide synthase and interferon-gamma mRNA both in vitro and in vivo. Treatment with IL-12 was also found to have significant therapeutic benefit by decreasing the percentage of MDSC in the tumour microenvironment and increasing the percentage of active CD8+ T cells. Treatment with IL-12 resulted in an increase in overall survival accompanied by a reduction in metastasis. The findings in this paper identify IL-12 as a modulator of immune suppression with significant potential as a therapeutic agent for metastatic breast cancer.
    Signal Transducers and Activators of Transcription 3 (STAT3) Directly Regulates Cytokine-induced Fascin Expression and Is Required for Breast Cancer Cell Migration
    Marylynn Snyder, The Journal of Biological Chemistry - 2011
    Background: The goal is to understand the molecular mechanism of metastasis and the roles of IL-6/OSM- STAT3- and fascin.- Results: STAT3 binds to the fascin promoter and is essential for its expression and cell migration in response to IL-6/OSM.- Conclusion: STAT3 plays a central role in cell migration through direct control of fascin expression.- Significance: Drug targets are identified to block tumor metastasis.- The cytokines oncostatin M (OSM) and IL-6 promote breast cancer cell migration and metastasis. Both cytokines activate STAT3- a member of the STAT (signal transducers and activators of transcription) family of transcription factors. Through transcriptional regulation of its target genes- STAT3 controls a wide range of cellular processes- including cellular proliferation- oncogenesis- and cancer metastasis. Fascin is an actin-bundling protein involved in cell migration. Elevated levels of fascin expression are found in many metastatic cancers- and inhibition of fascin function by small chemical compounds leads to a block of tumor metastasis. In this work- we demonstrate that fascin is a direct STAT3 target gene in response to OSM and IL-6 in both mouse and human breast cancer cells. We show that NFkB also binds to the fascin promoter in response to cytokine treatment and this binding is STAT3-dependent. Both STAT3 and NFkB are required for the cytokine-induced expression of fascin in cancer cells. Furthermore- we demonstrate that STAT3- in directly controlling fascin expression- is both necessary and sufficient for breast cancer cell migration.
    Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase
    Jonathan Grim, Journal of Cell Biology - 2008
    The SCFFBW7 ubiquitin ligase degrades proteins involved in cell division- growth- and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations- suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7\{alpha\} isoform accounts for almost all Fbw7 activity toward cyclin E- c-Myc- and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle- and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity- cyclin E autophosphorylation- and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover- they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.
    The Adenovirus E1B 55-Kilodalton and E4 Open Reading Frame 6 Proteins Limit Phosphorylation of eIF2alpha during the Late Phase of Infection
    Megan Spurgeon, Journal of Virology - 2009
    During a productive infection- species C adenovirus reprograms the host cell to promote viral translation at the expense of cellular translation. The E1B 55-kilodalton (E1B-55K) and E4 open reading frame 6 (E4orf6) proteins are important in this control of gene expression. As part of a ubiquitin-protein ligase- these viral proteins stimulate viral mRNA export- inhibit cellular mRNA export- promote viral gene expression- and direct the degradation of certain host proteins. We report here that the E1B-55K and E4orf6 proteins limited phosphorylation of eIF2\{alpha\} and the activation of the eIF2\{alpha\} kinase PKR. Phospho-eIF2\{alpha\} levels were observed to rise and fall at least twice during infection. The E1B-55K and E4orf6 proteins prevented a third increase at late times of infection. PKR appeared to phosphorylate eIF2\{alpha\} only in the absence of E1B-55K/E4orf6 function. PKR activation and eIF2\{alpha\} phosphorylation was unrelated to the cytoplasmic levels of the adenovirus inhibitor of PKR- VA-I RNA. Nonetheless- expression of a PKR inhibitor- the reovirus double-stranded RNA-binding protein sigma 3- prevented PKR activation and eIF2\{alpha\} phosphorylation. The sigma 3 protein largely corrected the defect in viral late protein synthesis associated with the E1B-55K and E4orf6 mutant viruses without affecting cytoplasmic levels of the late viral mRNA. The ubiquitin-protein ligase activity associated with the E1B-55K/E4orf6 complex was necessary to prevent activation of PKR and phosphorylation of eIF2\{alpha\}. These findings reveal a new contribution of the E1B-55K/E4orf6 complex to viral late protein synthesis and the existence of multiple layers of regulation imposed on eIF2\{alpha\} phosphorylation and PKR activation in adenovirus-infected cells.
    Beclin 1 Gene Transfer Activates Autophagy and Ameliorates the Neurodegenerative Pathology in alpha-Synuclein Models of Parkinson's and Lewy Body Diseases
    Brian Spencer, Journal of Neuroscience - 2009
    Accumulation of the synaptic protein \{alpha\}-synuclein (\{alpha\}-syn) is a hallmark of Parkinson's disease (PD) and Lewy body disease (LBD)- a heterogeneous group of disorders with dementia and parkinsonism- where Alzheimer's disease and PD interact. Accumulation of \{alpha\}-syn in these patients might be associated with alterations in the autophagy pathway. Therefore- we postulate that delivery of beclin 1- a regulator of the autophagy pathway- might constitute a strategy toward developing a therapy for LBD/PD. Overexpression of \{alpha\}-syn from lentivirus transduction in a neuronal cell line resulted in lysosomal accumulation and alterations in autophagy. Coexpression of beclin 1 activated autophagy- reduced accumulation of \{alpha\}-syn- and ameliorated associated neuritic alterations. The effects of beclin 1 overexpression on LC3 and \{alpha\}-syn accumulation were partially blocked by 3-MA and completely blocked by bafilomycin A1. In contrast- rapamycin enhanced the effects of beclin 1. To evaluate the potential effects of activating autophagy in vivo- a lentivirus expressing beclin 1 was delivered to the brain of a \{alpha\}-syn transgenic mouse. Neuropathological analysis demonstrated that beclin 1 injections ameliorated the synaptic and dendritic pathology in the tg mice and reduced the accumulation of \{alpha\}-syn in the limbic system without any significant deleterious effects. This was accompanied by enhanced lysosomal activation and reduced alterations in the autophagy pathway. Thus- beclin 1 plays an important role in the intracellular degradation of \{alpha\}-syn either directly or indirectly through the autophagy pathway and may present a novel therapeutic target for LBD/PD.
    GnRH-Regulated Expression of Jun and JUN Target Genes in Gonadotropes Requires a Functional Interaction between TCF/LEF Family Members and beta-Catenin
    Travis Salisbury, Molecular Endocrinology - 2009
    GnRH regulates gonadotrope function through a complex transcriptional network that includes three members of the immediate early gene family: Egr1- Jun- and Atf3. These DNA-binding proteins act alone or in pairs to confer hormonal responsiveness to Cga- Lhb- Fshb- and Gnrhr. Herein we suggest that the transcriptional response of Jun requires a functional interaction between the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of DNA-binding proteins and \{beta\}-catenin (officially CTNNB1)- a coactivator of TCF/LEF. Supporting data include demonstration that GnRH increases activity of TOPflash- a TCF/LEF-dependent luciferase reporter- in L\{beta\}T2 cells- a gonadotrope-derived cell line. Additional cotransfection experiments indicate that a dominant-negative form of TCF7L2 (TCFDN) that binds DNA- but not \{beta\}-catenin- blocks GnRH induction of TOPflash. Overexpression of AXIN- an inhibitor of \{beta\}-catenin- also reduces GnRH stimulation of TOPflash. Transduction of L\{beta\}T2 cells with TCFDN adenoviruses diminishes GnRH stimulation of Jun mRNA without altering expression of Egr1 and Atf3- two other immediate early genes that confer GnRH responsiveness. Reduction of \{beta\}-catenin in L\{beta\}T2 cells- through stable expression of short hairpin RNA- also selectively compromises GnRH regulation of Jun expression and levels of JUN protein. Finally- overexpression of TCFDN attenuates GnRH regulation of Cga promoter activity- a known downstream target of JUN. Together- these results indicate that GnRH regulation of Jun transcription requires a functional interaction between TCF/LEF and \{beta\}-catenin and that alteration of either impacts expression of JUN downstream targets such as Cga.
    Stem cell fate dictated solely by altered nanotube dimension
    Seunghan Oh, Proceedings of the National Academy of Sciences U.S.A. - 2009
    Two important goals in stem cell research are to control the cell proliferation without differentiation and to direct the differentiation into a specific cell lineage when desired. Here- we demonstrate such paths by controlling only the nanotopography of culture substrates. Altering the dimensions of nanotubular-shaped titanium oxide surface structures independently allowed either augmented human mesenchymal stem cell (hMSC) adhesion or a specific differentiation of hMSCs into osteoblasts by using only the geometric cues- absent of osteogenic inducing media. hMSC behavior in response to defined nanotube sizes revealed a very dramatic change in hMSC behavior in a relatively narrow range of nanotube dimensions. Small (\{approx\}30-nm diameter) nanotubes promoted adhesion without noticeable differentiation- whereas larger (\{approx\}70- to 100-nm diameter) nanotubes elicited a dramatic stem cell elongation (\{approx\}10-fold increased)- which induced cytoskeletal stress and selective differentiation into osteoblast-like cells- offering a promising nanotechnology-based route for uniqueorthopedics-related hMSC treatments.
    Differential Gene Expression of Resistant and Susceptible Sweetpotato Plants after Infection with the Causal Agents of Sweet Potato Virus Disease
    Cecilia McGregor, Journal of the American Society for Horticultural Science - 2009
    Sweet potato virus disease (SPVD) is one of the most devastating diseases affecting sweetpotato (Ipomoea batatas)- an important food crop in developing countries. SPVD develops when sweetpotato plants are dually infected with sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). To better understand the synergistic interaction between these viruses- global gene expression was previously studied in the susceptible cultivar Beauregard. In the current study- global gene expression between SPVD-affected plants and virus-tested control plants (VT) were compared in Beauregard' (Bx) and resistant NASPOT 1' (Nas) sweetpotato cultivars at 5- 9- 13- and 17 days post inoculation (DPI). Titer levels of SPFMV and SPCSV were significantly lower in inoculated resistant plants (Nas\_SPVD) than in susceptible plants (Bx\_SPVD) at most of the time points. Chloroplast genes and cell expansion-related genes (including xyloglucan endotransglucosylase/hydrolases) were suppressed in Bx\_SPVD- while stress-related genes were induced. This trend was not observed in resistant NAS\_SPVD. Genes related to protein synthesis (e.g.- ribosomal proteins and elongation factor genes) were induced in resistant NAS\_SPVD at 5 DPI before returning to levels comparable with NAS\_VT plants. At this time (5 DPI)- individual viruses could not be detected in NAS\_SPVD samples- and no symptoms were observed. Induction of protein synthesis-related genes is common in susceptible plants after virus infection and is generally in proportion to virus accumulation. Our results show that induction of protein synthesis genes also occurs early in the infection process in resistant plants- while virus titers were below the level of detection- suggesting that virus accumulation is not required for induction.
    Deep mRNA Sequencing for In Vivo Functional Analysis of Cardiac Transcriptional Regulators Application to Galphaq
    Scot Matkovich, Circulation Research - 2010
    Rationale: Transcriptional profiling can detect subclinical heart disease and provide insight into disease etiology and functional status. Current microarray-based methods are expensive and subject to artifact. Objective: To develop RNA sequencing methodologies using next generation massively parallel platforms for high throughput comprehensive analysis of individual mouse cardiac transcriptomes. To compare the results of sequencing- and array-based transcriptional profiling in the well-characterized Galphaq transgenic mouse hypertrophy/cardiomyopathy model. Methods and Results: The techniques for preparation of individually bar-coded mouse heart RNA libraries for Illumina Genome Analyzer II resequencing are described. RNA sequencing showed that 234 high-abundance transcripts ({\textgreater}60 copies/cell) comprised 55\% of total cardiac mRNA. Parallel transcriptional profiling of Galphaq transgenic and nontransgenic hearts by Illumina RNA sequencing and Affymetrix Mouse Gene 1.0 ST arrays revealed superior dynamic range for mRNA expression and enhanced specificity for reporting low-abundance transcripts by RNA sequencing. Differential mRNA expression in Galphaq and nontransgenic hearts correlated well between microarrays and RNA sequencing for highly abundant transcripts. RNA sequencing was superior to arrays for accurately quantifying lower-abundance genes- which represented the majority of the regulated genes in the Galphaq transgenic model. Conclusions: RNA sequencing is rapid- accurate- and sensitive for identifying both abundant and rare cardiac transcripts- and has significant advantages in time- and cost-efficiencies over microarray analysis.
    Cooperative Role of NF-kappaB and Poly(ADP-ribose) Polymerase 1 (PARP-1) in the TNF-induced Inhibition of PHEX Expression in Osteoblasts
    Pawel Majewski, Journal of Biological Chemistry - 2010
    Reduced bone mass is a common complication in chronic inflammatory diseases- although the mechanisms are not completely understood. The PHEX gene encodes a zinc endopeptidase expressed in osteoblasts and contributes to bone mineralization. The aim of this study was to determine the molecular mechanism involved in TNF-mediated down-regulation of Phex gene transcription. We demonstrate down-regulation of the Phex gene in two models of colitis: naive T-cell transfer and in gnotobiotic IL-10-/- mice. In vitro- TNF decreased expression of Phex in UMR106 cells and did not require de novo synthesis of a transrepressor. Transfecting UMR-106 cells with a series of deletion constructs of the proximal Phex promoter identified a region located within -74 nucleotides containing NF-\{kappa\}B and AP-1 binding sites. After TNF treatment- the RelA/p50 NF-\{kappa\}B complex interacted with two cis-elements at positions -70/-66 and -29/-25 nucleotides in the proximal Phex promoter. Inhibition of NF-\{kappa\}B signaling increased the basal level of Phex transcription and abrogated the effects of TNF- whereas overexpression of RelA mimicked the effect of TNF. We identified poly(ADP-ribose) polymerase 1 (PARP-1) binding immediately upstream of the NF-\{kappa\}B sites and showed that TNF induced poly(ADP-ribosyl)ation of RelA when bound to the Phex promoter. TNF-mediated Phex down-regulation was completely abrogated in vitro by PARP-1 inhibitor and overexpression of poly(ADP-ribose) glucohydrolase (PARG) and in vivo in PARP-1-/- mice. Our results suggest that NF-\{kappa\}B signaling and PARP-1 enzymatic activity cooperatively contribute to the constitutive and inducible suppression of Phex. The described phenomenon likely contributes to the loss of bone mass density in chronic inflammatory diseases- such as inflammatory bowel disease.
    Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation
    Kellie Jacks, Journal of Biological Chemistry - 2010
    Mitogen- and stress-activated protein kinases- MSK1 and the closely related isoform MSK2- are nuclear kinases that are activated following mitogen stimulation or cellular stress- including UV radiation- by the ERK1/2 and p38 MAPK signaling cascades- respectively. However- factors that differentially regulate MSK1 and MSK2 have not been well characterized. Here we report that the CK2 protein kinase- which contributes to NF-\{kappa\}B activation following UV radiation in a p38-dependent manner- physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue Ser324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells- but not MSK1 depletion- resulted in impaired UV-induced phosphorylation of NF-\{kappa\}B p65 at Ser276 in vivo- which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore- UV radiation led to the activation of NF-\{kappa\}B-responsive gene expression in MDA-MB-231 cells and induced p65 transactivation capacity that was dependent on MSK2- MSK2 residue Ser324- and p65-Ser276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at Ser276 that positively regulates NF-\{kappa\}B activity in MDA-MB-231 cells.
    Labour is associated with decreased expression of the PGF2alpha receptor (PTGFR) and a novel PTGFR splice variant in human myometrium but not decidua
    Andrea Hay, Molecular Human Reproduction - 2010
    The prostaglandin F2\{alpha\} receptor (PTGFR) is believed to play a role in the process of parturition. The main support for this comes from animal studies, however- in humans- the evidence is less clear. The gene coding for PTGFR may be subject to alternative splicing to generate alternate variants with different signalling pathways. We have determined regional (upper versus lower segment) and labour-associated expression of PTGFR mRNA and a recently identified splice variant of PTGFR in human myometrium and decidua. We also examined the effect of the inflammatory cytokine interleukin-1\{beta\} (IL-1\{beta\}) on PTGFR mRNA expression in a model of cultured human myometrial smooth muscle cells. We identified a PTGFR transcript variant 2 (PTGFR-v2) generated by alternate splicing in human myometrium and decidua. The PTGFR-v2 contains an additional 71 base pair exon- which results in a truncated protein at 297 amino acids compared with the PTGFR transcript variant 1 (PTGFR-v1) at 359 amino acids. In contrast to our hypothesis- we demonstrate that PTGFR-v1 and PTGFR-v2 mRNA expression is not significantly higher in upper segment compared with lower segment paired samples. We also show a labour-associated decrease in PTGFR-v1 and PTGFR-v2 mRNA expression in lower segment myometrial samples. IL-1\{beta\}-stimulated mRNA expression of both PTGFR variants in a distinct time-dependent manner in myometrial cell cultures. We suggest that the role of the PTGFR in the human uterus requires further validation prior to pursuing it as a target for the treatment of preterm labour. In addition- the presence of distinct variants suggests further levels of gene regulation within the pregnant uterus.
    Retinal Self-Antigen Induces a Predominantly Th1 Effector Response in Axl and Mertk Double-Knockout Mice
    Fei Ye, The Journal of Immunology - 2011
    The TAM family of receptors (Tyro3- Axl- and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli- and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues- including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye- we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response- as characterized by increased IFN-\{gamma\} production and higher frequency of IFN-\{gamma\}-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls- as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly- APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation- IL-12 and IL-18- was dramatically increased in dko DCs and macrophages- and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.
    Expression and Immunotherapeutic Targeting of the SSX Family of Cancer-Testis Antigens in Prostate Cancer
    Heath Smith, Cancer Research - 2011
    Recent U.S. Food and Drug Administration approval of the first immunotherapy for prostate cancer encourages efforts to improve immune targeting of this disease. The synovial sarcoma X chromosome breakpoint (SSX) proteins comprise a set of cancer-testis antigens that are upregulated in MHC class I-deficient germline cells and in various types of advanced cancers with a poor prognosis. Humoral and cell-mediated immune responses to the SSX family member SSX2 can arise spontaneously in prostate cancer patients. Thus- SSX2 and other proteins of the SSX family may offer useful targets for tumor immunotherapy. In this study- we evaluated the expression of SSX family members in prostate cancer cell lines and tumor biopsies to identify which members might be most appropriate for immune targeting. We found that SSX2 was expressed most frequently in prostate cell lines- but that SSX1 and SSX5 were also expressed after treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. Immunohistochemical analysis of microarrayed tissue biopsies confirmed a differential level of SSX protein expression in human prostate cancers. Notably- SSX expression in patient tumor samples was restricted to metastatic lesions (5/22, 23\%) and no expression was detected in primary prostate tumors examined (0/73, P {\textless} 0.001). We determined that cross-reactive immune responses to a dominant HLA-A2-specific SSX epitope (p103-111) could be elicited by immunization of A2/DR1 transgenic mice with SSX vaccines. Our findings suggest that multiple SSX family members are expressed in metastatic prostate cancers which are amenable to simultaneous targeting. Cancer Res, 71(21), 6785-95. (C)2011 AACR.
    Corticospinal-specific HCN expression in mouse motor cortex: Ih-dependent synaptic integration as a candidate microcircuit mechanism involved in motor control
    Patrick Sheets, Journal of Neurophysiology - 2011
    Motor cortex is a key brain center involved in motor control in rodents and other mammals- but specific intracortical mechanisms at the microcircuit level are largely unknown. Neuronal expression of hyperpolarization-activated current (Ih) is cell class specific throughout the nervous system- but in neocortex- where pyramidal neurons are classified in various ways- a systematic pattern of expression has not been identified. We tested whether Ih is differentially expressed among projection classes of pyramidal neurons in mouse motor cortex. Ih expression was high in corticospinal neurons and low in corticostriatal and corticocortical neurons- a pattern mirrored by mRNA levels for HCN1 and Trip8b subunits. Optical mapping experiments showed that Ih attenuated glutamatergic responses evoked across the apical and basal dendritic arbors of corticospinal but not corticostriatal neurons. Due to Ih- corticospinal neurons resonated- with a broad peak at [{\textasciitilde}]4 Hz- and were selectively modulated by -adrenergic stimulation. Ih reduced the summation of short trains of artificial excitatory postsynaptic potentials (EPSPs) injected at the soma- and similar effects were observed for short trains of actual EPSPs evoked from layer 2/3 neurons. Ih narrowed the coincidence detection window for EPSPs arriving from separate layer 2/3 inputs- indicating that the dampening effect of Ih extended to spatially disperse inputs. To test the role of corticospinal Ih in transforming EPSPs into action potentials- we transfected layer 2/3 pyramidal neurons with channelrhodopsin-2 and used rapid photostimulation across multiple sites to synaptically drive spiking activity in postsynaptic neurons. Blocking Ih increased layer 2/3-driven spiking in corticospinal but not corticostriatal neurons. Our results imply that Ih-dependent synaptic integration in corticospinal neurons constitutes an intracortical control mechanism- regulating the efficacy with which local activity in motor cortex is transferred to downstream circuits in the spinal cord. We speculate that modulation of Ih in corticospinal neurons could provide a microcircuit-level mechanism involved in translating action planning into action execution.
    Placenta growth factor expression is regulated by hydrogen peroxide in vascular smooth muscle cells
    Jennifer Shaw, Cell Physiology - 2011
    When supply arteries become occluded- blood is diverted through preexisting collateral vessels. Shear stress arising from this increase in blood flow provides the initial physiological stimulus for expansion of the collateral circulation- a process termed arteriogenesis. Endothelial cells (EC) respond to increased shear stress by releasing a variety of mediators that can act on underlying smooth muscle cells (SMC). Placenta growth factor (PLGF) is known to mediate certain aspects of arteriogenesis- such as recruitment of monocytes to the vessel wall. Therefore- we tested whether SMC PLGF expression is influenced by mediators released by EC. We used A10 SMC cultured with medium that had been conditioned by EOMA EC for 4 days as a model. We found that EC-conditioned medium is able to upregulate PLGF gene expression in A10 SMC. Further experiments identified hydrogen peroxide (H2O2) as a key mediator of this response. We confirmed the physiological relevance of this mechanism in primary human coronary artery SMCs by demonstrating that exogenous H2O2 specifically upregulates PLGF gene and protein expression. We also demonstrated that the physiological stimulus of shear stress raises endogenous H2O2 levels in media into the range found to increase PLGF expression. In this study- we demonstrate that EC-released H2O2 acts as a positive regulator of PLGF gene and protein expression in vascular SMC. To our knowledge- this is the first study to describe H2O2 as a regulator of PLGF expression and therefore an upstream mediator of PLGF-driven arteriogenesis.
    DeltaNp63alpha Confers Tumor Cell Resistance to Cisplatin through the AKT1 Transcriptional Regulation
    Tanusree Sen, Cancer Research - 2011
    Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g.- ovarian- head/neck- and lung) where platins are used widely and resistance occurs commonly. We found that upon \{Delta\}Np63 overexpression- AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift- chromatin immunoprecipitation and functional reporter assays implicated \{Delta\}Np63 in positive regulation of AKT1 transcription. Importantly- we found that \{Delta\}Np63- AKT1- and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of \{Delta\}Np63 expression dramatically decreased AKT1 expression- whereas knockdown of either \{Delta\}Np63 or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely- enforced expression of \{Delta\}Np63 increased cancer cell proliferation and reduced apoptosis. Together- our findings define a novel \{Delta\}Np63-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells. Cancer Res, 71(3), 1167-76. (C)2011 AACR.
    XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes
    Huseyin Saribasak, Journal of Experimental Medicine - 2011
    Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis- we decreased the level of x-ray cross-complementing 1 (XRCC1)- a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells- and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1+/- splenic B cells- the length of microhomology at the switch junctions decreased- suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore- Xrcc1+/- B cells had reduced Igh/c-myc translocations during CSR- supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.
    Ku Must Load Directly onto the Chromosome End in Order to Mediate Its Telomeric Functions
    Christopher Lopez, PLoS Genetics - 2011
    The Ku heterodimer associates with the Saccharomyces cerevisiae telomere- where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel- its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions- we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners- such as Sir4- a factor required for telomeric silencing- and TLC1- the RNA component of telomerase. Despite these interactions- we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection- length regulation- and silencing functions. In contrast to Ku null strains- the DNA end binding defective Ku mutants resulted in increased- rather than markedly decreased- imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap- where it promotes a specific architecture that is central to telomere function and maintenance.- The telomeric cap modulates telomere replication and prevents natural chromosome ends from being processed as DNA double-strand breaks (DSBs). In multiple species- including budding yeast- a detailed picture exists of the factors that comprise the telomeric cap and how they associate with telomeric DNA. It is less clear where to place Ku- a conserved heterodimer involved in multiple aspects of telomere biology and DSB repair. Although Ku avidly binds DNA ends- its access to telomeric ends might be restricted by telomere binding proteins and/or higher-order telomere structure. Ku might also be recruited to telomeres via its telomere-associated binding partners. Here- we address whether Ku loads directly onto telomeric ends and whether direct DNA binding is crucial for its telomeric functions. Using structure-guided mutagenesis- we generated end binding-defective yeast Ku heterodimers that retained the ability to associate with Ku's known telomeric binding partners. These end binding-defective heterodimers showed a dramatic reduction in telomere association and were defective for all of Ku's telomeric functions. Our findings indicate that Ku is indeed a component of the telomere cap and that its loading onto telomeric ends is crucial for its telomeric functions and- perhaps- a specific telomere architecture.
    Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance
    J. Butler, RNA - 2012
    RNA surveillance systems function at critical steps during the formation and function of RNA molecules in all organisms. The RNA exosome plays a central role in RNA surveillance by processing and degrading RNA molecules in the nucleus and cytoplasm of eukaryotic cells. The exosome functions as a complex of proteins composed of a nine-member core and two ribonucleases. The identity of the molecular determinants of exosome RNA substrate specificity remains an important unsolved aspect of RNA surveillance. In the nucleus of Saccharomyces cerevisiae- TRAMP complexes recognize and polyadenylate RNAs- which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA-binding factors called Air proteins. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. Experiments reported here show that the Air proteins govern separable functions. Phenotypic analysis and RNA deep-sequencing results from air mutants reveal specific requirements for each Air protein in the regulation of the levels of noncoding and coding RNAs. Loss of these regulatory functions results in specific metabolic and plasmid inheritance defects. These findings reveal differential functions for Air proteins in RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.
    O-GlcNAc cycling mutants modulate proteotoxicity in Caenorhabditis elegans models of human neurodegenerative diseases
    Peng Wang, Proceedings of the National Academy of Sciences U.S.A. - 2012
    O-GlcNAcylation is an abundant posttranslational modification in the brain implicated in human neurodegenerative diseases. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized Caenorhabditis elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the phenotype in transgenic models of tauopathy- amyloid \{beta\}-peptide- and polyglutamine expansion. Intriguingly- loss of function of O-GlcNAc transferase alleviated- whereas loss of O-GlcNAcase enhanced- the phenotype of multiple neurodegenerative disease models. The O-GlcNAc cycling mutants act in part by altering DAF-16-dependent transcription and modulating the protein degradation machinery. These findings suggest that O-GlcNAc levels may directly influence neurodegenerative disease progression- thus making the enzymes of O-GlcNAc cycling attractive targets for neurodegenerative disease therapies.
    IRES-mediated translation of cellular messenger RNA operates in eIF2?- independent manner during stress
    Nehal Thakor, Nucleic Acids Research - 2012
    Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2alpha. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2alpha phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia- serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However- the molecular mechanism that allows IRES-mediated translation to continue in an eIF2alpha-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis- XIAP- IRES to address this question. Using toeprinting assay- western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2alpha is phosphorylated both in vitro and in vivo. During normal growth condition eIF2alpha-dependent translation on the IRES is preferred. However- IRES-mediated translation switches to eIF5B-dependent mode when eIF2alpha is phosphorylated as a consequence of cellular stress.
    Production of Transgenic Calves Expressing an shRNA Targeting Myostatin
    K Tessanne, Molecular Reproduction and Development - 2012
    Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype- a desirable agricultural trait. Increased neonatal morbidity is common- however- resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end- we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo- a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term- but died following delivery by Caesarean-section. As an alternative strategy- microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows- resulting in 7 pregnancies. Five transgenic calves were produced- of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus- and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.
    Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation
    Ricardo Soto-Rifo, Nucleic Acids Research - 2012
    The 5′-untranslated region (5′-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here- we have analyzed and compared the properties by which the viral 5′-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5′-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA- which was much inhibitory for HIV-2 translation. Altogether- these data highlight important structural and functional differences between these two human pathogens.
    Two Independent Mechanisms Promote Expression of an N-terminal Truncated USP18 Isoform with Higher DeISGylation Activity in the Nucleus
    Christoph Burkart, Journal of Biological Chemistry - 2012
    Expression of the ISG15 specific protease USP18 is highly induced by type I interferons. The two main functions of USP18- i.e. its enzymatic activity and down-regulation of type I interferon signaling- are well characterized. However- to date all functional studies focused on full-length USP18. Here- we report that translation of human USP18 is initiated by a rare start codon (CUG). Usage of this non-canonical initiation site with its weak translation initiation efficiency promotes expression of an N-terminal truncated isoform (USP18-sf). In addition- an internal ribosome entry site (IRES) located in the 5'-coding region of USP18 also contributes to translation of USP18-sf. Functionally- both isoforms exhibit enzymatic activity and interfere with type I interferon signaling. However- USP18-sf shows different subcellular distribution compared with the full-length protein and enhanced deISGylation activity in the nucleus. Taken together- we report the existence of an N-terminal truncated isoform of USP18- whose expression is controlled on translational level by two independent mechanisms providing translational flexibility as well as cell type-specific resistance to inhibition of cap-dependent translation.
    ATR cooperates with CTC1 and STN1 to maintain telomeres and genome integrity in Arabidopsis
    Kara Boltz, Molecular Biology of the Cell - 2012
    The CTC1/STN1/TEN1 (CST) complex is an essential constituent of plant and vertebrate telomeres. Here we show that CST and ATR (ataxia telangiectasia mutated [ATM] and Rad3-related) act synergistically to maintain telomere length and genome stability in Arabidopsis. Inactivation of ATR- but not ATM- temporarily rescued severe morphological phenotypes associated with ctc1 or stn1. Unexpectedly- telomere shortening accelerated in plants lacking CST and ATR. In first-generation (G1) ctc1 atr mutants- enhanced telomere attrition was modest- but in G2 ctc1 atr- telomeres shortened precipitously- and this loss coincided with a dramatic decrease in telomerase activity in G2 atr mutants. Zeocin treatment also triggered a reduction in telomerase activity- suggesting that the prolonged absence of ATR leads to a hitherto-unrecognized DNA damage response (DDR). Finally- our data indicate that ATR modulates DDR in CST mutants by limiting chromosome fusions and transcription of DNA repair genes and also by promoting programmed cell death in stem cells. We conclude that the absence of CST in Arabidopsis triggers a multifaceted ATR-dependent response to facilitate maintenance of critically shortened telomeres and eliminate cells with severe telomere dysfunction.
    Shox2 regulates progression through chondrogenesis in the mouse proximal limb
    Brent Bobick, Journal of Cell Science - 2012
    In humans- loss of SHOX gene function is responsible for the mesomelic short stature characteristic of Turner syndrome- Leri-Weill dyschondrosteosis- and Langer dysplasia. In a mouse model of SHOX deficiency- Prrx1-Cre-driven limb-specific deletion of the paralogous gene Shox2 results in severe rhizomelia. In this study- we show that Col2a1-Cre-driven deletion of Shox2 in developing chondrocytes also results in shortening of the stylopodial skeleton (i.e. humerus- femur) and that this rhizomelia is due to precocious chondrocyte maturation and hypertrophy. We demonstrate- using the micromass culture model system- that increased BMP activity triggers accelerated maturation and hypertrophy in Col2a1-Cre Shox2 mutant chondrocytes and we confirm in vivo that elevated transcript levels and expanded expression domains of Bmp2 and 4 are associated with premature formation of the hypertrophic zone in mutant humeri. In micromass cultures of Prrx1-Cre Shox2 mutant limb cells- we find that Shox2 deletion in undifferentiated mesenchymal cells results in increased BMP activity that enhances early chondrogenesis- but is insufficient to provoke chondrocyte maturation and hypertrophy. Similarly- shRNA-mediated Shox2 knockdown in multipotent C3H10T1/2 cells and primary mouse bone marrow mesenchymal stem cells results in spontaneous chondrogenesis in the absence of chondrostimulation- but again fails to induce progression through the later stages of chondrogenic differentiation. Importantly- exogenous BMP supplementation can overcome the block to maturation and hypertrophy caused by Shox2 depletion prior to overt chondrogenesis. Thus- we provide evidence that Shox2 regulates progression through chondrogenesis at two distinct stages - the onset of early differentiation and the transition to maturation and hypertrophy.
    GnRH Regulation of Jun and Atf3 Requires Calcium- Calcineurin- and NFAT
    April Binder, Molecular Endocrinology - 2012
    GnRH binds to its receptor on gonadotropes and activates multiple members of the MAPK signaling family that in turn regulates the expression of several immediate early genes (IEGs) including Jun- Fos- Atf3- and Egr1. These IEGs confer hormonal responsiveness to gonadotrope-specific genes including Gnrhr- Cga- Fshb- and Lhb. In this study we tested the hypothesis that GnRH specifically regulates the accumulation of Jun and Atf3 mRNA through a pathway that includes intracellular Ca2+- calcineurin- and nuclear factor of activated T cells (NFAT). Our results indicate that pretreatment of murine L\{beta\}T2 cells with 1- 2-bis-(o-aminophenoxy)-ethane-N-N-N'-N'-tetraacetic acid tetra(acetoxymethyl)-ester- a Ca2+ chelator- reduced the expression of all the IEGs to varying degrees- whereas treatment with thapsigargin- an intracellular Ca2+ protein pump inhibitor- increased the expression of the IEG. Furthermore- cyclosporin A- a calcineurin-specific inhibitor- reduced the ability of GnRH to regulate accumulation of Jun and Atf3 mRNA and to a lesser extent Fos. In contrast- Egr1 mRNA was unaffected. NFATs are transcription factors regulated by calcineurin and were detected in L\{beta\}T2 cells. GnRH increased luciferase activity of an NFAT-dependent promoter reporter that was dependent on intracellular Ca2+ and calcineurin activity. Additionally- although small interfering RNA specific for Nfat4 only marginally reduced GnRH regulation of Jun- Fos- and Atf3 mRNA accumulation- activity of an activator protein-1-responsive reporter construct was reduced by 48\%. Together these data suggest that calcineurin and NFAT are new members of the gonadotrope transcriptional network that confer hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion.
    Impaired Auxin Biosynthesis in the defective endosperm18 Mutant Is Due to Mutational Loss of Expression in the ZmYuc1 Gene Encoding Endosperm-Specific YUCCA1 Protein in Maize
    Jamila Bernardi, Plant Physiology - 2012
    The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here- we characterized a seed-specific viable maize (Zea mays) mutant- defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40\% less dry mass- compared with De18. Cellular analyses showed lower total cell number- smaller cell volume- and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes- maize Yucca1 (ZmYuc1)- and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally- all three genes showed high expression coincident with high IAA levels, however- only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore- sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant- including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids- compared with the 400 amino acids in the De18. The putative- approximately 1.5-kb- Yuc1 promoter region also showed many rearrangements- including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10- contig 395- showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively- the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis- essential for normal endosperm development in maize.
    PTP1B Deficiency Exacerbates Inflammation and Accelerates Leukocyte Trafficking In Vivo
    Sergejs Berdnikovs, The Journal of Immunology - 2012
    It is reported that PTP1B limits cytokine signaling in vitro. However- PTP1B's function during inflammation in vivo is not known. In this report- we determined whether PTP1B deficiency affects allergic inflammation in vivo. Briefly- lungs of OVA-challenged PTP1B-/- mice had elevated numbers of eosinophils and eosinophil progenitors at 6 h after one OVA challenge and at 24 h after a third OVA challenge as compared with OVA-challenged wild-type mice. There was also an increase in numbers of CD11b+SiglecF+CD34+IL-5R+ eosinophil progenitors in the bone marrow- peripheral blood- and spleens of OVA-challenged PTP1B-/- mice. Intravital microscopy revealed that- in OVA-challenged PTP1B-/- mice- blood leukocytes rapidly bound to endothelium (5-30 min)- whereas- in wild-type mice- blood leukocytes bound to endothelium at the expected 6-18 h. Consistent with early recruitment of leukocytes- lung eotaxin and Th2 cytokine levels were elevated early in the PTP1B-/- mice. Interestingly- spleen leukocytes from PTP1B-/- mice exhibited an increased chemotaxis- chemokinesis- and transendothelial migration in vitro. In summary- PTP1B functions as a critical negative regulator to limit allergic responses.
    The Role of EZH2 in the Regulation of the Activity of Matrix Metalloproteinases in Prostate Cancer Cells
    Yong Shin, PLoS ONE - 2012
    Degradation of the extracellular matrix (ECM)- a critical step in cancer metastasis- is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells- this balance is shifted towards MMPs- promoting ECM degradation. Here- we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation of the TIMP gene promoters. Overexpression of EZH2 confers an invasive phenotype on benign prostate epithelial cells, however- this phenotype is suppressed by cooverexpression of TIMP3. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9- thereby decreasing the invasive activity of prostate cancer cells. These results suggest that the transcriptional repression of the TIMP genes by EZH2 may be a major mechanism to shift the MMPs/TIMPs balance in favor of MMP activity and thus to promote ECM degradation and subsequent invasion of prostate cancer cells.
    Stress-Induced GSK3 Regulates the Redox Stress Response by Phosphorylating Glucose-6-Phosphate Dehydrogenase in Arabidopsis
    Silvia Santo, The Plant Cell Online - 2012
    Diverse stresses such as high salt conditions cause an increase in reactive oxygen species (ROS)- necessitating a redox stress response. However- little is known about the signaling pathways that regulate the antioxidant system to counteract oxidative stress. Here- we show that a Glycogen Synthase Kinase3 from Arabidopsis thaliana (ASKalpha) regulates stress tolerance by activating Glc-6-phosphate dehydrogenase (G6PD)- which is essential for maintaining the cellular redox balance. Loss of stress-activated ASKalpha leads to reduced G6PD activity- elevated levels of ROS- and enhanced sensitivity to salt stress. Conversely- plants overexpressing ASKalpha have increased G6PD activity and low levels of ROS in response to stress and are more tolerant to salt stress. ASKalpha stimulates the activity of a specific cytosolic G6PD isoform by phosphorylating the evolutionarily conserved Thr-467- which is implicated in cosubstrate binding. Our results reveal a novel mechanism of G6PD adaptive regulation that is critical for the cellular stress response.
    Diazocyte development in the marine diazotrophic cyanobacterium Trichodesmium
    Gustaf Sandh, Microbiology - 2012
    The establishment of non-diazotrophic cultures of the filamentous marine cyanobacterium Trichodesmium erythraeum IMS101 enabled the first detailed investigation of the process leading to the development of its unique nitrogen-fixing cell type- the diazocyte. Trichome heterogeneity was apparent already within 3-8 h- while the differentiation of mature diazocytes- containing the nitrogenase enzyme- required 27 h after the removal of combined nitrogen. The distribution of pro-diazocytes' within the trichomes correlates with the localization of mature diazocytes- which suggests that pattern regulation is an early event during diazocyte development. The development was initially identified as changes in the subcellular ultrastructure- most notably the degradation of glycogen granules and gas vacuoles. These changes were preceded by the induced expression of the global nitrogen regulator ntcA at an early stage of combined nitrogen deprivation- followed by elevated expression of genes related to nitrogen metabolism and their corresponding proteins. The strongest induction (10-fold) was related to the transcription of the respiratory gene coxB2- apparent already at an early stage- which suggests an important role for respiration and the subsequent energy generation in the subcellular changes found- and in the creation of the reducing environment required for nitrogen fixation in diazocytes.
    Aberrant growth and lethality of Arabidopsis deficient in nonsense-mediated RNA decay factors is caused by autoimmune-like response
    Nina Riehs-Kearnan, Nucleic Acids Research - 2012
    Nonsense-mediated RNA decay (NMD) is an evolutionarily conserved RNA quality control mechanism that eliminates transcripts containing nonsense mutations. NMD has also been shown to affect the expression of numerous genes- and inactivation of this pathway is lethal in higher eukaryotes. However- despite relatively detailed knowledge of the molecular basis of NMD- our understanding of its physiological functions is still limited and the underlying causes of lethality are unknown. In this study- we examined the importance of NMD in plants by analyzing an allelic series of Arabidopsis thaliana mutants impaired in the core NMD components SMG7 and UPF1. We found that impaired NMD elicits a pathogen defense response which appears to be proportional to the extent of NMD deficiency. We also demonstrate that developmental aberrations and lethality of the strong smg7 and upf1 alleles are caused by constitutive pathogen response upregulation. Disruption of pathogen signaling suppresses the lethality of the upf1-3 null allele and growth defects associated with SMG7 dysfunction. Interestingly- infertility and abortive meiosis observed in smg7 mutants is not coupled with impaired NMD suggesting a broader function of SMG7 in cellular metabolism. Taken together- our results uncover a major physiological consequence of NMD deficiency in Arabidopsis and revealed multifaceted roles of SMG7 in plant growth and development.
    The transcription of the alarmin cytokine interleukin-1 alpha is controlled by hypoxia inducible factors 1 and 2 alpha in hypoxic cells
    Peleg Rider, Frontiers in Immunology - 2012
    During hypoxia- cells undergo transcriptional changes to adjust to metabolic stress- to promote cell survival- and to induce pro-angiogenic factors. Hypoxia-induced factors (HIFs) regulate these transcriptional alterations. Failure to restore oxygen levels results in cell death by necrosis. IL-1alpha is one of the most important mediators of sterile inflammation following hypoxia-mediated necrosis. During hypoxia- IL-1alpha is up-regulated and released from necrotic cells- promoting the initiation of sterile inflammation. This study examined the role of IL-1alpha transcription in initiation of hypoxic stress and the correlation between IL-1alpha transcription and HIFalpha factors. In an epithelial cell line cultured under hypoxic conditions- IL-1alpha transcription was up-regulated in a process mediated and promoted by HIFalpha factors. IL-1alpha transcription was also up-regulated in hypoxia in a fibroblast cell line- however- in these cells- HIFalpha factors inhibited the elevation of transcription. These data suggest that HIFalpha factors play a significant role in initiating sterile inflammation by controlling IL-1alpha transcription during hypoxia in a differential manner- depending on the cell type.
    The Subcellular Localization of Tubby-Like Proteins and Participation in Stress Signaling and Root Colonization by the Mutualist Piriformospora indica
    Marco Reitz, Plant Physiology - 2012
    Tubby and Tubby-like proteins (TLPs) were first discovered in mammals- where they are involved in the development and function of neuronal cells. Due to their importance as plasma membrane (PM)-tethered transcription factors or mediators of vesicle trafficking- their lack causes obesity and other disease syndromes. Phosphatidylinositol 4-5-bisphosphate binding of the carboxyl-terminal Tubby domain attaches these proteins to the PM and vesicles and is essential for function. TLPs are conserved across eukaryotic kingdoms including plants- suggesting fundamental biological functions of TLPs. Plant TLPs possess an amino-terminal F-box domain that distinguishes them from other eukaryotic TLPs. Arabidopsis (Arabidopsis thaliana) encodes 11 AtTLPs that fall into six phylogenetic clades. We identified the significance of AtTLPs for root colonization of Arabidopsis by the mutualistic fungus Piriformospora indica. Our results further indicate conserved phosphatidylinositol 4-5-bisphosphate-binding sites in the Tubby domains that are required for PM anchoring of AtTLPs. More detailed studies revealed phospholipase C-triggered release of AtTLP3 from the PM- indicating a conserved mechanism as reported for mammalian Tubby and TLP3. We further show that hydrogen peroxide stimulates the release of AtTLP3 from the PM- presumably for activating downstream events. Different from mammalian homologs- the amino-terminal part of almost all AtTLPs has nucleocytosolic and plastidial localization patterns. Thus- it is tempting to assume that TLPs translate reactive oxygen species currents into signaling not only for transcriptional regulation in the nucleus but also affect plastid-associated functions after release from the PM.
    Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection
    Jessica Queen, Infection and Immunity - 2012
    Cholera is classically considered a noninflammatory diarrheal disease- in comparison to invasive enteric organisms- although there is a low-level proinflammatory response during early infection with Vibrio cholerae and a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model- this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1 V. cholerae infection is characterized by the upregulation of interleukin-6 (IL-6)- IL-10- and macrophage inflammatory protein 2 alpha in the intestine- indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils- the infection spread to the spleen and led to increased systemic levels of IL-1\{beta\} and tumor necrosis factor alpha- suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However- when genes for secreted accessory toxins were deleted- the colonizing bacteria were cleared from the intestine- and this clearance is dependent upon neutrophils. Thus- the requirement for accessory toxins in virulence is negated in neutropenic mice- which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall- these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins.
    The Mutualistic Fungus Piriformospora indica Colonizes Arabidopsis Roots by Inducing an Endoplasmic Reticulum Stress-Triggered Caspase-Dependent Cell Death
    Xiaoyu Qiang, PLANT CELL - 2012
    In Arabidopsis thaliana roots- the mutualistic fungus Piriformospora indica initially colonizes living cells- which die as the colonization proceeds. We aimed to clarify the molecular basis of this colonization-associated cell death. Our cytological analyses revealed endoplasmic reticulum (ER) swelling and vacuolar collapse in invaded cells- indicative of ER stress and cell death during root colonization. Consistent with this- P. indica-colonized plants were hypersensitive to the ER stress inducer tunicamycin. By clear contrast- ER stress sensors bZIP60 and bZIP28 as well as canonical markers for the ER stress response pathway- termed the unfolded protein response (UPR)- were suppressed at the same time. Arabidopsis mutants compromised in caspase 1-like activity- mediated by cell death-regulating vacuolar processing enzymes (VPEs)- showed reduced colonization and decreased cell death incidence. We propose a previously unreported microbial invasion strategy during which P. indica induces ER stress but inhibits the adaptive UPR. This disturbance results in a VPE/caspase 1-like-mediated cell death- which is required for the establishment of the symbiosis. Our results suggest the presence of an at least partially conserved ER stress-induced caspase-dependent cell death pathway in plants as has been reported for metazoans.
    Activation of NF-kappaB Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish
    Ajay Pradhan, Journal of Biological Chemistry - 2012
    Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-\{kappa\}B is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals- leading to the suppression of anti-Mullerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development- NF-\{kappa\}B-mediated induction of anti-apoptotic genes could- therefore- also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-\{kappa\}B in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-\{kappa\}B pathways and resulted in an increased ratio of females from 30 to 85\%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-\{kappa\}B-regulated genes. To confirm the involvement of NF-\{kappa\}B-induced anti-apoptotic effects- zebrafish were treated with sodium deoxycholate- a known inducer of NF-\{kappa\}B or NF-\{kappa\}B activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45\%- whereas the inhibition of NF-\{kappa\}B using NAI resulted in a decrease in females from 45 to 20\%. This study provides proof for an essential role of NF-\{kappa\}B in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.
    Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression
    Alireza Baradaran-Heravi, Human Molecular Genetics - 2012
    Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related- matrix-associated- actin-dependent regulator of chromatin- subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD- MIM 242900)- an incompletely penetrant autosomal recessive disorder. Using human- Drosophila and mouse models- we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that- as found in SIOD patients- deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice- although such deficiency causes modest diffuse alterations in gene expression. Rather- disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.
    Inhibition of allergic inflammation by supplementation with 5-hydroxytryptophan
    Hiam Abdala-Valencia, Lung Cellular and Molecular Physiology - 2012
    Clinical reports indicate that patients with allergy/asthma commonly have associated symptoms of anxiety/depression. Anxiety/depression can be reduced by 5-hydroxytryptophan (5-HTP) supplementation. However- it is not known whether 5-HTP reduces allergic inflammation. Therefore- we determined whether 5-HTP supplementation reduces allergic inflammation. We also determined whether 5-HTP decreases passage of leukocytes through the endothelial barrier by regulating endothelial cell function. For these studies- C57BL/6 mice were supplemented with 5-HTP- treated with ovalbumin fraction V (OVA)- house dust mite (HDM) extract- or IL-4- and examined for allergic lung inflammation and OVA-induced airway responsiveness. To determine whether 5-HTP reduces leukocyte or eosinophil transendothelial migration- endothelial cells were pretreated with 5-HTP- washed and then used in an in vitro transendothelial migration assay under laminar flow. Interestingly- 5-HTP reduced allergic lung inflammation by 70-90\% and reduced antigen-induced airway responsiveness without affecting body weight- blood eosinophils- cytokines- or chemokines. 5-HTP reduced allergen-induced transglutaminase 2 (TG2) expression and serotonylation (serotonin conjugation to proteins) in lung endothelial cells. Consistent with the regulation of endothelial serotonylation in vivo- in vitro pretreatment of endothelial cells with 5-HTP reduced TNF--induced endothelial cell serotonylation and reduced leukocyte transendothelial migration. Furthermore- eosinophil and leukocyte transendothelial migration was reduced by inhibitors of transglutaminase and by inhibition of endothelial cell serotonin synthesis- suggesting that endothelial cell serotonylation is key for leukocyte transendothelial migration. In summary- 5-HTP supplementation inhibits endothelial serotonylation- leukocyte recruitment- and allergic inflammation. These data identify novel potential targets for intervention in allergy/asthma.
    Role of sepsis in the development of limb muscle weakness in a porcine intensive care unit model
    Sudhakar Aare, Physiological Genomics - 2012
    Severe muscle wasting and loss of muscle function in critically ill mechanically ventilated intensive care unit (ICU) patients have significant negative consequences on their recovery and rehabilitation that persist long after their hospital discharge, moreover- the underlying mechanisms are unclear. Mechanical ventilation (MV) and immobilization-induced modifications play an important role in these consequences- including endotoxin-induced sepsis. The present study aims to investigate how sepsis aggravates ventilator and immobilization-related limb muscle dysfunction. Hence- biceps femoris muscle gene expression was investigated in pigs exposed to ICU intervention- i.e.- immobilization- sedation- and MV- alone or in combination with sepsis- for 5 days. In previous studies- we have shown that ICU intervention alone or in combination with sepsis did not affect muscle fiber size on day 5- but a significant decrease was observed in single fiber maximal force normalized to cross-sectional area (specific force) when sepsis was added to the ICU intervention. According to microarray data- the addition of sepsis to the ICU intervention induced a deregulation of {\textgreater}500 genes- such as an increased expression of genes involved in chemokine activity- kinase activity- and transcriptional regulation. Genes involved in the regulation of the oxidative stress response and cytoskeletal/sarcomeric and heat shock proteins were on the other hand downregulated when sepsis was added to the ICU intervention. Thus- sepsis has a significant negative effect on muscle function in critically ill ICU patients- and chemokine activity and heat shock protein genes are forwarded to play an instrumental role in this specific muscle wasting condition.
    Forebrain gene expression predicts deficits in sensorimotor gating after isolation rearing in male rats
    Neal Swerdlow, Behavioural Brain Research - 2013
    Abstract Compared to socially housed (SH) rats- adult isolation-reared (IR) rats exhibit phenotypes relevant to schizophrenia (SZ)- including reduced prepulse inhibition (PPI) of startle. PPI is normally regulated by the medial prefrontal cortex (mPFC) and nucleus accumbens (NAC). We assessed PPI- auditory-evoked local field potentials (LFPs) and expression of seven PPI- and SZ-related genes in the mPFC and NAC- in IR and SH rats. Buffalo (BUF) rats were raised in same-sex groups of 2-3 (SH) or in isolation (IR). PPI was measured early (d53) and later in adulthood (d74), LFPs were measured approximately on d66. Brains were processed for RT-PCR measures of mPFC and NAC expression of Comt- Erbb4- Grid2- Ncam1- Slc1a2- Nrg1 and Reln. Male IR rats exhibited PPI deficits- most pronounced at d53, male and female IR rats had significantly elevated startle magnitude on both test days. Gene expression levels were not significantly altered by IR. PPI levels (d53) were positively correlated with mPFC expression of several genes- and negatively correlated with NAC expression of several genes- in male IR but not SH rats. Late (P90) LFP amplitudes correlated significantly with expression levels of 6/7 mPFC genes in male rats- independent of rearing. After IR that disrupts early adult PPI in male BUF rats- expression levels of PPI- and SZ-associated genes in the mPFC correlate positively with PPI- and levels in the NAC correlate negatively with PPI. These results support the model that specific gene-behavior relationships moderate the impact of early-life experience on SZ-linked behavioral and neurophysiological markers.
    Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing
    Nathalie Piazzon, Journal of Molecular Cell Biology - 2012
    Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules- at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1-/- knockout mice. Bicc1- a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM)- is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer- whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKI by miR-27a. Thus- Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.
    Strain-Specific Regulation of Striatal Phenotype in Drd2-eGFP BAC Transgenic Mice
    Jayms Peterson, Journal of Neuroscience - 2012
    Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists- providing a powerful means of dissecting complex neural circuits in the brain. Recently- it was reported that one popular line of these mice--mice possessing a BAC transgene with a D2 dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter--had abnormal striatal gene expression- physiology- and motor behavior. Unlike most of the work using BAC mice- this interesting study relied upon mice backcrossed on the outbred Swiss Webster (SW) strain that were homozygous for the Drd2-eGFP BAC transgene. The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported- SW mice were very sensitive to transgene expression. However- in more commonly used inbred strains of mice (C57BL/6- FVB/N) that were hemizygous for the transgene- the Drd2-eGFP BAC transgene did not alter striatal gene expression- physiology- or motor behavior. Thus- the use of inbred strains of mice that are hemizygous for the Drd2 BAC transgene provides a reliable tool for studying basal ganglia function.
    Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways
    Erick Perera, Journal of Experimental Biology - 2012
    The effects of pelleted diets with different protein composition (fish- squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time- fish- and squid-based diets induced trypsin secretion- as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response- soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However- the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland- suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However- other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency.
    A Transcriptional Regulatory Role of the THAP11-HCF-1 Complex in Colon Cancer Cell Function
    J. Parker, Molecular and Cellular Biology - 2012
    The recently identified Thanatos-associated protein (THAP) domain is an atypical zinc finger motif with sequence-specific DNA-binding activity. Emerging data suggest that THAP proteins may function in chromatin-dependent processes- including transcriptional regulation- but the roles of most THAP proteins in normal and aberrant cellular processes remain largely unknown. In this work- we identify THAP11 as a transcriptional regulator differentially expressed in human colon cancer. Immunohistochemical analysis of human colon cancers revealed increased THAP11 expression in both primary tumors and metastases. Knockdown of THAP11 in SW620 colon cancer cells resulted in a significant decrease in cell proliferation- and profiling of gene expression in these cells identified a novel gene set composed of 80 differentially expressed genes- 70\% of which were derepressed by THAP11 knockdown. THAP11 was found to associate physically with the transcriptional coregulator HCF-1 (host cell factor 1) and recruit HCF-1 to target promoters. Importantly- THAP11-mediated gene regulation and its chromatin association require HCF-1- while HCF-1 recruitment at these genes requires THAP11. Collectively- these data provide the first characterization of THAP11-dependent gene expression in human colon cancer cells and suggest that the THAP11-HCF-1 complex may be an important transcriptional and cell growth regulator in human colon cancer.
    Relationship between asparagine metabolism and protein concentration in soybean seed
    Sudhakar Pandurangan, Journal of Experimental Botany - 2012
    The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration- including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat- levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight- suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern- being low at 50 mg and progressively increased until 150 mg- coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars- [{\textasciitilde}]2-fold higher levels of asparaginase B1 protein and activity in seed coat- were associated with high protein concentration- reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene- ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.
    Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels
    D.H. Rosenzweig, Acta Biomaterialia - 2013
    Abstract Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However- standard culture techniques require cell passaging- leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes- grown either in CE culture or passaged twice on static silicone dishes (SS culture, comparable to standard methods)- were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes- CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture- correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition- yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties- making them more suitable than SS cultured cells for tissue engineering applications.
    The Brominated flame retardant TBECH activates the zebrafish (Danio rerio) androgen receptor- alters gene transcription and causes developmental disturbances
    Ajay Pradhan, Aquatic Toxicology - 2013
    Abstract Tetrabromoethylcyclohexane (TBECH) is a brominated flame retardant that has been shown to be a potent agonist to the human androgen receptor (AR). However- while it is present in the environment- it is not known if it interacts with AR from aquatic species. The present study was therefore aimed at improving our understanding of how TBECH affects aquatic animals using zebrafish as a model organism. In silico modeling demonstrated that TBECH diastereomers bind to the zebrafish androgen receptor (zAR) and in vitro and in vivo data showed that TBECH has androgenic properties. Deleterious effects of TBECH were studied on embryonic and juvenile zebrafish and qRT-PCR analysis in vitro and in vivo was performed to determine TBECH effects on gene regulation. TBECH was found to delay hatching at 1 μM and 10 μM doses while morphological abnormalities and juvenile mortality was observed at 10 μM. The qRT-PCR analysis showed alterations of multiple genes involved in chondrogenesis (cartilage development)- metabolism and stress response. Thus- TBECH induces androgenic activity and has negative effects on zebrafish physiology and therefore its impact on the environment should be carefully monitored.
    Different early weaning protocols in common sole (Solea solea L.) larvae: Implications on the performances and molecular ontogeny of digestive enzyme precursors
    Luca Parma, Aquaculture - 2013
    Abstract The effects of early weaning on the performances and molecular ontogeny of digestive enzyme precursors in common sole (Solea solea) larvae were studied. Four experimental weaning time protocols (WPs) were performed in triplicate. One protocol was used for the control group where larvae were fed live feed until 27 days post-hatching (dph) (WP-27) and weaned with dry feed. The other groups provided only with dry feed (WP-4)- live feed until 13 dph (WP-13) and live feed until 18 dph (WP-18)- followed by weaning on to the same commercial microdiet (MD) used in the WP-27 group (AgloNorse K/S Tromsø Fiskeindustry A/S &, Co- Norway). The trial lasted until 33 dph. The larvae of the WP-4 group displayed a significantly lower survival rate- lower growth increase and a delay in the onset of metamorphosis when compared with the other groups. The WP-13 and WP-18 groups displayed significantly lower weight increases when compared with the WP-27 group- whereas metamorphosis development and survival were not influenced. Partial transcripts of trypsinogen 1 (tryp1)- trypsinogen 3 (tryp3)- trypsinogen Y (trypY)- chymotrypsinogen (chymt)- amylase (amy) and hepatic lipase (HL) were cloned and analysed by quantitative real-time PCR (qPCR). The tryp1 and chymt mRNA levels displayed a rapid increase in all the treatments between 4 and 8 dph and thereafter displayed a homogeneous fluctuating pattern with a higher increasing tendency for chymt at the end of the trial. The amount of tryp3 and trypY mRNA remained stable or slightly increased until 27 dph in all WP groups and thereafter displayed a general up-regulation trend. The amy transcript levels displayed a gradual increase throughout the study in all WP groups. The HL mRNA levels increased until 13 dph in the WP-27- WP-18 and WP-13 groups and thereafter remained generally stable until 27 dph- whereas in the WP-4 group- there was a linear time-related increasing trend until the end of the trial. This study supports previous results on the feasibility of early weaning of common sole larvae using a MD before the end of metamorphosis. The molecular ontogeny of some important digestive enzyme precursors is described for the first time in this species. The transcripts analysed were not greatly influenced by the WPs.
    Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction
    C. Janzen, Placenta - 2013
    AbstractIntroduction Transport of glucose from maternal blood across the placental trophoblastic tissue barrier is critical to sustain fetal growth. The mechanism by which GLUTs are regulated in trophoblasts in response to ischemic hypoxia encountered with intrauterine growth restriction (IUGR) has not been suitably investigated. Objective To investigate placental expression of GLUT1- GLUT3 and GLUT4 and possible mechanisms of GLUT regulation in idiopathic IUGR. Methods We analyzed clinical- biochemical and histological data from placentas collected from women affected by idiopathic full-term IUGR (n = 10) and gestational age-matched healthy controls (n = 10). Results We found increased GLUT3 protein expression in the trophoblast (cytotrophoblast greater than syncytiotrophoblast) on the maternal aspect of the placenta in IUGR compared to normal placenta- but no differences in GLUT1 or GLUT4 were found. No differential methylation of the GLUT3 promoter between normal and IUGR placentas was observed. Increased GLUT3 expression was associated with an increased nuclear concentration of HIF-1alpha- suggesting hypoxia may play a role in the up-regulation of GLUT3. Discussion Further studies are needed to elucidate whether increased GLUT3 expression in IUGR is a marker for defective villous maturation or an adaptive response of the trophoblast in response to chronic hypoxia. Conclusions Patients with IUGR have increased trophoblast expression of GLUT3- as found under the low-oxygen conditions of the first trimester.
    A diet high in alpha-linolenic acid and monounsaturated fatty acids attenuates hepatic steatosis and alters hepatic phospholipid fatty acid profile in diet-induced obese rats
    Danielle Hanke, Prostaglandins- Leukotrienes and Essential Fatty Acids (PLEFA) - 2013
    Abstract This study investigated the efficacy of the plant-based n-3 fatty acid- alpha-linolenic acid (ALA)- a dietary precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)- for modulating hepatic steatosis. Rats were fed high fat (55\% energy) diets containing high oleic canola oil- canola oil- a canola/flax oil blend (C/F- 3:1)- safflower oil- soybean oil- or lard. After 12 weeks- C/F and weight-matched (WM) groups had 20\% less liver lipid. Body mass- liver weight- glucose and lipid metabolism- inflammation and molecular markers of fatty acid oxidation- synthesis- desaturation and elongation did not account for this effect. The C/F group had the highest total n-3 and EPA in hepatic phospholipids (PL)- as well as one of the highest DHA and lowest arachidonic acid (n-6) concentrations. In conclusion- the C/F diet with the highest content of the plant-based n-3 ALA attenuated hepatic steatosis and altered the hepatic PL fatty acid profile.
    Clonal Immortalized Human Glial Cell Lines Support Varying Levels of JC Virus Infection due to Differences in Cellular Gene Expression
    Michael Ferenczy, Journal of Neuroimmune Pharmacology - 2013
    JC virus (JCV) is a ubiquitous human polyomavirus that causes the demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). JCV replicates in limited cell types in culture- predominantly in human glial cells. Following introduction of a replication defective SV40 mutant that expressed large T protein into a heterogeneous culture of human fetal brain cells- multiple phenotypes became immortalized (SVG cells). A subset of SVG cells could support JCV replication. In the current study- clonal cell lines were selected from the original SVG cell culture. The 5F4 clone showed low levels of viral growth. The 10B1 clone was highly permissive for JCV DNA replication and gene expression and supported persistent and stable JCV infection over months in culture. Microarray analysis revealed that viral infection did not significantly change gene expression in these cells. More resistant 5F4 cells expressed high levels of transcription factors known to inhibit JCV transcription. Interestingly- 5F4 cells expressed high levels of RNA of markers of radial glia and 10B1 cells had high expression of markers of immature glial cells and activation of transcription regulators important for stem/progenitor cell self-renewal. These SVG-derived clonal cell lines provide a biologically relevant model to investigate cell type differences in JCV host range and pathogenesis- as well as neural development. Several transcription regulators were identified which may be targets for therapeutic modulation of expression to abrogate JCV replication in PML patients. Additionally- these clonal cell lines can provide a consistent culture platform for testing therapies against JCV infection of the central nervous system.
    Early Growth Response 3 (Egr-3) Is Induced by Transforming Growth Factor-Beta and Regulates Fibrogenic Responses
    Feng Fang, The American Journal of Pathology - 2013
    Members of the early growth response (Egr) gene family of transcription factors have nonredundant biological functions. Although Egr-3 is implicated primarily in neuromuscular development and immunity- its regulation and role in tissue repair and fibrosis has not been studied. We now show that in normal skin fibroblasts- Egr-3 was potently induced by transforming growth factor-Beta via canonical Smad3. Moreover- transient Egr-3 overexpression was sufficient to stimulate fibrotic gene expression- whereas deletion of Egr-3 resulted in substantially attenuated transforming growth factor-Beta responses. Genome-wide expression profiling in fibroblasts showed that genes associated with tissue remodeling and wound healing were prominently up-regulated by Egr-3. Notably- <,5\% of fibroblast genes regulated by Egr-1 or Egr-2 were found to be coregulated by Egr-3- revealing substantial functional divergence among these Egr family members. In a mouse model of scleroderma- development of dermal fibrosis was accompanied by accumulation of Egr-3-positive myofibroblasts in the lesional tissue. Moreover- skin biopsy samples from patients with scleroderma showed elevated Egr-3 levels in the dermis- and Egr-3 mRNA levels correlated with the extent of skin involvement. These results provide the first evidence that Egr-3- a functionally distinct member of the Egr family with potent effects on inflammation and immunity- is up-regulated in scleroderma and is necessary and sufficient for profibrotic responses- suggesting important and distinct roles in the pathogenesis of fibrosis.
    Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes.pdf
    Brandon Markway, Arthritis Research & Therapy - 2013
    Introduction: Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However- hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus- we assessed whether healthy and OA chondrocytes have distinct responses to oxygen- particularly with regard to hypertrophy and degradation during redifferentiation. Methods: Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression- matrix quality and quantity- degradative enzyme expression and HIF expression were measured. Results: In hypoxia- both healthy and OA chondrocytes had higher human collagen type II- a1 gene (COL2A1)- and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation- concomitant with lower human collagen type X- a1 gene (COL10A1)- and human collagen type I- a1 gene (COL1A1)- expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes- but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1- MMP2- MMP3 and MMP13 in hypoxia and less active MMP2 enzyme- consistent with lower MMP14 expression. However- aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells- and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1a and HIF-2a had similar expression profiles in healthy and OA cells- increasing to maximal levels early in hypoxia and decreasing over time. Conclusions: Hypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation- but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins- combined with decreased expression of numerous degradative enzymes by hypoxia- minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore- though HIFs may be involved in the pathogenesis of OA- conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy- even in cells from OA joints.
    Cytokinin Response Factor 6 Negatively Regulates Leaf Senescence and is Induced in Response to Cytokinin and Numerous Abiotic Stresses
    Paul Zwack, Plant and Cell Physiology - 2013
    Cytokinin response factor 6 (CRF6) is an Arabidopsis AP2/ERF transcription factor which is transcriptionally induced by cytokinin. Cytokinin is known to delay leaf senescence in wild-type (WT) plants- for example in dark-incubated detached leaves. This response is mediated by the cytokinin receptor Arabidopsis histidine kinase receptor 3 (AHK3). Similar to ahk3 mutants- crf6 leaves show decreased sensitivity to this cytokinin effect. Leaves overexpressing CRF6 retain more Chl than those of the WT under these conditions without exogenous cytokinin. It therefore appears that an increase in expression of CRF6 downstream of the perception of cytokinin by AHK3 is involved in the delay of leaf senescence. Intact crf6 plants also begin to undergo monocarpic senescence sooner than WT plants. Interestingly- plants overexpressing CRF6 display a more extreme acceleration of development than crf6 mutants- suggesting that a specific expression level or localization of CRF6 is necessary to prevent premature senescence. Expression analyses indicate that CRF6 is highly expressed in the veins of mature leaves and that this expression decreases with age. CRF6 expression is shown to be induced by abiotic stress- in addition to increased cytokinin. Together- these findings suggest that CRF6 functions to regulate developmental senescence negatively and may have a similar role in response to stress. CRF6 may therefore be involved in fine-tuning the timing of developmental and stress-induced senescence. CRF6 functioning in negative regulation of senescence is significant in that it is the first process known to be regulated by cytokinin- in which a CRF can be placed specifically downstream of the cytokinin signaling pathway.
    Expression Profiling of Nuclear Receptors Identifies Key Roles of NR4A Subfamily in Uterine Fibroids
    Hanwei Yin, Molecular Endocrinology - 2013
    Uterine fibroids (UFs)- also known as uterine leiomyomas- are benign- fibrotic smooth muscle tumors. Although the GnRH analog leuprolide acetate that suppresses gonadal steroid hormones is used as a treatment- it has significant side effects- thereby limiting its use. Availability of more effective therapy is limited because of a lack of understanding of molecular underpinnings of the disease. Although ovarian steroid hormones estrogen and progesterone and their receptors are clearly involved- the role of other nuclear receptors (NRs) in UFs is not well defined. We used quantitative real-time PCR to systematically profile the expression of 48 NRs and identified several NRs that were aberrantly expressed in UFs. Among others- expression of NR4A subfamily members including NGFIB (NR4A1)- NURR1 (NR4A2)- and NOR1 (NR4A3) were dramatically suppressed in leiomyoma compared with the matched myometrium. Restoration of expression of each of these NR4A members in the primary leiomyoma smooth muscle cells decreased cell proliferation. Importantly- NR4As regulate expressions of the profibrotic factors including TGF\{beta\}3 and SMAD3- and several collagens that are key components of the extracellular matrix. Finally- we identify NR4A members as targets of leuprolide acetate treatment. Together- our results implicate several NRs including the NR4A subfamily in leiomyoma etiology and identify NR4As as potential therapeutic targets for treating fibrotic diseases.
    Role of LytF and AtlS in eDNA Release by Streptococcus gordonii
    Yifan Xu, PLoS ONE - 2013
    Extracellular DNA (eDNA) is an important component of the biofilm matrix produced by many bacteria. In general- the release of eDNA is associated with the activity of muralytic enzymes leading to obvious cell lysis. In the Gram-positive oral commensal Streptococcus gordonii- eDNA release is dependent on pyruvate oxidase generated hydrogen peroxide (H2O2). Addition of H2O2 to cells grown under conditions non-permissive for H2O2 production causes eDNA release. Furthermore- eDNA release is maximal under aerobic growth conditions known to induce pyruvate oxidase gene expression and H2O2 production. Obvious cell lysis- however- does not occur. Two enzymes have been recently associated with eDNA release in S. gordonii. The autolysin AtlS and the competence regulated murein hydrolase LytF. In the present report- we investigated the role of both proteins in the H2O2 dependent eDNA release process. Single and double mutants in the respective genes for LytF and AtlS released less eDNA under normal growth conditions- but the AtlS mutant was still inducible for eDNA release by external H2O2. Moreover- we showed that the AtlS mutation interfered with the ability of S. gordonii to produce eDNA release inducing amounts of H2O2. Our data support a role of LytF in the H2O2 eDNA dependent release of S. gordonii as part of the competence stress pathway responding to oxidative stress.
    Surveillance of the tumor mutanome by T cells during progression from primary to recurrent ovarian cancer
    Darin Wick, Clinical Cancer Research - 2013
    Purpose: Cancers accumulate mutations over time- each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in ovarian cancer patients undergoing standard treatment. Experimental Design: Tumor-associated T cells from three ovarian cancer patients were assessed by ELISPOT for recognition of non-synonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence. Results: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells, however a highly specific CD8+ T cell response to the mutation HSDL1L25V was detected in one patient. In the primary tumor- the HSDL1L25V mutation had low prevalence and expression- and a corresponding T-cell response was undetectable. At first recurrence- there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope- and this was accompanied by the emergence of the HSDL1L25V-specific CD8+ T cell response. At second recurrence- the HSDL1L25V mutation and epitope continued to be expressed, however- the corresponding T-cell response was no longer detectable. Conclusions: The immune system can respond to the evolving ovarian cancer genome. However- the T-cell response detected here was rare- transient and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy.
    The retinoic acid receptor-alpha modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
    Daniel Wansley, Clinical and Molecular Allergy - 2013
    Th2 cytokine responses are enhanced by all trans retinoic acid (ATRA)- the bioavailable form of vitamin A. Retinoic acid receptor alpha (RARalpha) is the high affinity receptor for ATRA that mediates these pro-Th2 effects. We have previously characterized two major human Th2 subpopulations: IL-5- Th2 (IL-5-- IL-4+- IL-13+) and IL-5+ Th2 cells (IL-5+- IL-4+- IL-13+)- which represent less and more highly differentiated Th2 cells- respectively. We hypothesized that the pro-Th2 effects of ATRA may differentially affect these Th2 subpopulations. PMID: 24314292
    GNC and CGA1 Modulate Chlorophyll Biosynthesis and Glutamate Synthase (GLU1/Fd-GOGAT) Expression in Arabidopsis
    Darryl Hudson, PLoS ONE - 2011
    Chloroplast development is an important determinant of plant productivity and is controlled by environmental factors including amounts of light and nitrogen as well as internal phytohormones including cytokinins and gibberellins (GA). The paralog GATA transcription factors GNC and CGA1/GNL up-regulated by light- nitrogen and cytokinin while also being repressed by GA signaling. Modifying the expression of these genes has previously been shown to influence chlorophyll content in Arabidopsis while also altering aspects of germination- elongation growth and flowering time. In this work- we also use transgenic lines to demonstrate that GNC and CGA1 exhibit a partially redundant control over chlorophyll biosynthesis. We provide novel evidence that GNC and CGA1 influence both chloroplast number and leaf starch in proportion to their transcript level. GNC and CGA1 were found to modify the expression of chloroplast localized GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT)- which is the primary factor controlling nitrogen assimilation in green tissue. Altering GNC and CGA1 expression was also found to modulate the expression of important chlorophyll biosynthesis genes (GUN4- HEMA1- PORB- and PORC). As previously demonstrated- the CGA1 transgenic plants demonstrated significantly altered timing to a number of developmental events including germination- leaf production- flowering time and senescence. In contrast- the GNC transgenic lines we analyzed maintain relatively normal growth phenotypes outside of differences in chloroplast development. Despite some evidence for partial divergence- results indicate that regulation of both GNC and CGA1 by light- nitrogen- cytokinin- and GA acts to modulate nitrogen assimilation- chloroplast development and starch production. Understanding the mechanisms controlling these processes is important for agricultural biotechnology.
    A chronic high-cholesterol diet paradoxically suppresses hepatic CYP7A1 expression in FVB/NJ mice
    Anne Henkel, Journal of Lipid Research - 2011
    Cholesterol 7-hydroxylase (CYP7A1) encodes for the rate-limiting step in the conversion of cholesterol to bile acids in the liver. In response to acute cholesterol feeding- mice upregulate CYP7A1 via stimulation of the liver X receptor (LXR) . However- the effect of a chronic high-cholesterol diet on hepatic CYP7A1 expression in mice is unknown. We demonstrate that chronic cholesterol feeding (0.2\% or 1.25\% w/w cholesterol for 12 weeks) in FVB/NJ mice results in a {\textgreater}60\% suppression of hepatic CYP7A1 expression associated with a {\textgreater}2-fold increase in hepatic cholesterol content. In contrast- acute cholesterol feeding induces a {\textgreater}3-fold upregulation of hepatic CYP7A1 expression. We show that chronic- but not acute- cholesterol feeding increases the expression of hepatic inflammatory cytokines- tumor necrosis factor (TNF)- and interleukin (IL)-1\{beta\}- which are known to suppress hepatic CYP7A1 expression. Chronic cholesterol feeding also results in activation of the mitogen activated protein (MAP) kinases- c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore- we demonstrate in vitro that suppression of CYP7A1 by TNF and IL-1\{beta\} is dependent on JNK and ERK signaling. We conclude that chronic high-cholesterol feeding suppresses CYP7A1 expression in mice. We propose that chronic cholesterol feeding induces inflammatory cytokine activation and liver damage- which leads to suppression of CYP7A1 via activation of JNK and ERK signaling pathways.
    Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia
    Lynn Heltemes-Harris, Journal of Experimental Medicine - 2011
    As STAT5 is critical for the differentiation- proliferation- and survival of progenitor B cells- this transcription factor may play a role in acute lymphoblastic leukemia (ALL). Here- we show increased expression of activated signal transducer and activator of transcription 5 (STAT5)- which is correlated with poor prognosis- in ALL patient cells. Mutations in EBF1 and PAX5- genes critical for B cell development have also been identified in human ALL. To determine whether mutations in Ebf1 or Pax5 synergize with STAT5 activation to induce ALL- we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice heterozygous for Ebf1 or Pax5. Haploinsufficiency of either Pax5 or Ebf1 synergized with Stat5b-CA to rapidly induce ALL in 100\% of the mice. The leukemic cells displayed reduced expression of both Pax5 and Ebf1- but this had little effect on most EBF1 or PAX5 target genes. Only a subset of target genes was deregulated, this subset included a large percentage of potential tumor suppressor genes and oncogenes. Further- most of these genes appear to be jointly regulated by both EBF1 and PAX5. Our findings suggest a model whereby small perturbations in a self-reinforcing network of transcription factors critical for B cell development- specifically PAX5 and EBF1- cooperate with STAT5 activation to initiate ALL.
    Yin-Yang1 Is Required in the Mammalian Oocyte for Follicle Expansion
    Gillian Griffith, Biology of Reproduction - 2011
    The multifaceted polycomb group gene Yin-Yang1 (Yy1) has been implicated in a variety of transcriptional regulatory roles both as an activator and silencer of gene expression. Here we examine the role of Yy1 during oocyte growth by conditional deletion of the locus in the growing oocyte. Our results indicate that YY1 is required for oocyte maturation and granulosa cell expansion. In mutant oocytes- we observe severely reduced expression of both Gdf9 and Bmp15- suggesting a mechanism underlying the failure of granulosa cell expansion. Consequently- we observe infertility- failure of estrus cycling- and altered reproductive hormone levels in mutant females. Additionally- we find that YY1-deficient oocytes exhibit altered levels of several oocyte-specific factors- including Pou5f1- Figla- Lhx8- Oosp1- and Sohlh2. These results document YY1's involvement in folliculogenesis and ovarian function in the mouse and indicate that YY1 is required specifically in the oocyte for oocyte-granulosa cell communication.
    Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system
    David Graber, Journal of Neuroinflammation - 2011
    Background While it is clear that inbred strains of mice have variations in immunological responsiveness- the influence of genetic background following tissue damage in the central nervous system is not fully understood. A cortical explant system was employed as a model for injury to determine whether the immediate transcriptional response to tissue resection revealed differences among three mouse strains. Methods Immunological mRNAs were measured in cerebral cortex from SJL/J- C57BL/6J- and BALB/cJ mice using real time RT-PCR. Freshly isolated cortical tissue and cortical sections incubated in explant medium were examined. Levels of mRNA- normalized to Beta-actin- were compared using one way analysis of variance with pooled samples from each mouse strain. Results In freshly isolated cerebral cortex- transcript levels of many pro-inflammatory mediators were not significantly different among the strains or too low for comparison. Constitutive- baseline amounts of CD74 and antisecretory factor (ASF) mRNAs- however- were higher in SJL/J and C57BL/6J- respectively. When sections of cortical tissue were incubated in explant medium- increased message for a number of pro-inflammatory cytokines and chemokines occurred within five hours. Message for chemokines- IL-1alpha- and COX-2 transcripts were higher in C57BL/6J cortical explants relative to SJL/J and BALB/cJ. IL-1Beta- IL-12/23 p40- and TNF-alpha were lower in BALB/cJ explants relative to SJL/J and C57BL/6J. Similar to observations in freshly isolated cortex- CD74 mRNA remained higher in SJL/J explants. The ASF mRNA in SJL/J explants- however- was now lower than levels in both C57BL/6J and BALB/cJ explants. Conclusions The short-term cortical explant model employed in this study provides a basic approach to evaluate an early transcriptional response to neurological damage- and can identify expression differences in genes that are influenced by genetic background.
    Extraordinary Sequence Divergence at Tsga8- an X-linked Gene Involved in Mouse Spermiogenesis
    Jeffrey Good, Molecular Biology and Evolution - 2011
    The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8)- a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion-deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus- differing in length from the reference mouse genome by up to 89 amino acids (27\% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species- we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5' and 3' ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60\% of the rat-coding domain. Overall- Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice.
    S-Adenosylmethionine Decreases Lipopolysaccharide-Induced Phosphodiesterase 4B2 and Attenuates Tumor Necrosis Factor Expression via cAMP/Protein Kinase A Pathway
    Leila Gobejishvili, Journal of Pharmacology and Experimental Therapeutics - 2011
    S-Adenosylmethionine (SAM) treatment has anti-inflammatory- cytoprotective effects against endotoxin-induced organ injury. An important component of the anti-inflammatory action of SAM involves down-regulation of the lipopolysaccharide (LPS)-induced transcriptional induction of tumor necrosis factor- (TNF) expression by monocytes/macrophages. We examined the effect of SAM on expression and activity of LPS-induced up-regulation of phosphodiesterase 4 (PDE4)- which regulates cellular cAMP levels and TNF expression. LPS treatment of RAW 264.7- a mouse macrophage cell line- led to the induction of Pde4b2 mRNA expression with no effect on Pde4a or Pde4d. SAM pretreatment led to a significant decrease in LPS-induced up-regulation of Pde4b2 expression in both RAW 264.7 cells and primary human CD14+ monocytes. Of note- the decreased Pde4b2 mRNA expression correlated with the SAM-dependent increase in the transcriptionally repressive histone H3 lysine 9 trimethylation on the Pde4b2 intronic promoter region. The SAM-mediated decrease in LPS-inducible Pde4b2 up-regulation resulted in an increase in cellular cAMP levels and activation of cAMP-dependent protein kinase A (PKA)- which plays an inhibitory role in LPS-induced TNF production. In addition- SAM did not affect LPS-inducible inhibitor of nuclear factor-\{kappa\}B degradation or nuclear factor-\{kappa\}B (NF-\{kappa\}B)-p65 translocation into the nucleus but rather inhibited NF-\{kappa\}B transcriptional activity. These results demonstrate for the first time that inhibition of LPS-induced PDE4B2 up-regulation and increased cAMP-dependent PKA activation are significant mechanisms contributing to the anti-TNF effect of SAM. Moreover- these data also suggest that SAM may be used as an effective PDE4B inhibitor in the treatment of chronic inflammatory disorders in which TNF expression plays a significant pathogenic role.
    Polyubiquitin Binding to Optineurin Is Required for Optimal Activation of TANK-binding Kinase 1 and Production of Interferon ?
    Catherine Gleason, The Journal of Biological Chemistry - 2011
    Background: Optineurin is a polyubiquitin-binding protein of unknown function.- Result: Macrophages from mice expressing a polyubiquitin-binding defective mutant of optineurin show reduced activation of TANK-binding kinase 1 (TBK1) and reduced production of interferon Beta.- Conclusion: The binding of polyubiquitin to optineurin is required for optimal activation and function of TBK1.- Significance: This study identifies a new physiological role for optineurin.- TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) Beta gene in response to lipopolysaccharide (LPS) and double-stranded RNA- but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously- we identified the NF-kB essential modulator (NEMO)-related polyubiquitin-binding protein- optineurin (OPTN)- as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFNBeta production- we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant- OPTND477N/D477N. In this study- we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTND477N/D477N mice. Consistent with this- the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFNBeta mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN- which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical IkB kinases (IKKalpha/Beta) and the IKK-related kinases (TBK1/IKKϵ). In contrast- LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTND477N/D477N mice- and inhibition of the canonical IKKs alone prevented phosphorylation- providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKKBeta phosphorylated OPTN preferentially at Ser-177 and Ser-513- respectively- in vitro. In conclusion- our results suggest that OPTN binds to polyubiquitylated species formed in response to LPS and poly(I:C)- enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFNBeta.
    Mechanisms of oocyte development in European sea bass (Dicentrarchus labrax L.): investigations via application of unilateral ovariectomy
    Angel Garcia-Lopez, Reproduction - 2011
    Unilateral ovariectomy (ULO) was performed in European sea bass (Dicentrarchus labrax L.) during late pre-vitellogenesis/early vitellogenesis. Plasma steroid levels and the expression of a suite of potential oogenesis-relevant genes in the ovary- brain- and pituitary were evaluated with the aim of understanding their involvement in the compensatory oocyte development occurring within the remaining ovarian lobe. After 69 days of surgery the remaining ovarian lobe in ULO fish was gravimetrically equivalent to an intact-paired ovary of sham operated- control fish. This compensatory ovarian growth was based on an increased number of early perinucleolar oocytes and mid-late stage vitellogenic follicles without an apparent recruitment of primary oocytes into the secondary growth phase. Plasma steroid levels were similar in ULO and control females at all time points analyzed- suggesting an increased steroid production of the remaining ovarian lobe in hemi-castrated females. Results of the gene expression survey conducted indicate that the signaling pathways mediated by Fsh and Gnrh1 constitute the central axes orchestrating the observed ovarian compensatory growth. In addition- steroid receptors- Star protein- Igfs- and members of the transforming growth factor beta superfamily including anti-Mullerian hormone and bone morphogenetic protein 4 were identified as potentially relevant players within this process- although their specific actions and interactions remain to be established. Our results demonstrate that ULO provides an excellent in vivo model for elucidating the interconnected endocrine and molecular mechanisms controlling oocyte development in European sea bass.
    LFR1 Ferric Iron Reductase of Leishmania amazonensis Is Essential for the Generation of Infective Parasite Forms
    Andrew Flannery, The Journal of Biological Chemistry - 2011
    The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages- where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly- indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here- we identify and functionally characterize a second member of the Leishmania iron acquisition pathway- the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms- metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability- precluding its ability to rescue LFR1 null replication in macrophages. However- LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition- the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe3+ reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe2+ production- which impairs parasite viability after intracellular transport by LIT1.
    The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor ? protein
    JL Fiori, British Journal of Pharmacology - 2011
    BACKGROUND AND PURPOSE AM251 is an inverse agonist of the cannabinoid 1 receptor (CB1R) that can exert ‘off-target’ effects in vitro and in CB1R knock-out mice. AM251 is also potent at modulating tumour cell growth- suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis- we examined AM251 regulation of EGF receptor (EGFR) expression and function. EXPERIMENTAL APPROACH The various biological functions of AM251 were measured in CB1R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data. KEY RESULTS The mRNA levels for EGFR and its associated ligands- including HB-EGF- were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790- a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor alpha (ERRalpha)- also induced EGFR and HB-EGF expression to the same extent as AM251- whereas pretreatment with the ERRalpha-selective agonist- biochanin A- blunted AM251 actions. AM251 promoted the degradation of ERRalpha protein without loss of the corresponding mRNA. Knock-down of ERRalpha by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels- and eliminated the biological responses of AM251 and XCT790. Finally- AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRalpha. CONCLUSIONS AND IMPLICATIONS AM251 up-regulates EGFR expression and signalling via a novel non-CB1R-mediated pathway involving destabilization of ERRalpha protein in selected cancer cell lines.
    In vivo functional analysis reveals specific roles for the integrin-binding sites of talin
    Stephanie Ellis, Journal of Cell Science - 2011
    Adhesion receptors play diverse roles during animal development and require precise spatiotemporal regulation- which is achieved through the activity of their binding partners. Integrins- adhesion receptors that mediate cell attachment to the extracellular matrix (ECM)- connect to the intracellular environment through the cytoplasmic adapter protein talin. Talin has two essential functions: orchestrating the assembly of the intracellular adhesion complex (IAC)- which associates with integrin- and regulating the affinity of integrins for the ECM. Talin can bind to integrins through two different integrin-binding sites (IBS-1 and IBS-2- respectively). Here- we have investigated the roles of each in the context of Drosophila development. We find that although IBS-1 and IBS-2 are partially redundant- they each have specialized roles during development: IBS-1 reinforces integrin attachment to the ECM- whereas IBS-2 reinforces the link between integrins and the IAC. Disruption of each IBS has different developmental consequences- illustrating how the functional diversity of integrin-mediated adhesion is achieved.
    Chronic Social Isolation Is Associated with Metabolic Gene Expression Changes Specific to Mammary Adipose Tissue
    Paul Volden, Cancer Prevention Research - 2013
    Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen FVB/N (TAg) mouse model of "triple-negative" breast cancer- the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e.- during the in situ carcinoma stage). To further understand the mechanisms underlying how accelerated mammary tumor growth is associated with social isolation- we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically- increased expression of the key metabolic genes Acaca- Hk2- and Acly was found in the adipocyte- rather than the epithelial fraction. Surprisingly- metabolic gene expression was not significantly increased in visceral adipose depots of socially isolated female mice. As expected- increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism- lipid synthesis- and leptin secretion from this adipose depot. Furthermore- application of media that had been cultured with isolated mouse mammary adipose tissue (conditioned media) resulted in increased proliferation of mammary cancer cells relative to group-housed-conditioned media. These results suggest that exposure to a chronic stressor (social isolation) results in specific metabolic reprogramming in mammary gland adipocytes that in turn contributes to increased proliferation of adjacent preinvasive malignant epithelial cells. Metabolites and/or tumor growth-promoting proteins secreted from adipose tissue could identify biomarkers and/or targets for preventive intervention in breast cancer. Cancer Prev Res, 6(7), 634-45. (C)2013 AACR.
    CD24 Expression Identifies Teratogen-Sensitive Fetal Neural Stem Cell Subpopulations: Evidence from Developmental Ethanol Exposure and Orthotopic Cell Transfer Models
    Joseph Tingling, PLoS ONE - 2013
    BackgroundEthanol is a potent teratogen. Its adverse neural effects are partly mediated by disrupting fetal neurogenesis. The teratogenic process is poorly understood- and vulnerable neurogenic stages have not been identified. Identifying these is a prerequisite for therapeutic interventions to mitigate effects of teratogen exposures.MethodsWe used flow cytometry and qRT-PCR to screen fetal mouse-derived neurosphere cultures for ethanol-sensitive neural stem cell (NSC) subpopulations- to study NSC renewal and differentiation. The identity of vulnerable NSC populations was validated in vivo- using a maternal ethanol exposure model. Finally- the effect of ethanol exposure on the ability of vulnerable NSC subpopulations to integrate into the fetal neurogenic environment was assessed following ultrasound guided- adoptive transfer.ResultsEthanol decreased NSC mRNAs for c-kit- Musashi-1and GFAP. The CD24+ NSC population- specifically the CD24+CD15+ double-positive subpopulation- was selectively decreased by ethanol. Maternal ethanol exposure also resulted in decreased fetal forebrain CD24 expression. Ethanol pre-exposed CD24+ cells exhibited increased proliferation- and deficits in cell-autonomous and cue-directed neuronal differentiation- and following orthotopic transplantation into naïve fetuses- were unable to integrate into neurogenic niches. CD24depleted cells retained neurosphere regeneration capacity- but following ethanol exposure- generated increased numbers of CD24+ cells relative to controls.ConclusionsNeuronal lineage committed CD24+ cells exhibit specific vulnerability- and ethanol exposure persistently impairs this population’s cell-autonomous differentiation capacity. CD24+ cells may additionally serve as quorum sensors within neurogenic niches, their loss- leading to compensatory NSC activation- perhaps depleting renewal capacity. These data collectively advance a mechanistic hypothesis for teratogenesis leading to microencephaly.
    Reactivation of Estrogen Receptor alpha by Vorinostat Sensitizes Mesenchymal-Like Triple-Negative Breast Cancer to Aminoflavone- a Ligand of the Aryl Hydrocarbon Receptor
    Karri Stark, PLoS ONE - 2013
    ObjectiveAminoflavone (AF) acts as a ligand of the aryl hydrocarbon receptor (AhR). Expression of estrogen receptor alpha (ERalpha) and AhR-mediated transcriptional induction of CYP1A1 can sensitize breast cancer cells to AF. The objective of this study was to investigate the combined antitumor effect of AF and the histone deacetylase inhibitor vorinostat for treating mesenchymal-like triple-negative breast cancer (TNBC) as well as the underlying mechanisms of such treatment.MethodsIn vitro antiproliferative activity of AFP464 (AF prodrug) in breast cancer cell lines was evaluated by MTS assay. In vitro- the combined effect of AFP464 and vorinostat on cell proliferation was assessed by the Chou-Talalay method. In vivo- antitumor activity of AFP464- given alone and in combination with vorinostat- was studied using TNBC xenograft models. Knockdown of ERalpha was performed using specific- small-interfering RNA. Western blot- quantitative RT-PCR- immunofluorescence- and immunohistochemical staining were performed to study the mechanisms underlying the combined antitumor effect.ResultsLuminal and basal A subtype breast cancer cell lines were sensitive to AFP464- whereas basal B subtype or mesenchymal-like TNBC cells were resistant. Vorinostat sensitized mesenchymal-like TNBC MDA-MB-231 and Hs578T cells to AFP464. It also potentiated the antitumor activity of AFP464 in a xenograft model using MDA-MB-231 cells. In vitro and in vivo mechanistic studies suggested that vorinostat reactivated ERalpha expression and restored AhR-mediated transcriptional induction of CYP1A1.ConclusionThe response of breast cancer cells to AF or AFP464 was associated with their gene expression profile. Vorinostat sensitized mesenchymal-like TNBC to AF- at least in part- by reactivating ERalpha expression and restoring the responsiveness of AhR to AF.
    Discovery and Biological Characterization of 1-(1H-indol-3-yl)-9H-pyrido[3-4-b]indole as an Aryl Hydrocarbon Receptor Activator Generated by Photoactivation of Tryptophan by Sunlight
    Silvia Diani-Moore, Chemico-biological interactions - 2011
    Activation of the aryl hydrocarbon receptor (AHR) by 2-3-7-8-tetrachlorodibenzo-p-dioxin (TCDD) is required for AHR dependent transcriptional activation and TCDD toxicity. We previously reported that aqueous tryptophan exposed to sunlight through window glass (aTRP) contains multiple photoproducts- including the well characterized 6-formylindolo[3-2-b]carbazole (FICZ)- capable of activating the AHR and inducing CYP1A and CYP1A-mediated enzyme activities. We report here the isolation from aTRP and chemical characterization and synthesis of 1-(1H-indol-3-yl)-9H-pyrido[3-4-b]indole (IPI)- a compound previously identified as a natural product of marine ascidia and now shown to be a TRP photoproduct with AHR-inducing properties. IPI- FICZ and TCDD produced equieffective induction of CYP1A-mediated 7-ethoxyresorufin deethylase (EROD) activity in chick embryo primary hepatocytes and mammalian Hepa1c1c7 cells. EROD induction by IPI was markedly curtailed in AHR-defective c35 cells- supporting the AHR dependence of the IPI response. Although IPI had a higher EC50 for EROD induction than FICZ- the much larger amount of IPI than FICZ in aTRP makes IPI a prominent contributor to EROD induction in aTRP. IPI was detected in TRP-containing culture medium under ambient laboratory conditions but not in TRP-free medium- consistent with its production from TRP. Cotreatment of hepatocytes with submaximal EROD-inducing doses of IPI and FICZ or TCDD produced additive increases in EROD without synergistic or inhibitory interactions. IPI and FICZ were readily metabolized by cultured hepatocytes. In addition to increasing CYP1A4 mRNA and EROD- IPI and FICZ decreased hepatocyte phosphoenolpyruvate carboxykinase mRNA expression and glucose output- biological effects associated with TCDD metabolic dysregulation. The findings underscore a role for sunlight in generating AHR-activating bioactive molecules.
    Intrinsic expression of Nod2 in CD4+ T lymphocytes is not necessary for the development of cell-mediated immunity and host resistance to Toxoplasma gondii
    Braulia Caetano, European Journal of Immunology - 2011
    Nod2 belongs to the NLR family of proteins and senses bacterial cell wall components to initiate innate immune responses against various pathogens. Recently it has been reported that T cell-intrinsic expression of Nod2 promotes host defense against Toxoplasma gondii infection by inducing type 1 immunity. Here we present results that demonstrate that Nod2 does not play a role in the defense against T. gondii infection. Nod2-deficient mice were fully capable of inducing Th1 immune responses and did not show enhanced susceptibility to infection. Upon T cell receptor stimulation in vitro- Nod2-deficient CD4+ T cells showed normal activation- IL-2 production- proliferation and Th1/2 differentiation. Nod2 mRNA and protein are expressed in CD4+ T and CD8+ T cells at substantial levels. Therefore Nod2- although expressed in CD4+ T cells- does not have an intrinsic function in T cell activation and differentiation.
    FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development
    Aaron Brown, Development - 2011
    Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment- distinguished by expression of CITED1- possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood- and their elucidation will provide key insights into normal and dysregulated nephrogenesis- as well as into regenerative processes that follow kidney injury. In this study- we found that the mouse CITED1+ progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo- RAS inactivation by expression of sprouty 1 (Spry1) in CITED1+ nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation- infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.
    Host Defense Peptide LL-37 Selectively Reduces Proinflammatory Macrophage Responses
    Kelly Brown, The Journal of Immunology - 2011
    The human cathelicidin peptide- LL-37- is a host defense peptide with a wide range of immunomodulatory activities and modest direct antimicrobial properties. LL-37 can exert both pro- and anti-inflammatory effects and can modulate the proinflammatory responses of human peripheral blood monocytes and epithelial cells. In this study- we evaluated the effect of LL-37 on mouse bone marrow-derived macrophages (BMDM) and tissue macrophages in vitro and in vivo. LL-37 dramatically reduced TNF- and NO levels produced by LPS and IFN-\{gamma\}-polarized M1-BMDM and slightly reduced reactive oxygen species production by these cells. LL-37 did not affect the ability of IL-4-polarized M2-BMDM to upregulate arginase activity- although it did inhibit LPS-induced TNF- secretion in these cells. LL-37 did not compromise the ability of M1-polarized BMDM to phagocytose and kill bacteria and did not affect the uptake of apoptotic neutrophils by M2-polarized BMDM. However- LL-37-treated M1-BMDM were more efficient at suppressing tumor growth in vitro. LL-37 significantly reduced LPS-induced TNF- secretion in ex vivo alveolar macrophages- whereas its effect on peritoneal macrophages was much less dramatic. Effective inhibition of LPS-induced TNF- secretion by alveolar macrophages also occurred in vivo when LL-37 was administered by intratracheal injection. This demonstrates a selective ability of LL-37 to decrease M1-BMDM- M2-BMDM- and tissue macrophage production of the proinflammatory cytokine TNF- in response to LPS while leaving other crucial anti-inflammatory M1 and M2 macrophage functions unaltered.
    Constitutive HIF-1? Expression Blunts the Beneficial Effects of Cardiosphere-Derived Cell Therapy in the Heart by Altering Paracrine Factor Balance
    Michael Bonios, Journal of cardiovascular translational research - 2011
    Hypoxia-inducible factor-1alpha (HIF-1alpha) expression promotes angiogenesis and can influence stem cell engraftment. We investigated the effect of stable over-expression of constitutively active HIF-1alpha on cardiosphere-derived cell (CDC) engraftment and left ventricular function. CDCs were transduced with a lentivirus expressing a constitutively active mutant of human HIF-1alpha (LVHIF-1alpha). Two million male rat CDCs were injected into the infarct following ligation of the mid-LAD in female syngeneic rats. Left ventricular ejection fraction (EF) and circumferential strain were measured by echocardiography at 1 and 4 weeks post-MI in the following groups: PBS group (n=7)- CELL group (n=7)- and CELL-HIF group (n=7). HIF-1alpha- VEGF- endothelin-1 expression- and CDC engraftment were measured by quantitative PCR. At 30 days- EF was unchanged in the CELL-HIF group (p=NS)- increased in the CELL group (p=0.025)- and decreased in the PBS group (p=0.021)- but engraftment was similar (2.4\%±3.3\% vs 1.7\%±0.8\%- p=NS). Mean circumferential strain of the infarcted region was unchanged in the CELL-HIF group- but improved in the CELL group (p=0.02). Endothelin-1 and VEGF expression were higher in HIF-CDCs exposed to hypoxia- compared with non-transduced CDCs. HIF-1alpha expression in CDCs blunted the beneficial functional effects of CDC transplantation- suggesting that paracrine factor balance may play an important role in cardiac regeneration.
    TFEB regulates lysosomal proteostasis
    Wensi Song, Human Molecular Genetics - 2013
    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants- thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs)- a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure- resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native- biologically functional structure of these unstable mutated enzymes. However- proteostasis modulators specific for the lysosomal system are currently unknown. Here- we investigate the role of the transcription factor EB (TFEB)- a master regulator of lysosomal biogenesis and function- in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding- trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD)- the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking- thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a \{beta\}-hexosaminidase mutant associated with the development of another LSD- Tay-Sachs disease- thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary- our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs.
    Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation
    Mandeep Singh, Proceedings of the National Academy of Sciences U.S.A. - 2013
    One strategy to restore vision in retinitis pigmentosa and age-related macular degeneration is cell replacement. Typically- patients lose vision when the outer retinal photoreceptor layer is lost- and so the therapeutic goal would be to restore vision at this stage of disease. It is not currently known if a degenerate retina lacking the outer nuclear layer of photoreceptor cells would allow the survival- maturation- and reconnection of replacement photoreceptors- as prior studies used hosts with a preexisting outer nuclear layer at the time of treatment. Here- using a murine model of severe human retinitis pigmentosa at a stage when no host rod cells remain- we show that transplanted rod precursors can reform an anatomically distinct and appropriately polarized outer nuclear layer. A trilaminar organization was returned to rd1 hosts that had only two retinal layers before treatment. The newly introduced precursors were able to resume their developmental program in the degenerate host niche to become mature rods with light-sensitive outer segments- reconnecting with host neurons downstream. Visual function- assayed in the same animals before and after transplantation- was restored in animals with zero rod function at baseline. These observations suggest that a cell therapy approach may reconstitute a light-sensitive cell layer de novo and hence repair a structurally damaged visual circuit. Rather than placing discrete photoreceptors among preexisting host outer retinal cells- total photoreceptor layer reconstruction may provide a clinically relevant model to investigate cell-based strategies for retinal repair.
    The CYP27B1 variant associated with increased risk of autoimmune disease is underexpressed in tolerising dendritic cells
    Fernando Shahijanian, Human Molecular Genetics - 2013
    Genome-wide association studies have identified a linkage disequilibrium block on chromosome 12 associated with multiple sclerosis (MS)- type 1 diabetes and other autoimmune diseases. This block contains CYP27B1- which catalyses conversion of 25 Vitamin D3 to 1-25 Vitamin D3. Fine mapping analysis has failed to identify which of the 17 genes in this block is most associated with MS. We have previously used a functional approach to identify the causal gene. We showed that the expression of several genes in this block in whole blood is highly associated with the MS risk allele- but not CYP27B1. Here we show that CYP27B1 is predominantly expressed in dendritic cells (DCs). Its expression in these cells is necessary for their response to Vitamin D- which is known to upregulate pathways involved in generating a tolerogenic DC phenotype. Here we utilize a differentiation protocol to generate inflammatory (DC1) and tolerogenic (DC2) dendritic cells- and show that for the MS risk allele CYP27B1 is underexpressed in dendritic cells- especially DC2s. Of the other Chr12 LD block genes expressed in these cells- only METT21B expression was as affected by genotype. Another gene associated with autoimmune diseases- CYP24A1- catabolises 1-25 Vitamin D3- and is predominantly expressed in DCs- but equally between DC1s and DC2s. Overall- these data are consistent with the hypothesis that reduced vitamin D pathway gene upregulation in DC2s of carriers of the risk haplotype of CYP27B1 contributes to autoimmune diseases. These data support therapeutic approaches aimed at targeting Vitamin D effects on DCs.
    Salmonellae in Fish Feces Analyzed by In Situ Hybridization and Quantitative Polymerase Chain Reaction
    Qiong Sha, Journal of Aquatic Animal Health - 2013
    The potential of fish to transfer salmonellae from heterogeneous aquatic biofilms into feces was assessed in controlled aquarium studies with Suckermouth Catfish Hypostomus plecostomus and with biofilms inoculated with salmonellae. Neither the presence of catfish nor inoculation with salmonellae had detectable effects on the abundance of the microbial community. Densities of the microbial community were about 105 cells/mL in the water during a 1-week period- whereas densities of the microbial community increased 10-fold (106 to 107 cells/mg) in catfish feces during the same period. Salmonellae were detected by both quantitative polymerase chain reaction (qPCR) and situ hybridization in water samples immediately after inoculation- in numbers of about 104 cells/mL- representing up to 20\% of the cells of the microbial community. Numbers decreased by three orders of magnitude within the first 3 d of the study- which represented only 0.01\% of the community- and became undetectable after day 5. In catfish feces- numbers of Salmonella initially increased to up to 6\% of the cells of the community but then declined. These results suggest that Salmonella are not biomagnified during gut passage- and thus- fish only provide a means for the translocation of this pathogen. Received November 25- 2012, accepted March 25- 2013
    Marianne Seney, Biology of Sex Differences - 2013
    BACKGROUND: Studies on major depressive and anxiety disorders suggest dysfunctions in brain corticolimbic circuits- including altered gamma-aminobutyric acid (GABA) and modulatory (serotonin and dopamine) neurotransmission. Interestingly- sexual dimorphisms in GABA- serotonin- and dopamine systems are also reported. Understanding the mechanisms behind these sexual dimorphisms may help unravel the biological bases of the heightened female vulnerability to mood disorders. Here- we investigate the contribution of sex-related factors (sex chromosome complement- developmental gonadal sex- or adult circulating hormones) to frontal cortex expression of selected GABA-- serotonin-- and dopamine-related genes. METHODS: As gonadal sex is determined by sex chromosome complement- the role of sex chromosomes cannot be investigated individually in humans. Therefore- we used the Four Core Genotypes (FCG) mouse model- in which sex chromosome complement and gonadal sex are artificially decoupled- to examine the expression of 13 GABA-related genes- 6 serotonin- and dopamine-related genes- and 8 associated signal transduction genes under chronic stress conditions. Results were analyzed by three-way ANOVA (sex chromosome complement × gonadal sex × circulating testosterone). A global perspective of gene expression changes was provided by heatmap representation and gene co-expression networks to identify patterns of transcriptional activities related to each main factor. RESULTS: We show that under chronic stress conditions- sex chromosome complement influenced GABA/serotonin/dopamine-related gene expression in the frontal cortex- with XY mice consistently having lower gene expression compared to XX mice. Gonadal sex and circulating testosterone exhibited less pronounced- more complex- and variable control over gene expression. Across factors- male conditions were associated with a tightly co-expressed set of signal transduction genes. CONCLUSIONS: Under chronic stress conditions- sex-related factors differentially influence expression of genes linked to mood regulation in the frontal cortex. The main factor influencing expression of GABA-- serotonin-- and dopamine-related genes was sex chromosome complement- with an unexpected pro-disease effect in XY mice relative to XX mice. This effect was partially opposed by gonadal sex and circulating testosterone- although all three factors influenced signal transduction pathways in males. Since GABA- serotonin- and dopamine changes are also observed in other psychiatric and neurodegenerative disorders- these findings have broader implications for the understanding of sexual dimorphism in adult psychopathology.
    Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFN?-Induced STAT1 Transcriptional Activity
    Anne Schneider, PLoS ONE - 2013
    The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host- including blocking IFNgamma-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined- yet is known to serve as an early target of in vivo infection. Unexpectedly- we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16- recently shown to mediate STAT3 and STAT6 phosphorylation- was not required for STAT1 phosphorylation. In combination with IFNgamma- T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNgamma consensus sequence oligonucleotides. Despite these findings- parasite infection blocked STAT1 binding to the native promoters of the IFNgamma-inducible genes Irf-1 and Lrg47- along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNgamma responses in dendritic cells- while simultaneously showing that T. gondii alone induces STAT1 phosphorylation.
    Aryl Hydrocarbon Receptor Activation by TCDD Reduces Inflammation Associated with Crohn's Disease
    Jenna Benson, Toxicological Sciences - 2011
    Crohn's disease results from a combination of genetic and environmental factors that trigger an inappropriate immune response to commensal gut bacteria. The aryl hydrocarbon receptor (AhR) is well known for its involvement in the toxicity of 2-3-7-8-tetrachlorodibenzo-p-dioxin (TCDD)- an environmental contaminant that affects people primarily through the diet. Recently- TCDD was shown to suppress immune responses by generating regulatory T cells (Tregs). We hypothesized that AhR activation dampens inflammation associated with Crohn's disease. To test this hypothesis- we utilized the 2-4-6-trinitrobenzenesulfonic acid (TNBS) murine model of colitis. Mice were gavaged with TCDD prior to colitis induction with TNBS. Several parameters were examined including colonic inflammation via histological and flow cytometric analyses. TCDD-treated mice recovered body weight faster and experienced significantly less colonic damage. Reduced levels of interleukin (IL) 6- IL-12- interferon-gamma- and tumor necrosis factor-[alpha] demonstrated suppression of inflammation in the gut following TCDD exposure. Forkhead box P3 (Foxp3)egfp mice revealed that TCDD increased the Foxp3+ Treg population in gut immune tissue following TNBS exposure. Collectively- these results suggest that activation of the AhR by TCDD decreases colonic inflammation in a murine model of colitis in part by generating regulatory immune cells. Ultimately- this work may lead to the development of more effective therapeutics for the treatment of Crohn's disease.
    Chronic Maternal Protein Deprivation in Mice Is Associated with Overexpression of the Cohesin-Mediator Complex in Liver of Their Offspring123
    Alfred Balasa, The Journal of Nutrition - 2011
    Epigenetic mechanisms may play an important role in the developmental programming of adult-onset chronic metabolic diseases resulting from suboptimal fetal nutrition- but the exact molecular mechanisms are incompletely understood. Given the central role of the liver in metabolic regulation- we investigated whether chronic maternal dietary protein restriction has long-term effects on liver gene expression in the offspring. We fed adult C57BL/6J dams ad libitum an 8\% maternal low-protein (MLP) or 20\% protein control diet (C) from 4 wk prior to mating until the end of lactation. Male pups were weaned to standard nonpurified diet and singly housed at 21 d of age (d 21). Body weights were followed to 1 y of age (1 y). At d 21 and 1 y- organs were quantitatively dissected and analyzed. MLP offspring had significantly lower body weights at all ages and significantly lower serum activity of alanine aminotransferase and lactate dehydrogenase at 1 y. Gene expression profiling of liver at 1 y showed 521 overexpressed and 236 underexpressed genes in MLP compared to C offspring. The most important novel finding was the overexpression of genes found in liver that participate in organization and maintenance of higher order chromatin architecture and regulation of transcriptional activation. These included members of the cohesin-mediator complex- which regulate gene expression by forming DNA loops between promoters and enhancers in a cell type-specific fashion. Thus- our findings of increased expression of these factors in liver of MLP offspring implicate a possible novel epigenetic mechanism in developmental programming.
    A bioresponsive hydrogel tuned to chondrogenesis of human mesenchymal stem cells
    Chelsea Bahney, FASEB Journal - 2011
    Cartilage tissue engineering aims to replace damaged or diseased tissue with a functional regenerate that restores joint function. Scaffolds are used to deliver cells and facilitate tissue development- but they can also interfere with the structural assembly of the cartilage matrix. Biodegradable scaffolds have been proposed as a means to improve matrix deposition and the biomechanical properties of neocartilage. The challenge is designing scaffolds with appropriate degradation rates- ideally such that scaffold degradation is proportional to matrix deposition. In this study- we developed a bioresponsive hydrogel with cell-mediated degradation aligned to the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). We identified matrix metalloproteinase 7 (MMP7) as an enzyme with a temporal expression pattern that corresponded with cartilage development. By embedding MMP7 peptide substrates within a poly(ethylene glycol) diacrylate backbone- we built MMP7-sensitive hydrogels with distinct degradation rates. When MMP7-sensitive scaffolds were compared with nondegradable scaffolds in vitro- photoencapsulated hMSCs produced neocartilage constructs with more extensive collagenous matrices- as demonstrated through immunohistochemistry and biochemical quantification of matrix molecules. Furthermore- these changes translated into an increased dynamic compressive modulus. This work presents a practical strategy for designing biomaterials uniquely tuned to individual biological processes.--Bahney- C. S.- Hsu- C.-W.- Yoo- J. U.- West- J. L.- Johnstone- B. A bioresponsive hydrogel tuned to chondrogenesis of human mesenchymal stem cells.
    Isogenic Pairs of Wild Type and Mutant Induced Pluripotent Stem Cell (iPSC) Lines from Rett Syndrome Patients as In Vitro Disease Model
    Gene Ananiev, PLoS ONE - 2011
    Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms- leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes- better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2- and drug testing in RTT mice and clinical trials in human RTT patients. However- because of potential differences in the underlying biology between humans and common research animals- there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC)- we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5-6\% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency- and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology- screening for novel drugs- and testing toxicology.
    Pancreatic beta-cell Raf-1 is required for glucose tolerance- insulin secretion- and insulin 2 transcription
    Emilyn Alejandro, FASEB Journal - 2011
    Regulation of glucose homeostasis by insulin depends on pancreatic \{beta\}-cell growth- survival- and function. Raf-1 kinase is a major downstream target of several growth factors that promote proliferation and survival of many cell types- including the pancreatic \{beta\} cells. We have previously reported that insulin protects \{beta\} cells from apoptosis and promotes proliferation by activating Raf-1 signaling in cultured human islets- mouse islets- and MIN6 cells. As Raf-1 activity is critical for basal apoptosis and insulin secretion in vitro- we hypothesized that Raf-1 may play an important role in glucose homeostasis in vivo. To test this hypothesis- we utilized the Cre-loxP recombination system to obtain a pancreatic \{beta\}-cell-specific ablation of Raf-1 kinase gene (RIPCre+/+:Raf-1flox/flox) and a complete set of littermate controls (RIPCre+/+:Raf-1wt/wt). RIPCre+/+:Raf-1flox/flox mice were viable- and no effects on weight gain were observed. RIPCre+/+:Raf-1flox/flox mice had increased fasting blood glucose levels and impaired glucose tolerance but normal insulin tolerance compared to littermate controls. Insulin secretion in vivo and in isolated islets was markedly impaired- but there was no apparent effect on the exocytosis machinery. However- islet insulin protein and insulin 2 mRNA- but not insulin 1 mRNA- were dramatically reduced in Raf-1-knockout mice. Analysis of insulin 2 knockout mice demonstrated that this reduction in mRNA was sufficient to impair in vivo insulin secretion. Our data further indicate that Raf-1 specifically and acutely regulates insulin 2 mRNA via negative action on Foxo1- which has been shown to selectively control the insulin 2 gene. This work provides the first direct evidence that Raf-1 signaling is essential for the regulation of basal insulin transcription and the supply of releasable insulin in vivo.--Alejandro- E. U.- Lim- G. E.- Mehran- A. E.- Hu- X.- Taghizadeh- F.- Pelipeychenko- D.- Baccarini- M.- Johnson- J. D. Pancreatic \{beta\}-cell Raf-1 is required for glucose tolerance- insulin secretion- and insulin 2 transcription.
    NADPH-Oxidase 4 Protects against Kidney Fibrosis during Chronic Renal Injury
    Stellor Nlandu Khodo, Journal of the American Society of Nephrology - 2012
    NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys- with the former being the major renal isoform- but their contribution to renal disease is not well understood. Here- we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type- NOX4-- NOX2-- and NOX4/NOX2-deficient mice. Compared with wild-type mice- NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction- with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1 and vascular endothelial growth factor in obstructed kidneys. Furthermore- NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency- renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCDcl1) cell line increased TGF-\{beta\}1-induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition- NOX4 silencing decreased hypoxia-inducible factor-1 and expression of its target genes in response to hypoxia. In summary- these results demonstrate that the absence of NOX4 promotes kidney fibrosis- independent of NOX2- through enhanced tubular cell apoptosis- decreased microvascularization- and enhanced oxidative stress. Thus- NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.
    Stromal TIMP3 Regulates Liver Lymphocyte Populations and Provides Protection against Th1 T Cell-Driven Autoimmune Hepatitis
    Aditya Murthy, The Journal of Immunology - 2012
    Lymphocyte infiltration into epithelial tissues and proinflammatory cytokine release are key steps in autoimmune disease. Although cell-autonomous roles of lymphocytes are well studied in autoimmunity- much less is understood about the stromal factors that dictate immune cell function. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls systemic cytokine bioavailability and signaling by inhibiting the ectodomain shedding of cytokines and their receptors. The role of TIMP3 in cytokine biology is emerging, however- its contribution to cellular immunology remains unknown. In this study- we show that TIMP3 produced by the hepatic stroma regulates the basal lymphocyte populations in the liver and prevents autoimmune hepatitis. TIMP3 deficiency in mice led to spontaneous accumulation and activation of hepatic CD4+- CD8+- and NKT cells. Treatment with Con A in a model of polyclonal T lymphocyte activation resulted in a greatly enhanced Th1 cytokine response and acute liver failure- which mechanistically depended on TNF signaling. Bone marrow chimeras demonstrated that TIMP3 derived from the stromal rather than hematopoietic compartment provided protection against autoimmunity. Finally- we identified hepatocytes as the major source of Timp3 in a resting liver- whereas significant Timp3 gene transcription was induced by hepatic stellate cells in the inflamed liver. These results uncover metalloproteinase inhibitors as critical stromal factors in regulating cellular immunity during autoimmune hepatitis.
    Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors
    Andrea Mosher, Biology of Reproduction - 2012
    Inflammatory mediators- including prostaglandins- cytokines- and chemokines- are strongly implicated in the mechanism of human labor- though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8)- monocyte chemotactic protein-1 (CCL2)- and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However- this effect was repressed by prostaglandin E2 (PGE2). As PGE2 can activate four distinct PGE2 receptors (EP1- EP2- EP3- and EP4) to elicit various responses- we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP4 antagonist- we show that PGE2 mediates the repression of IL-1beta-induced release of CXCL8- CCL2- and CSF2 via activation of the EP2 and EP4 receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP2 was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells- and immunocytochemistry showed that EP2 protein is abundantly present throughout the cells. Interestingly- PGE2 does not appear to reduce mRNA expression of CXCL8- CCL2- and CSF2. Our results demonstrate that PGE2 can elicit anti-inflammatory responses via activation of the EP2 and EP4 receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy- induction of labor or indeed treatment of preterm labor.
    Functional analysis of arylamine N-acetyltransferase 1 (NAT1) NAT1*10 haplotypes in a complete NATb mRNA construct
    Lori Millner, Carcinogenesis - 2012
    N-acetyltransferase 1 (NAT1) catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1*10 is the most common variant haplotype and is associated with increased risk for numerous cancers. NAT1 is transcribed from a major promoter- NATb- and an alternative promoter- NATa- resulting in messenger RNAs (mRNAs) with distinct 5'-untranslated regions (UTRs). To best mimic in vivo metabolism and the effect of NAT1*10 polymorphisms on polyadenylation usage- pcDNA5/Flp recombination target plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATb- the open reading frame and 888 nucleotides of the 3'-UTR. Following stable transfection of NAT1*4- NAT1*10 and an additional NAT1*10 variant (termed NAT1*10B) into nucleotide excision repair-deficient Chinese hamster ovary cells- N- and O-acetyltransferase activity (in vitro and in situ)- mRNA and protein expression were higher in cells transfected with NAT1*10 and NAT1*10B than in cells transfected with NAT1*4 (P {\textless} 0.05). Consistent with NAT1 expression and activity- cytotoxicity and hypoxanthine phosphoribosyl transferase mutants following 4-aminobiphenyl exposures were higher in NAT1*10 than in NAT1*4 transfected cells. Ribonuclease protection assays showed no difference between NAT1*4 and NAT1*10. However- protection of one probe by NAT1*10B was not observed with NAT1*4 or NAT1*10- suggesting additional mechanisms that regulate NAT1*10B. The higher mutants in cells transfected with NAT1*10 and NAT1*10B are consistent with an increased cancer risk for individuals possessing NAT1*10 haplotypes.
    Arthritogenic Self-Reactive CD4+ T Cells Acquire an FR4hiCD73hi Anergic State in the Presence of Foxp3+ Regulatory T Cells
    Ryan Martinez, The Journal of Immunology - 2012
    Rheumatoid arthritis develops in association with a defect in peripheral CD4+ T cell homeostasis. T cell lymphopenia has also been shown to be a barrier to CD4+ T cell clonal anergy induction. We therefore explored the relationship between clonal anergy induction and the avoidance of autoimmune arthritis by tracking the fate of glucose-6-phosphate isomerase (GPI)-reactive CD4+ T cells in the setting of selective T cell lymphopenia. CD4+ T cell recognition of self-GPI peptide/MHC class II complexes in normal murine hosts did not lead to arthritis and instead caused those T cells to develop a Folate receptor 4hiCD73hi anergic phenotype. In contrast- hosts selectively depleted of polyclonal Foxp3+CD4+ regulatory T cells could not make GPI-specific CD4+ T cells anergic and failed to control arthritis. This suggests that autoimmune arthritis develops in the setting of lymphopenia when Foxp3+CD4+ regulatory T cells are insufficient to functionally inactivate all autoreactive CD4+ T cells that encounter self-Ag.
    Identification of axon-enriched microRNAs localized in growth cones of cortical neurons
    Yukio Sasaki, Developmental Neurobiology - 2013
    There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. A few studies have revealed the presence of microRNAs (miRNAs) in dendrites and axons- which may control local protein synthesis during axon development. However- so far- there has been no attempt to screen for axon-enriched miRNAs and validate their possible localization in growth cones of developing axons from neurons of the central nervous system. In this study- we examined the localization of miRNAs in axons and growth cones in cortical neurons using a “neuron ball� culture method that is suitable to prepare axonal miRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball cultures of mouse cortical neurons were analyzed by quantitative real-time RT-PCR. Among 375 miRNAs that were analyzed- 105 miRNAs were detected in axons- and 6 miRNAs were significantly enriched in axonal fractions compared to cell body fractions. Fluorescent in situ hybridization revealed that two axon-enriched miRNAs- miR-181a-1* and miR-532- localized as distinct granules in distal axons and growth cones. Association of these miRNAs with the RNA-induced silencing complex (RISC) further supported their function to regulate mRNA levels or translation in the brain. These results suggest a mechanism to localize specific miRNAs to distal axons and growth cones- where they could be involved in local mRNA regulation. Our findings provide new insight into the presence of axonal miRNAs and motivate further analysis on their function in local protein synthesis underlying axon guidance. © 2013 Wiley Periodicals- Inc. Develop Neurobiol- 2013
    Members of the Francisella tularensis Phagosomal Transporter Subfamily of Major Facilitator Superfamily Transporters Are Critical for Pathogenesis
    Mark Marohn, Infection and Immunity - 2012
    Francisella tularensis is the causative agent of tularemia. Due to its aerosolizable nature and low infectious dose- F. tularensis is classified as a category A select agent and- therefore- is a priority for vaccine development. Survival and replication in macrophages and other cell types are critical to F. tularensis pathogenesis- and impaired intracellular survival has been linked to a reduction in virulence. The F. tularensis genome is predicted to encode 31 major facilitator superfamily (MFS) transporters- and the nine-member Francisella phagosomal transporter (Fpt) subfamily possesses homology with virulence factors in other intracellular pathogens. We hypothesized that these MFS transporters may play an important role in F. tularensis pathogenesis and serve as good targets for attenuation and vaccine development. Here we show altered intracellular replication kinetics and attenuation of virulence in mice infected with three of the nine Fpt mutant strains compared with wild-type (WT) F. tularensis LVS. The vaccination of mice with these mutant strains was protective against a lethal intraperitoneal challenge. Additionally- we observed pronounced differences in cytokine profiles in the livers of mutant-infected mice- suggesting that alterations in in vivo cytokine responses are a major contributor to the attenuation observed for these mutant strains. These results confirm that this subset of MFS transporters plays an important role in the pathogenesis of F. tularensis and suggest that a focus on the development of attenuated Fpt subfamily MFS transporter mutants is a viable strategy toward the development of an efficacious vaccine.
    Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS- a LOB domain protein regulating shoot-borne root initiation in maize (Zea mays L.)
    Christine Majer, Philosophical Transactions of the Royal Society B: Biological Sciences - 2012
    Rootless concerning crown and seminal roots (Rtcs) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize (Zea mays L.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover- enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally- reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition- comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. Consistently- luciferase reporter assays illustrated the capacity of RTCS- RTCL and ZmARF34 to activate downstream gene expression. Finally- RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments- suggesting a role of these complexes in downstream gene regulation. In summary- the data provide novel insights into the molecular interactions resulting in crown root initiation in maize.
    Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2
    Urszula Liwak, Molecular and Cellular Biology - 2012
    Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2)- which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2-S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2-S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression.
    Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components
    Kyoung-Hee Lee, Journal of Biological Chemistry - 2012
    The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process- their excessive activation is hazardous to hosts, thus- tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However- the mechanism underlying tolerance is not understood. Here- we show that activation of Nod2 by its ligand- muramyl dipeptide (MDP) in the bacterial cell wall- induces rapid degradation of Nod2- which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein- Hsp90- which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation- Hsp90 rapidly dissociates from Nod2- which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore- Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock.
    Expression of PaNAC01- a Picea abies CUP-SHAPED COTYLEDON orthologue- is regulated by polar auxin transport and associated with differentiation of the shoot apical meristem and formation of separated cotyledons
    Emma Larsson, Annals of Botany - 2012
    Background and AimsDuring embryo development in most gymnosperms- the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant- Arabidopsis thaliana- the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes- which belong to the large NAC gene family. The aim of this study was to characterize CUC-like genes in a gymnosperm- and to elucidate their expression during SAM and cotyledon differentiation- and in response to PAT. MethodsSixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant. Key ResultsTwo full-length CUC-like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01- but not PaNAC02- harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore- PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant. ConclusionsThe results show that CUC-like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago- and suggest a conserved function between PaNAC01 and CUC1/CUC2.
    Mechanisms in Decorin Regulation of Vascular Endothelial Growth Factor-Induced Human Trophoblast Migration and Acquisition of Endothelial Phenotype
    Neena Lala, Biology of Reproduction - 2012
    Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor- decorin- a transforming growth factor (TGF)-beta binding- leucine-rich proteoglycan produced by the decidua- negatively regulates EVT proliferation- migration- and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors- including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line- HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK- MEK3/6- and ERK1/2) in EVT cells- and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors- we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells- and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia- a hypoinvasive trophoblast disorder in pregnancy.
    Effects of Auraptene on IGF-1 Stimulated Cell Cycle Progression in the Human Breast Cancer Cell Line- MCF-7
    Prasad Krishnan, International Journal of Breast Cancer - 2012
    Auraptene is being investigated for its chemopreventive effects in many models of cancer including skin- colon- prostate- and breast. Many mechanisms of action including anti-inflammatory- antiproliferative- and antiapoptotic effects are being suggested for the chemopreventive properties of auraptene. We have previously shown in the N-methylnitrosourea induced mammary carcinogenesis model that dietary auraptene (500 ppm) significantly delayed tumor latency. The delay in time to tumor corresponded with a significant reduction in cyclin D1 protein expression in the tumors. Since cyclin D1 is a major regulator of cell cycle- we further studied the effects of auraptene on cell cycle and the genes related to cell cycle in MCF-7 cells. Here we show that auraptene significantly inhibited IGF-1 stimulated S phase of cell cycle in MCF-7 cells and significantly changed the transcription of many genes involved in cell cycle.
    Control of viral latency in neurons by axonal mTOR signaling and the 4E-BP translation repressor
    Mariko Kobayashi, Genes & Development - 2012
    Latent herpes simplex virus-1 (HSV1) genomes in peripheral nerve ganglia periodically reactivate- initiating a gene expression program required for productive replication. Whether molecular cues detected by axons can be relayed to cell bodies and harnessed to regulate latent genome expression in neuronal nuclei is unknown. Using a neuron culture model- we found that inhibiting mTOR- depleting its regulatory subunit raptor- or inducing hypoxia all trigger reactivation. While persistent mTORC1 activation suppressed reactivation- a mutant 4E-BP (eIF4E-binding protein) translational repressor unresponsive to mTORC1 stimulated reactivation. Finally- inhibiting mTOR in axons induced reactivation. Thus- local changes in axonal mTOR signaling that control translation regulate latent HSV1 genomes in a spatially segregated compartment.
    alpha6beta4 Integrin- a Master Regulator of Expression of Integrins in Human Keratinocytes
    Kristina Kligys, Journal of Biological Chemistry - 2012
    Three major laminin and collagen-binding integrins in skin (6\{beta\}4- 3\{beta\}1- and 2\{beta\}1) are involved in keratinocyte adhesion to the dermis and dissemination of skin cells during wound healing and/or tumorigenesis. Knockdown of 6 integrin in keratinocytes not only results in motility defects but also leads to decreased surface expression of the 2- 3- and \{beta\}4 integrin subunits. Whereas 2 integrin mRNA levels are decreased in 6 integrin knockdown cells- 3 and \{beta\}4 integrin mRNAs levels are unaffected. Expression of either 6 or 3 integrin in 6 integrin knockdown cells restores 2 integrin mRNA levels. Moreover- re-expression of 6 integrin increases \{beta\}4 integrin protein at the cell surface- which results in an increase in 3 integrin expression via activation of initiation factor 4E-binding protein 1. Our data indicate that the 6\{beta\}4 integrin is a master regulator of transcription and translation of other integrin subunits and underscore its pivotal role in wound healing and cancer.
    The Type of Dietary Fat Modulates Intestinal Tight Junction Integrity- Gut Permeability- and Hepatic Toll-Like Receptor Expression in a Mouse Model of Alcoholic Liver Disease
    Irina Kirpich, Alcoholism- Clinical and Experimental Research - 2012
    Background Interactions between the gut- immune system- and the liver- as well as the type of fat in the diet- are critical components of alcoholic liver disease (ALD). The goal of the present study was to determine the effects of saturated and unsaturated fat on ethanol-induced gut-liver interactions in a mouse model of ALD. Methods C57BL/6N mice were fed Lieber-DeCarli liquid diets containing EtOH and enriched in unsaturated (USF- corn oil) or saturated fat (SF- MCT:beef tallow). Control mice were pair-fed on an isocaloric basis. Liver injury and steatosis- blood endotoxin levels- intestinal permeability and tight junction (TJ) integrity- as well as hepatic Toll-like receptor (TLR) gene expression were evaluated. Results After 8 weeks of EtOH feeding liver injury and steatosis were observed in USF+EtOH group compared to control and SF+EtOH. Significantly increased intestinal permeability in conjunction with elevated blood endotoxin levels were observed in the ileal segments of the mice fed USF+EtOH. USF diet alone resulted in down-regulation of intestinal TJ protein mRNA expression compared to SF. Importantly- alcohol further suppressed TJ proteins in USF+EtOH but did not affect intestinal TJ in SF+EtOH group. The type of fat in the diet alone did not affect hepatic TLR expression. Compared to control animals- hepatic TLR (TLR 1- 2- 3- 4- 7- 8- 9) mRNA expression was significantly (p{\textless}0.05) increased in USF+EtOH- but not in SF+EtOH group. Notably- TLR5 was the only up-regulated TLR in both SF+EtOH and USF+EtOH groups. Conclusions Dietary fat is an important cofactor in alcohol-associated liver injury. We demonstrate that unsaturated fat (corn oil/linoleic acid) by itself results in dysregulation of intestinal TJ integrity leading to increased gut permeability- and alcohol further exacerbates these alterations. We postulate that elevated blood endotoxin levels in response to unsaturated fat and alcohol in conjunction with up-regulation of hepatic TLRs combine to cause hepatic injury in ALD.
    Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs
    Franceline Juillard, Nucleic Acids Research - 2012
    The Epstein-Barr Virus (EBV) protein EB2 (also called Mta- SM and BMLF1)- is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability- splicing- nuclear export and translation. Using a yeast two-hybrid screen- we have identified three SR proteins- SF2/ASF- 9G8 and SRp20- as cellular partners of EB2. Then- by using siRNA to deplete cells of specific SR proteins- we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA- the human \{beta\}-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here- we show that SRp20 depletion results in an increase in the accumulation of these mRNAs- which correlates with an absence of additive effect of EB2- suggesting that EB2 functions by antagonizing SRp20. Moreover- by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the \{beta\}-globin transcript suggesting that EB2 acts by stabilizing SRp20's labile interactions with the RNA.
    Docosahexaenoic Acid Supplementation Does Not Improve Western Diet-Induced Cardiomyopathy in Rats
    Kimberly Jeckel, PLoS ONE - 2012
    Obesity increases risk for cardiomyopathy in the absence of hypertension- diabetes or ischemia. The fatty acid milieu- modulated by diet- may modify myocardial structure and function- lending partial explanation for the array of cardiomyopathic phenotypy. We sought to identify gross- cellular and ultrastructural myocardial changes associated with Western diet intake- and subsequent modification with docosahexaenoic acid (DHA) supplementation. Wistar and Sprague-Dawley (SD) rats received 1 of 3 diets: control (CON), Western (WES), Western + DHA (WES+DHA). After 12 weeks of treatment- echocardiography was performed and myocardial adiponectin- fatty acids- collagen- area occupied by lipid and myocytes- and ultrastructure were determined. Strain effects included higher serum adiponectin in Wistar rats- and differences in myocardial fatty acid composition. Diet effects were evident in that both WES and WES+DHA feeding were associated with similarly increased left ventricular (LV) diastolic cranial wall thickness (LVWcr/d) and decreased diastolic internal diameter (LVIDd)- compared to CON. Unexpectedly- WES+DHA feeding was associated additionally with increased thickness of the LV cranial wall during systole (LVWcr/s) and the caudal wall during diastole (LVWca/d) compared to CON, this was observed concomitantly with increased serum and myocardial adiponectin. Diastolic dysfunction was present in WES+DHA rats compared to both WES and CON. Myocyte cross sectional area (CSA) was greater in WES compared to CON rats. In both fat-fed groups- transmission electron microscopy (TEM) revealed myofibril degeneration- disorganized mitochondrial cristae- lipid inclusions and vacuolation. In the absence of hypertension and whole body insulin resistance- WES+DHA intake was associated with more global LV thickening and with diastolic dysfunction- compared to WES feeding alone. Myocyte hypertrophy- possibly related to subcellular injury- is an early change that may contribute to gross hypertrophy. Strain differences in adipokines and myocardial fatty acid accretion may underlie heterogeneous data from rodent studies.
    Borrelia burgdorferi Harbors a Transport System Essential for Purine Salvage and Mammalian Infection
    Sunny Jain, Infection and Immunity - 2012
    Borrelia burgdorferi is the tick-borne bacterium that causes the multistage inflammatory disease Lyme disease. B. burgdorferi has a reduced genome and lacks the enzymes required for de novo synthesis of purines for synthesis of RNA and DNA. Therefore- this obligate pathogen is dependent upon the tick vector and mammalian host environments for salvage of purine bases for nucleic acid biosynthesis. This pathway is vital for B. burgdorferi survival throughout its infectious cycle- as key enzymes in the purine salvage pathway are essential for the ability of the spirochete to infect mice and critical for spirochete replication in the tick. The transport of preformed purines into the spirochete is the first step in the purine salvage pathway and may represent a novel therapeutic target and/or means to deliver antispirochete molecules to the pathogen. However- the transport systems critical for purine salvage by B. burgdorferi have yet to be identified. Herein- we demonstrate that the genes bbb22 and bbb23- present on B. burgdorferi's essential plasmid circular plasmid 26 (cp26)- encode key purine transport proteins. BBB22 and/or BBB23 is essential for hypoxanthine transport and contributes to the transport of adenine and guanine. Furthermore- B. burgdorferi lacking bbb22-23 was noninfectious in mice up to a dose of 1 × 107 spirochetes. Together- our data establish that bbb22-23 encode purine permeases critical for B. burgdorferi mammalian infectivity- suggesting that this transport system may serve as a novel antimicrobial target for the treatment of Lyme disease.
    NLRP3 inflammasome induces chemotactic immune cell migration to the CNS in experimental autoimmune encephalomyelitis
    Makoto Inoue, Proceedings of the National Academy of Sciences U.S.A. - 2012
    The NLRP3 inflammasome is a multiprotein complex consisting of three kinds of proteins- NLRP3- ASC- and pro-caspase-1- and plays a role in sensing pathogens and danger signals in the innate immune system. The NLRP3 inflammasome is thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE)- an animal model of multiple sclerosis (MS). However- the mechanism by which the NLRP3 inflammasome induces EAE is not clear. In this study- we found that the NLRP3 inflammasome played a critical role in inducing T-helper cell migration into the CNS. To gain migratory ability- CD4+ T cells need to be primed by NLRP3 inflammasome-sufficient antigen-presenting cells to up-regulate chemotaxis-related proteins- such as osteopontin- CCR2- and CXCR6. In the presence of the NLRP3 inflammasome- dendritic cells and macrophages also induce chemotactic ability and up-regulate chemotaxis-related proteins- such as 4\{beta\}1 integrin- CCL7- CCL8- and CXCL16. On the other hand- reduced Th17 cell population size in immunized Nlrp3-/- and Asc-/- mice is not a determinative factor for their resistance to EAE. As currently applied in clinical interventions of MS- targeting immune cell migration molecules may be an effective approach in treating MS accompanied by NLRP3 inflammasome activation.
    Inducible IL-33 Expression by Mast Cells Is Regulated by a Calcium-Dependent Pathway
    Chia-Lin Hsu, The Journal of Immunology - 2012
    IL-33 is an IL-1 family cytokine that displays dual functions: a cytokine via its receptor- T1/ST2- or a chromatin-binding factor within the nucleus. Functionally- it promotes Th2-associated immunity by enhancing the activation and survival of several cell types. However- the pathways regulating IL-33 expression are still unclear. Although several cells display constitutive expression of IL-33- we showed previously that mast cells expressed low levels of IL-33 constitutively but that IL-33 was induced upon IgE-mediated activation. This was mediated via a calcium-dependent mechanism. In this study- we define the pathway through which this inducible IL-33 is regulated. Importantly- this pathway does not alter expression in cells with high constitutive IL-33 expression- such as epithelial cells or fibroblasts. Our data show that- upstream of calcium- inhibition of PI3K and Sphk activity decreases inducible IL-33 expression to IgE/Ag activation. Additionally- expression of Sphk1 short hairpin RNA prevents upregulation of IL-33 expression. Downstream of calcium- NFAT activity is necessary and sufficient for inducible IL-33 expression. We also demonstrate calcium-dependent transcription from two regions of the IL-33 gene that contain putative NFAT-binding sites- one upstream of exon 1 and one upstream of the start site. Interestingly- we show that blocking other calcium pathways- including inositol triphosphate receptor- or NF-\{kappa\}B inhibits IgE-driven IL-1\{beta\}- another IL-1 family cytokine- but it has no influence on inducible IL-33 expression. In summary- our data demonstrate cell-specific differences in the regulation of IL-33 expression and define a pathway critical for the expression of inducible IL-33 by mast cells upon their activation.
    PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells
    David Finlay, Journal of Experimental Medicine - 2012
    mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems- mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However- PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells- and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters- multiple rate-limiting glycolytic enzymes- cytolytic effector molecules- and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.
    Proliferation of External Globus Pallidus-Subthalamic Nucleus Synapses following Degeneration of Midbrain Dopamine Neurons
    Kai Fan, Journal of Neuroscience - 2012
    The symptoms of Parkinson's disease (PD) are related to changes in the frequency and pattern of activity in the reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN). In idiopathic and experimental PD- the GPe and STN exhibit hypoactivity and hyperactivity- respectively- and abnormal synchronous rhythmic burst firing. Following lesion of midbrain dopamine neurons- abnormal STN activity emerges slowly and intensifies gradually until it stabilizes after 2-3 weeks. Alterations in cellular/network properties may therefore underlie the expression of abnormal firing. Because the GPe powerfully regulates the frequency- pattern- and synchronization of STN activity- electrophysiological- molecular- and anatomical measures of GPe-STN transmission were compared in the STN of control and 6-hydroxydopamine-lesioned rats and mice. Following dopamine depletion: (1) the frequency (but not the amplitude) of mIPSCs increased by [{\textasciitilde}]70\%, (2) the amplitude of evoked IPSCs and isoguvacine-evoked current increased by [{\textasciitilde}]60\% and [{\textasciitilde}]70\%- respectively, (3) mRNA encoding 1- \{beta\}2- and \{gamma\}2 GABAA receptor subunits increased by 15-30\%, (4) the density of postsynaptic gephyrin and \{gamma\}2 subunit coimmunoreactive structures increased by [{\textasciitilde}]40\%- whereas the density of vesicular GABA transporter and bassoon coimmunoreactive axon terminals was unchanged, and (5) the number of ultrastructurally defined synapses per GPe-STN axon terminal doubled with no alteration in terminal/synapse size or target preference. Thus- loss of dopamine leads- through an increase in the number of synaptic connections per GPe-STN axon terminal- to substantial strengthening of the GPe-STN pathway. This adaptation may oppose hyperactivity but could also contribute to abnormal firing patterns in the parkinsonian STN.
    Pellino1 Is Required for Interferon Production by Viral Double-stranded RNA
    Karine Enesa, Journal of Biological Chemistry - 2012
    Viral double-stranded RNA- a ligand for Toll-like Receptor 3 (TLR3) and the cytoplasmic RNA receptors RIG1 and MDA5- activate a signaling network in which the IKK-related protein kinase TBK1 phosphorylates the transcription factor Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1. IRF3 then translocates to the nucleus where it stimulates transcription of the interferon\{beta\} (IFN\{beta\}) gene- but the function of Pellino1 in this pathway is unknown. Here- we report that myeloid cells and embryonic fibroblasts from knock-in mice expressing an E3 ligase-deficient mutant of Pellino1 produce reduced levels of IFN\{beta\} mRNA and secrete much less IFN\{beta\} in response to viral double-stranded RNA because the interaction of IRF3 with the IFN\{beta\} promoter is impaired. These results identify Pellino1 as a novel component of the signal transduction network by which viral double-stranded RNA stimulates IFN\{beta\} gene transcription.
    Estrous cycle-dependent changes of Fas expression in the bovine corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines
    Alice Duncan, Reproductive Biology and Endocrinology : RB&E - 2012
    Background Fas expression and Fas-induced apoptosis are mechanisms attributed to the selective destruction of cells of the corpus luteum (CL) during luteal regression. In certain cell-types- sensitivity to these death-inducing mechanisms is due to the loss or cleavage of keratin-containing intermediate filaments. Specifically- keratin 8/18 (K8/K18) filaments are hypothesized to influence cell death in part by regulating Fas expression at the cell surface. Methods Here- Fas expression on bovine luteal cells was quantified by flow cytometry during the early (Day 5- postovulation) and late stages (Days 16-18- postovulation) of CL function- and the relationship between Fas expression- K8/K18 filament expression and cytokine-induced cell death in vitro was evaluated. Results Both total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89\% vs. 44\% of cells for total Fas, 65\% vs.18\% of cells for cell surface Fas, respectively- P{\textless}0.05- n=6-9 CL/stage). A similar increase in the steady-state concentration of mRNA for Fas- as detected by quantitative real-time polymerase chain reaction- however- was not observed. Transient disruption of K8/K18 filaments in the luteal cells with acrylamide (5 mM)- however- had no effect on the surface expression of Fas (P{\textgreater}0.05- n=4 CL/stage)- despite evidence these conditions increased Fas expression on HepG2 cells (P{\textless}0.05- n= 3 expts). Exposure of the luteal cells to cytokines induced cell death (P{\textless}0.05) as expected- but there was no effect of K8/K18 filament disruption by acrylamide (P{\textgreater}0.05) or stage of CL (P{\textgreater}0.05- n= 4 CL/stage) on this outcome. Conclusion In conclusion- we rejected our null hypothesis that the cell surface expression of Fas does not differ between luteal cells of early and late stage CL. The results also did not support the idea that K8/K18 filaments influence the expression of Fas on the surface of bovine luteal cells. Potential downstream effects of these filaments on death signaling- however- remain a possibility. Importantly- the elevated expression of Fas observed on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s) of Fas in the corpus luteum- particularly during early luteal development.
    Characterization of a PDK1 Homologue from the Moss Physcomitrella patens
    Anna Dittrich, Plant Physiology - 2012
    The serine/threonine protein kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a highly conserved eukaryotic kinase that is a central regulator of many AGC kinase subfamily members. Through its regulation of AGC kinases- PDK1 controls many basic cellular processes- from translation to cell survival. While many of these PDK1-regulated processes are conserved across kingdoms- it is not well understood how PDK1 may have evolved within kingdoms. In order to better understand PDK1 evolution within plants- we have isolated and characterized the PDK1 gene from the moss Physcomitrella patens (PpPDK1)- a nonvascular representative of early land plants. PpPDK1 is similar to other plant PDK1s in that it can functionally complement a yeast PDK1 knockout line. However- unlike PDK1 from other plants- the P. patens PDK1 protein does not bind phospholipids due to a lack of the lipid-binding pleckstrin homology domain- which is used for lipid-mediated regulation of PDK1 activity. Sequence analysis of several PDK1 proteins suggests that lipid regulation of PDK1 may not commonly occur in algae and nonvascular land plants. PpPDK1 can phosphorylate AGC kinase substrates from tomato (Solanum lycopersicum) and P. patens at the predicted PDK1 phosphorylation site- indicating that the PpPDK1 substrate phosphorylation site is conserved with higher plants. We have also identified residues within the PpPDK1 kinase domain that affect kinase activity and show that a mutant with highly reduced kinase activity can still confer cell viability in both yeast and P. patens. These studies lay the foundation for further analysis of the evolution of PDK1 within plants.
    The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease
    Erin Daugherity, Cell Cycle - 2012
    Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death- and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H2O2- superoxide production- and expression of oxidative stress response genes- confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis- hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress- ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore- high fat diet fed Atm-/- mice had significantly less hepatic fibrosis than Atm+/+ or Atm+/- mice fed the same diet. Together- these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis- key features of NAFLD progression.
    Mechanisms underlying the sparing of masticatory versus limb muscle function in an experimental critical illness model
    Sudhakar Aare, Physiological Genomics - 2011
    Acute quadriplegic myopathy (AQM) is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients that is characterized by tetraplegia/generalized weakness of limb and trunk muscles. Masticatory muscles- on the other hand- are typically spared or less affected- yet the mechanisms underlying this striking muscle-specific difference remain unknown. This study aims to evaluate physiological parameters and the gene expression profiles of masticatory and limb muscles exposed to factors suggested to trigger AQM- such as mechanical ventilation- immobilization- neuromuscular blocking agents- corticosteroids (CS)- and sepsis for 5 days by using a unique porcine model mimicking the ICU conditions. Single muscle fiber cross-sectional area and force-generating capacity- i.e.- maximum force normalized to fiber cross-sectional area (specific force)- revealed maintained masseter single muscle fiber cross-sectional area and specific-force after 5 days' exposure to all triggering factors. This is in sharp contrast to observations in limb and trunk muscles- showing a dramatic decline in specific force in response to 5 days' exposure to the triggering factors. Significant differences in gene expression were observed between craniofacial and limb muscles- indicating a highly complex and muscle-specific response involving transcription and growth factors- heat shock proteins- matrix metalloproteinase inhibitor- oxidative stress responsive elements- and sarcomeric proteins underlying the relative sparing of cranial vs. spinal nerve innervated muscles during exposure to the ICU intervention.
    Negative Regulation of Pancreatic and Duodenal Homeobox-1 by Somatostatin Receptor Subtype 5
    Guisheng Zhou, Molecular Endocrinology - 2012
    Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin and its analogs on insulin expression/secretion and islet cell proliferation. We provide biochemical and genetic evidence that SSTR5 exerted its physiological actions via down-regulating pancreatic and duodenal homeobox-1 (PDX-1)- a \{beta\}-cell-specific homeodomain-containing transcription factor. Cotransfection of SSTR5 with PDX-1 resulted in dose-dependent inhibition of PDX-1 expression in human embryonic kidney 293 cells. SSTR5 agonist RPL-1980 inhibited PDX-1 expression and abolished glucagon-like peptide 1-stimulated PDX-1 expression in mouse insulinoma \{beta\}-TC-6 cells. SSTR5 knockdown by short hairpin RNA led to increased PDX-1 expression that was accompanied by enhanced insulin secretion stimulated by high glucose in \{beta\}-TC6 cells and alternated expressions of cell cycle proteins that favor cell proliferation in mouse insulinoma MIN6 cells. Quantitative RT-PCR analysis showed that cotransfected SSTR5 inhibited PDX-1 mRNA expression- whereas knockdown of SSTR5 increased PDX-1 mRNA expression. In addition- we found that cotransfected wild-type SSTR5 increased PDX-1 ubiquitination in human embryonic kidney 293 cells- whereas SSTR5 P335L- a hypofunctional single nucleotide polymorphism of SSTR5- inhibited PDX-1 ubiquitination. SSTR5 knockout resulted in increased expression of PDX-1- insulin- and proliferating cell nuclear antigen in the islets of sstr-/- mice. Immunohistochemistry analysis showed that SSTR5 P335L was associated with elevated expression of PDX-1 in human pancreatic neuroendocrine tumor. Taken together- our studies demonstrated that SSTR5 is a negative regulator for PDX-1 expression and that SSTR5 may mediate the inhibitory effects of somatostatin and its analogs on insulin expression/secretion and cell proliferation via down-regulating PDX-1 at both transcriptional and posttranslational levels.
    MeCP2 Phosphorylation Is Required for Modulating Synaptic Scaling through mGluR5
    Xiaofen Zhong, Journal of Neuroscience - 2012
    MeCP2 (methyl CpG binding protein 2) is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. The functional significance of MeCP2 to the mammalian nervous system is highlighted by the discovery that mutations in the MECP2 gene cause Rett syndrome (RTT)- a devastating neurological disease that shares many features with autism. Synaptic scaling is a form of non-Hebbian homeostatic plasticity that allows neurons to regulate overall excitability in response to changes in network neuronal activity levels. While it is known that neuronal activity can induce phosphorylation of MeCP2 and that MeCP2 can regulate synaptic scaling- the molecular link between MeCP2 phosphorylation and synaptic scaling remains undefined. We show here that MeCP2 phosphorylation is specifically required for bicuculline-induced synaptic scaling down in mouse hippocampal neurons and this phenotype is mediated by mGluR5 (metabotropic glutamate receptor 5). Our results reveal an important function of MeCP2 in regulating neuronal homeostasis and may eventually help us understand how MECP2 mutations cause RTT.
    Trpc1 Ion Channel Modulates Phosphatidylinositol 3-Kinase/Akt Pathway during Myoblast Differentiation and Muscle Regeneration
    Nadege Zanou, The Journal of Biological Chemistry - 2012
    Background: The PI3K/Akt pathway is involved in muscle development and regeneration.- Results: Knocking out Trpc1 channels or inhibiting Ca2+ fluxes decreases PI3K/Akt activation- slows down myoblasts migration and impairs muscle regeneration.- Conclusion: Trpc1-mediated Ca2+ influx enhances PI3K/Akt pathway during muscle regeneration.- Significance: The activity of PI3K/Akt pathway is modulated by intracellular Ca2+.- We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work- we used primary cell cultures and isolated muscles from Trpc1-/- and Trpc1+/+ murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models- we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1-/- muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD- Myf5- and myogenin and of one of their targets- the developmental MHC (MHCd). Consequently- muscle tension was systematically lower in muscles from Trpc1-/- mice. Importantly- the PI3K/Akt/mTOR/p70S6K pathway- which plays a crucial role in muscle growth and regeneration- was down-regulated in regenerating Trpc1-/- muscles. Indeed- phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K- the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1-/- primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca2+ or pretreated with EGTA-AM or wortmannin- suggesting that the entry of Ca2+ through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo- and identify a Ca2+-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.
    Smith-Magenis Syndrome Results in Disruption of CLOCK Gene Transcription and Reveals an Integral Role for RAI1 in the Maintenance of Circadian Rhythmicity
    Stephen Williams, American Journal of Human Genetics - 2012
    Haploinsufficiency of RAI1 results in Smith-Magenis syndrome (SMS)- a disorder characterized by intellectual disability- multiple congenital anomalies- obesity- neurobehavioral abnormalities- and a disrupted circadian sleep-wake pattern. An inverted melatonin rhythm (i.e.- melatonin peaks during the day instead of at night) and associated sleep-phase disturbances in individuals with SMS- as well as a short-period circadian rhythm in mice with a chromosomal deletion of Rai1- support SMS as a circadian-rhythm-dysfunction disorder. However- the molecular cause of the circadian defect in SMS has not been described. The circadian oscillator temporally orchestrates metabolism- physiology- and behavior largely through transcriptional modulation. Data support RAI1 as a transcriptional regulator- but the genes it might regulate are largely unknown. Investigation into the role that RAI1 plays in the regulation of gene transcription and circadian maintenance revealed that RAI1 regulates the transcription of circadian locomotor output cycles kaput (CLOCK)- a key component of the mammalian circadian oscillator that transcriptionally regulates many critical circadian genes. Data further show that haploinsufficiency of RAI1 and Rai1 in SMS fibroblasts and the mouse hypothalamus- respectively- results in the transcriptional dysregulation of the circadian clock and causes altered expression and regulation of multiple circadian genes- including PER2- PER3- CRY1- BMAL1- and others. These data suggest that heterozygous mutation of RAI1 and Rai1 leads to a disrupted circadian rhythm and thus results in an abnormal sleep-wake cycle- which can contribute to an abnormal feeding pattern and dependent cognitive performance. Finally- we conclude that RAI1 is a positive transcriptional regulator of CLOCK- pinpointing a novel and important role for this gene in the circadian oscillator.
    Oncostatin M suppresses oestrogen receptor-alpha expression and is associated with poor outcome in human breast cancer
    Nathan West, Endocrine Related Cancer - 2012
    The most important clinical biomarker for breast cancer management is oestrogen receptor alpha (ER). Tumours that express ER are candidates for endocrine therapy and are biologically less aggressive- while ER-negative tumours are largely treated with conventional chemotherapy and have a poor prognosis. Despite its significance- the mechanisms regulating ER expression are poorly understood. We hypothesised that the inflammatory cytokine oncostatin M (OSM) can downregulate ER expression in breast cancer. Recombinant OSM potently suppressed ER protein and mRNA expression in vitro in a dose- and time-dependent manner in two human ER+ breast cancer cell lines- MCF7 and T47D. This was dependent on the expression of OSM receptor beta (OSMR\{beta\}) and could be blocked by inhibition of the MEKK1/2 mitogen-activated protein kinases. ER loss was also necessary for maximal OSM-induced signal transduction and migratory activity. In vivo- high expression of OSM and OSMR mRNA (determined by RT-PCR) was associated with reduced ER (P{\textless}0.01) and progesterone receptor (P{\textless}0.05) protein levels in a cohort of 70 invasive breast cancers. High OSM and OSMR mRNA expression was also associated with low expression of ESR1 (ER- P{\textless}0.0001) and ER-regulated genes in a previously published breast cancer gene expression dataset (n=321 cases). In the latter cohort- high OSMR expression was associated with shorter recurrence-free and overall survival in univariate (P{\textless}0.0001) and multivariate (P=0.022) analyses. OSM signalling may be a novel factor causing suppression of ER and disease progression in breast cancer.
    Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes
    Deanna Howarth, Alcoholism- Clinical and Experimental Research - 2012
    Background Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis- which are associated with alcoholic liver disease. We investigated whether alcohol affects ER structure- function and UPR activation in hepatocytes in vitro and in vivo. Methods HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days post fertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR- in situ hybridization and Western blotting. Mobility of the major ER chaperone- BIP- was monitored in cells by fluorescence recovery after photobleaching (FRAP). Results VL-17A cells metabolized alcohol- yet only had slight activation of some UPR target genes following ethanol treatment. However- ER fragmentation- crowding and accumulation of unfolded proteins as detected by immunofluorescence and FRAP- demonstrating that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol- however- showed modest ER dilation and several UPR targets were significantly induced. Conclusion Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying alcoholic liver disease.
    Reducing endoplasmic reticulum stress does not improve steatohepatitis in mice fed a methionine- and choline-deficient diet
    Anne Henkel, Gastrointestinal and Liver Physiology - 2012
    Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of nonalcoholic steatohepatitis. The ER stress response is activated in the livers of mice fed a methionine- and choline-deficient (MCD) diet- yet the role of ER stress in the pathogenesis of MCD diet-induced steatohepatitis is unknown. Using chemical chaperones on hepatic steatosis and markers of inflammation and fibrosis in mice fed a MCD diet- we aim to determine the effects of reducing ER stress. C57BL/6J mice were fed a MCD diet with or without the ER chemical chaperones 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) for 2 wk. TUDCA and PBA effectively attenuated the ER stress response in MCD diet-fed mice- as evidenced by reduced protein levels of phosphorylated eukaryotic initiation factor 2 and phosphorylated JNK and suppression of mRNA levels of CCAAT/enhancer binding protein homologous protein- glucose-regulated protein 78 kDa- and X-box binding protein 1. However- PBA and TUDCA did not decrease MCD diet-induced hepatic steatosis. MCD diet-induced hepatic inflammation- as evidenced by increased plasma alanine aminotransferase and induction of hepatic TNF expression- was also not reduced by PBA or TUDCA. PBA and TUDCA did not attenuate MCD diet-induced upregulation of the fibrosis-associated genes tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9. ER chemical chaperones reduce MCD diet-induced ER stress- yet they do not improve MCD diet-induced hepatic steatosis- inflammation- or activation of genes associated with fibrosis. These data suggest that although the ER stress response is activated by the MCD diet- it does not have a primary role in the pathogenesis of MCD diet-induced steatohepatitis.
    p53 Basic C Terminus Regulates p53 Functions through DNA Binding Modulation of Subset of Target Genes
    Pierre-Jacques Hamard, Journal of Biological Chemistry - 2012
    The p53 gene encodes a transcription factor that is composed of several functional domains: the N-terminal transactivation domain- the central sequence-specific DNA binding domain- the tetramerization domain- and the highly basic C-terminal regulatory domain (CTD). The p53 CTD is a nonspecific DNA binding domain that is subject to extensive post-translational modifications. However- the functional significance of the p53 CTD remains unclear. The role of this domain in the regulation of p53 functions is explored by comparing the activity of ectopically expressed wild-type (WT) p53 protein to that of a truncated mutant lacking the 24 terminal amino acids (\{Delta\}24). Using quantitative real time PCR and chromatin Immuno-Precipitation experiments- a p53 CTD deletion is shown to alter the p53-dependent induction of a subset of its target genes due to impaired specific DNA binding. Moreover- p53-induced growth arrest and apoptosis both require an intact p53 CTD. These data indicate that the p53 CTD is a positive regulator of p53 tumor suppressor functions.
    DeltaNp63alpha represses anti-proliferative genes via H2A.Z deposition
    Corrie Gallant-Behm, Genes & Development - 2012
    \{Delta\} Np63 is a member of the p53 family of transcription factors that functions as an oncogene in squamous cell carcinomas (SCCs). Because \{Delta\}Np63 and p53 bind virtually identical DNA sequence motifs- it has been proposed that \{Delta\}Np63 functions as a dominant-negative inhibitor of p53 to promote proliferation and block apoptosis. However- most SCCs concurrently overexpress \{Delta\}Np63 and inactivate p53- suggesting the autonomous action of these oncogenic events. Here we report the discovery of a novel mechanism of transcriptional repression by \{Delta\}Np63 that reconciles these observations. We found that although both proteins bind the same genomic sites- they regulate largely nonoverlapping gene sets. Upon activation- p53 binds all enhancers regardless of \{Delta\}Np63 status but fails to transactivate genes repressed by \{Delta\}Np63. We found that \{Delta\}Np63 associates with the SRCAP chromatin regulatory complex involved in H2A/H2A.Z exchange and mediates H2A.Z deposition at its target loci. Interestingly- knockdown of SRCAP subunits or H2A.Z leads to specific induction of \{Delta\}Np63-repressed genes. We identified SAMD9L as a key anti-proliferative gene repressed by \{Delta\}Np63 and H2A.Z whose depletion suffices to reverse the arrest phenotype caused by \{Delta\}Np63 knockdown. Collectively- these results illuminate a molecular pathway contributing to the autonomous oncogenic effects of \{Delta\}Np63.
    SlrA/SlrR/SinR inhibits motility gene expression upstream of a hypersensitive and hysteric switch at the level of ?D in Bacillus subtilis
    Loralyn Cozy, Molecular Microbiology - 2012
    Exponentially growing Bacillus subtilis cultures are epigenetically differentiated into two subpopulations in which cells are either ON or OFF for σD-dependent gene expression: a pattern suggestive of bistability. The gene encoding σD- sigD- is part of the 31-gene fla/che operon where its location at the 3€² end- 25 kb away from the strong Pfla/che promoter- determines its expression level relative to a threshold. Here we show that addition of a single extra copy of the slrA gene in the chromosome inhibited σD-dependent gene expression. SlrA together with SinR and SlrR reduced sigD transcript by potentiating a distance-dependent decrease in fla/che operon transcript abundance that was not mediated by changes in expression from the Pfla/che promoter. Consistent with acting upstream of σD- SlrA/SinR/SlrR could be bypassed by artificial ectopic expression of sigD that hysteretically maintained for 20 generations by engaging the sigD gene at the native locus. SlrA/SinR/SlrR was also bypassed by increasing fla/che transcription and resulted a hypersensitive output in flagellin expression. Thus- flagellin gene expression demonstrated hypersensitivity and hysteresis and we conclude that σD-dependent gene expression is bistable.
    Transcript and metabolite signature of maize source leaves suggests a link between transitory starch to sucrose balance and the autonomous floral transition
    Viktoriya Coneva, Journal of Experimental Botany - 2012
    Little is known about the nature of floral inductive cues in day-neutral plants that are insensitive to photoperiod variations and- therefore- rely on endogenous signals to initiate reproductive growth. The INDETERMINATE1 (ID1) transcription factor is a key regulator of the transition to flowering in day-neutral maize. The ID1 gene is expressed exclusively in developing leaves- where it controls the production or transmission of leaf-derived florigenic signals. Florigen-producing source leaves were compared with mature leaves of late-flowering id1 plants- and metabolite and gene expression differences associated with the floral transition in maize were observed. While id1 mutants have a similar capacity for photosynthesis to wild-type siblings- id1 source leaves show quantitative differences in carbohydrate allocation prior to the floral transition stage- with a marked increase in sucrose and other soluble sugars- accompanied by a decrease in tricarboxylic acid (TCA) cycle organic acids. Importantly- source leaves of autonomous-flowering maize are typified by a higher transitory starch to sucrose ratio and a transcript profile enriched for sucrose synthesis and starch metabolism-related gene function. Finally- similar changes in transitory starch and sucrose are not observed in teosinte- the tropical progenitor of maize that requires short-day photoperiods to induce flowering. Together- these data define a transcript and metabolite signature associated with the autonomous floral transition in temperate maize leaves.
    Tacaribe Virus Causes Fatal Infection of An Ostensible Reservoir Host- the Jamaican Fruit Bat
    Ann Cogswell-Hawkinson, Journal of Virology - 2012
    Tacaribe virus (TCRV) was first isolated from 11 Artibeus species bats captured in Trinidad in the 1950s during a rabies virus surveillance program. Despite significant effort- no evidence of infection of other mammals- mostly rodents- was found- suggesting that no other vertebrates harbored TCRV. For this reason- it was hypothesized that TCRV was naturally hosted by artibeus bats. This is in stark contrast to other arenaviruses with known hosts- all of which are rodents. To examine this hypothesis- we conducted experimental infections of Jamaican fruit bats (Artibeus jamaicensis) to determine whether they could be persistently infected without substantial pathology. We subcutaneously or intranasally infected bats with TCRV strain TRVL-11573- the only remaining strain of TCRV- and found that low-dose (104 50\% tissue culture infective dose [TCID50]) inoculations resulted in asymptomatic and apathogenic infection and virus clearance- while high-dose (106 TCID50) inoculations caused substantial morbidity and mortality as early as 10 days postinfection. Uninoculated cage mates failed to seroconvert- and viral RNA was not detected in their tissues- suggesting that transmission did not occur. Together- these data suggest that A. jamaicensis bats may not be a reservoir host for TCRV.
    Adenosine A2B Receptor Blockade Slows Growth of Bladder and Breast Tumors
    Caglar Cekic, The Journal of Immunology - 2012
    The accumulation of high levels of adenosine in tumors activates A2A and A2B receptors on immune cells and inhibits their ability to suppress tumor growth. Deletion of adenosine A2A receptors (A2AARs) has been reported to activate antitumor T cells- stimulate dendritic cell (DC) function- and inhibit angiogenesis. In this study- we evaluated the effects of intermittent intratumor injection of a nonselective adenosine receptor antagonist- aminophylline (AMO, theophylline ethylenediamine) and- for the first time to our knowledge- a selective A2BAR antagonist- ATL801. AMO and ATL801 slowed the growth of MB49 bladder and 4T1 breast tumors in syngeneic mice and reduced by 85\% metastasizes of breast cancer cells from mammary fat to lung. Based on experiments with A2AAR-/- or adenosine A2B receptor-/- mice- the effect of AMO injection was unexpectedly attributed to A2BAR and not to A2AAR blockade. AMO and ATL801 significantly increased tumor levels of IFN-\{gamma\} and the IFN-inducible chemokine CXCL10- which is a ligand for CXCR3. This was associated with an increase in activated tumor-infiltrating CXCR3+ T cells and a decrease in endothelial cell precursors within tumors. Tumor growth inhibition by AMO or ATL801 was eliminated in CXCR3-/- mice and RAG1-/- mice that lack mature T cells. In RAG1-/- mice- A2BAR deletion enhanced CD86 expression on CD11b- DCs. Bone marrow chimera experiments demonstrated that CXCR3 and A2BAR expression on bone marrow cells is required for the antitumor effects of AMO. The data suggest that blockade of A2BARs enhances DC activation and CXCR3-dependent antitumor responses.
    MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action
    Elena Caproni, The Journal of Immunology - 2012
    The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG- resiquimod- and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT- only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine- we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines- we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast- resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb- we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.
    Differential Gene Expression Profile in Bovine Blastocysts Resulting from Hyperglycemia Exposure During Early Cleavage Stages
    Gael Cagnone, Biology of Reproduction - 2012
    To understand the compromised survival of embryos derived from assisted reproductive techniques- transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However- how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival- in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage- and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatment's being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling- calcium signaling- and energy metabolism. On a pathophysiological level- higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect- i.e.- emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.
    Diets high in mono-and polyunsaturated fatty acids decrease fatty acid synthase protein levels in adipose tissue but do not alter other markers of adipose function and inflammation in diet-induced obese rats
    Jennifer Enns, Prostaglandins- Leukotrienes and Essential Fatty Acids (PLEFA) - 2014
    This study investigates the effects of monounsaturated and polyunsaturated fatty acids from different fat sources (High Oleic Canola- Canola- Canola-Flaxseed (3:1 blend)- Safflower- or Soybean Oil- or a Lard-based diet) on adipose tissue function and markers of inflammation in Obese Prone rats fed high-fat (55\% energy) diets for 12 weeks. Adipose tissue fatty acid composition reflected the dietary fatty acids profiles. Protein levels of fatty acid synthase- but not mRNA levels- were lower in adipose tissue of all groups compared to the Lard group. Adiponectin and fatty acid receptors GPR41 and GPR43 protein levels were also altered- but other metabolic and inflammatory mediators in adipose tissue and serum were unchanged among groups. Overall- rats fed vegetable oil- or lard-based high-fat diets appear to be largely resistant to major phenotypic changes when the dietary fat composition is altered- providing little support for the importance of specific fatty acid profiles in the context of a high-fat diet.
    Heterozygous Igf1r deletion does not ameliorate pathological features associated with polyglutamine-containing huntingtin fragment
    Miriam Jakubik, Neuroscience Letters - 2014
    The evolutionarily conserved insulin/IGF-1 signaling pathway has pleiotropic effects on various cellular processes. Hypomorphic alleles of the insulin/IGF-1 receptor enhance catabolic processes as well as stress resistance- which ultimately lead to lifespan extension in invertebrates. Moreover- decreased insulin/IGF-1 signaling promotes the maintenance of protein quality control and suppresses the onset of cellular toxicity caused by aggregate-prone proteins. As loss of protein homeostasis is a feature of many sporadic and inherited forms of neurodegenerative disorders- the pharmacological inhibition of the IGF-1 receptor represents a promising potential therapeutic strategy for currently untreatable neurodegenerative disorders. However- additional studies are required to determine whether this approach is suitable to delay pathology in clinically relevant models of neurodegenerative disorders. Here we show that- in a mouse model of Huntington's disease- heterozygous knockout of the Igf1r does not prevent premature lethality of mice expressing a short fragment of the mutant human huntingtin. Moreover- Igf1r haploinsufficiency does not suppress the formation of huntingtin-containing aggregates. Thus- partial genetic manipulation of the insulin/IGF-1 signaling pathways does not seem sufficient to counteract protein toxicity and extend animal survival.
    Identification of heat-shock protein 10 (Hsp10) within the equine embryo- endometrium and maternal peripheral blood mononuclear cells
    J. Hatzel, Theriogenology - 2014
    Early Pregnancy Factor (EPF) has been identified as a 10 kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice- sheep- pigs- horses- cows and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and RT-qPCR. Additionally- analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both pregnant and non-pregnant mares was evaluated to determine expression levels of HSP10. Embryos were collected from Quarter Horse mares by uterine lavage at either 8 or 25 days after ovulation. Collection and separation of PBMCs occurred on day 8 for both pregnant and non-pregnant mares. Immunohistochemistry revealed cytoplasmic localization of HSP10 throughout the single layer of ectodermal cells forming the trophoblast in day 8 embryos. Day 25 embryos demonstrated intense localization focally along the apical boarder of ectodermal cells forming the trophoblast layer of the developing chorion. There was no nuclear staining in either embryonic population. RT-qPCR detected the presence of mRNA for HSP10 in both 8 d and 25 d equine embryos. Day 25 embryos exhibited an elevated degree of expression (P = 0.006) compared to the 8 d embryos for HSP10. Endometrial samples did not display any significant difference in degree of expression for HSP10 (P = 0.10). Finally- PBMCs from pregnant mares demonstrated elevated (P = 0.03) expression of HSP10 compared to the non-pregnant mares on day 8 of the estrous cycle. This study confirmed the presence of HSP10 protein and mRNA expression of HSP10 in equine embryos at two maturation stages. Additionally- the presence of increased gene expression within PBMCs of pregnant mares suggests communication- possibly leading to necessary immunomodulatory effects between the embryo and mare.
    Transcriptome analysis of bovine granulosa cells of preovulatory follicles harvested 30- 60- 90- and 120 days postpartum
    V. Golini, Theriogenology - 2014
    The nutritional requirements of high-producing dairy cows increase rapidly in the weeks postpartum due to elevated milk production- and most cows enter a state of negative energy balance (NEB). Cows in this condition may preferentially divert nutrients away from reproduction- thereby experiencing a period of anovulatory anestrus- delayed ovulation of large follicles- and impaired fertility. To better understand the changes occurring in these large follicles as a function of time postpartum- granulosa cells from preovulatory follicles were collected at different times after calving: 30 days- 60 days- 90 days- and 120 days from a minimum of six animals at each time point. Three subjects for each period were selected for the analysis of the transcriptome- which was performed using a global bovine oligoarray microarray to identify differences in gene expression and cellular functions that occur in the follicular microenvironment during the progressive recovery from NEB in dairy cows. The time point 120 days was chosen as a reference period- as cows are then fully recovered from NEB. Data clustering indicated that the most different period from 120 days was 60 days with several 100 genes deregulated at that time. The principal cellular functions affected were cell proliferation- which was reduced- and deregulation of steroidogenesis- indicating immaturity of the follicles from the 60-day period relative to 120 days. One of the most interesting downregulated pathways was p38 mitogen-activated protein kinase- which is sensitive to the amount of retinoic acid or vitamin A- suggesting a possible treatment to improve postpartum fertility.
    Reverse Transcription PCR (RT-PCR)
    Quantitative RT-qPCR
    The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification - viruses-09-00207-v2.pdf
    Leanne M. Bilawchuk, viruses - 2017
    Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition ( p=0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p=0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition;p=0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses.
    Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method
    Irit Davidson, Avian Pathology - 2016
    The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, dropping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA was now upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the env and the NS5 genes, increasing the sensitivity by 10-100 fold compared to the previously existing assays. Based on the recent complete sequences of 5 TMEV isolates we now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009-2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV d