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  • PCR & qPCR
    Real-Time Quantitative PCR
    SYBR Green Detection
    Jean Mawuena Branttie, - 2021
    Paramyxoviruses, pneumoviruses, and other non-segmented negative sense (NNS) RNA viruses have historically been of public health concern. Although their genomes are typically small (up to 19kbs) they are able to inflict large-scale detrimental pathologies on host cells. Human metapneumovirus (HMPV) is a widespread pathogen and is a NNS RNA virus. HMPV results respiratory tract infections and is particularly dangerous for preterm infants, the elderly, and immunocompromised individuals. Other viruses within the NNS RNA virus order include the deadly Ebola, Hendra, and Nipah viruses (EBOV, HeV, and NiV), as well as the re-emerging measles virus (MeV). Despite their public impact, there are currently very limited available FDA-approved therapeutics and antivirals against NNS RNA viruses. During the infectious cycle, viral surface glycoproteins play critical roles in establishing infection. For most NNS RNA viruses, the attachment protein is important for the tethering of a viral membrane to host cells, while the fusion protein is responsible for the membrane merger of the virus and host. The fusion protein of paramyxo-and pneumovirus proteins are class I proteins that are folded into trimers, must be proteolytically cleaved to be functional, and are held in a metastable prefusion conformation until the signal for fusion occurs. Upon being signaled, the fusion protein undergoes dramatic essentially irreversible conformational changes for membrane mixing. Because of its important role in starting infection, F has garnered interest as a potentially powerful target against infection. For paramyxoviruses, the ectodomain regions of F have been wellstudied; however, the hydrophobic nature of the transmembrane domain (TMD) of the protein has resulted in difficulties in crystallization. To address this, several biochemical assays have been utilized to address the function of the TMDs of paramyxo-and pneumovirus fusion proteins. Although initially thought to be solely a membrane anchor, the transmembrane domains of several viruses have been shown to be important for the functionality of fusion proteins. For some paramyxoviruses, replacement of the proteinaceous TMD resulted in the premature triggering. Further studies showed that the TMDs of paramyxoviruses and several other viral F proteins exist in isolation as trimers, and these trimeric associations in turn drive trimeric associations of the full protein. Studies of the HeV F TMD in isolation identified a leucine/isoleucine (L/I) zipper as an important motif for TMD-TMD trimerization. Mutations to this L/I zipper motif in the context of the full protein resulted in reduced surface expression, and a loss of functionality. The L/I zipper was found to be present in 140 paramyxo- and pneumovirus fusion protein TMDs. This work examines whether the importance of the wh iimporether L/Izipper in the context of another paramyxvovirus. Weused the model system, PIV5 F to dissect the role of the TMD L/I zipper fusogenic activity. We found that the (L/I) zipper plays important roles in in expression and functionality of the PIV5 F protein, but not surface expression of the protein. Following membrane merging, a series of events occur that facilitate the release of viral contents into the host cell. The NNS RNA carried by the virus into the cell is used as a template for viral replication and transcription; two important steps in generation of viral progeny. In the life cycle of NNS viruses, viral proteins assume multi-functional roles to optimize their replication and spread. One of the key players during the course of infection is the matrix protein (M). The matrix protein has been identified as a master regulator of viral infection with most studies focusing on its roles in late-stage infection, during assembly and budding of viral progeny. The matrix proteins of many enveloped viruses have been shown to associate in high order oligomers to form a gridlike array underneath the plasma membrane, where they can induce membrane curvature to allow for the budding of viral particles. Not surprisingly, the absence of M in some NNS RNA viruses results in a significant viral titer decrease. Interestingly, some recent studies show that the matrix protein has other critical roles in viral infection such as immune modulation and host cell translation antagonism. One of these newly uncovered roles for viral matrix proteins involves the regulation of viral RNA synthesis. Studies with EBOV and MeV demonstrate that the matrix protein is involved in early infection events, as inhibits viral replication. To study the roles of the HMPV M protein in early infection, we performed a spatiotemporal analysis of M in HMPV-infected cells. We noted the presence of HMPV M within the nucleus during early infection. Our knockdown studies of HMPV M indicate that HMPV M is a positive regulator of viral replication and transcription, as in its absence, the rates of mRNA and viral genomic RNA synthesis are dramatically reduced. Additionally, within the NNS RNA virus order, HMPV M is the only matrix protein found to bind calcium. We created alanine mutants to the calcium coordinating residues of HMPV M and found that these residues were important in properly folding the protein. Together, these findings contribute to our understanding of the mechanisms of NNS RNA viral infection
    Polycomb-group recruitment to a Drosophila target gene is the default state that is inhibited by a transcriptional activator
    Elnaz Ghotbi, Science Advances - 2021
    Polycomb-group (PcG) proteins are epigenetic regulators that maintain the transcriptional repression of target genes following their initial repression by transcription factors. PcG target genes are repressed in some cells, but active in others. Therefore, a mechanism must exist by which PcG proteins distinguish between the repressed and active states and only assemble repressive chromatin environments at target genes that are repressed. Here, we present experimental evidence that the repressed state of a Drosophila PcG target gene, giant (gt), is not identified by the presence of a repressor. Rather, de novo establishment of PcG-mediated silencing at gt is the default state that is prevented by the presence of an activator or coactivator, which may inhibit the catalytic activity of Polycomb-repressive complex 2 (PRC2).
    SIRT4 is an early regulatorof branched-chain amino acid catabolismthat promotes adipogenesis
    Elma Zaganjor, Cell Reports - 2021
    Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytescapable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolicdrivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumptionof numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and pro-motes peroxisome proliferator-activated receptor gamma (PPARg), a key regulator of adipogenesis. In earlyadipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcro-tonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxyl-ation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adi-pose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes,suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. Insummary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolicevents that stimulate transcriptional reprogramming.
    Novel Role of Tieg1 in Muscle Metabolism and Mitochondrial Oxidative Capacities
    Malek Kammoun, HAL - 2021
    Aim: Tieg1 is involved in multiple signaling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. Methods: We have utilized Tieg1 KO mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and EDL muscles. RNA sequencing, immunoblotting, TEM, MRI, NMR, histochemical and mitochondrial function assays were performed. Results: Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of SDH staining and a decrease in COX enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in citrate synthase and respiratory chain complex activities were identified in KO soleus. 1H-NMR spectra revealed no significant metabolic difference between WT and KO muscles. However, 31P spectra revealed a significant decrease in phosphocreatine and ATP. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. Conclusion: Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.
    Cardiomyocytes Derived from Induced Pluripotent Stem Cells as a Disease Model for Propionic Acidemia
    Esmeralda Alonso-Barroso, International Journal of Molecular Sciences - 2021
    Propionic acidemia (PA), one of the most frequent life-threatening organic acidemias, is caused by mutations in either the PCCA or PCCB genes encoding both subunits of the mitochondrial propionyl-CoA carboxylase (PCC) enzyme. Cardiac alterations (hypertrophy, dilated cardiomyopathy, long QT) are one of the major causes of mortality in patients surviving the neonatal period. To overcome limitations of current cellular models of PA, we generated induced pluripotent stem cells (iPSCs) from a PA patient with defects in the PCCA gene, and successfully differentiated them into cardiomyocytes. PCCA iPSC-derived cardiomyocytes exhibited reduced oxygen consumption, an accumulation of residual bodies and lipid droplets, and increased ribosomal biogenesis. Furthermore, we found increased protein levels of HERP, GRP78, GRP75, SIG-1R and MFN2, suggesting endoplasmic reticulum stress and calcium perturbations in these cells. We also analyzed a series of heart-enriched miRNAs previously found deregulated in the heart tissue of a PA murine model and confirmed their altered expression. Our novel results show that PA iPSC-cardiomyocytes represent a promising model for investigating the pathological mechanisms underlying PA cardiomyopathies, also serving as an ex vivo platform for therapeutic evaluation.
    An ionising radiation-induced specic transcriptional signature of inammationassociated genes in human whole blood: a pilot study
    Lourdes Cruz-Garcia, Research Square - 2021
    This communication reports the identication of a new panel of transcriptional changes in inammationassociated genes observed in response to ionising radiation received by radiotherapy patients. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 hours post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Statistical analysis with BRB Array Tools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specic responses in support of personalised approaches to radiation medicine.
    SIRT7 deficiency suppresses inflammation, induces EndoMT, and increases vascular permeability in primary pulmonary endothelial cells
    Anne E. Wyman, Scientific Reports - 2020
    Acute lung injury (ALI), a common condition in critically ill patients, has limited treatments and high mortality. Aging is a risk factor for ALI. Sirtuins (SIRTs), central regulators of the aging process, decrease during normal aging and in aging-related diseases. We recently showed decreased SIRT7 expression in lung tissues and fibroblasts from patients with pulmonary fibrosis compared to controls. To gain insight into aging-related mechanisms in ALI, we investigated the effects of SIRT7 depletion on lipopolysaccharide (LPS)-induced inflammatory responses and endothelial barrier permeability in human primary pulmonary endothelial cells. Silencing SIRT7 in pulmonary artery or microvascular endothelial cells attenuated LPS-induced increases in ICAM1, VCAM1, IL8, and IL6 and induced endomesenchymal transition (EndoMT) with decreases in VE-Cadherin and PECAM1 and increases in collagen, alpha-smooth muscle actin, TGFβ receptor 1, and the transcription factor Snail. Loss of endothelial adhesion molecules was accompanied by increased F-actin stress fibers and increased endothelial barrier permeability. Together, these results show that an aging phenotype induced by SIRT7 deficiency promotes EndoMT with impaired inflammatory responses and dysfunction of the lung vascular barrier.
    CTCF loss mediates unique DNA hypermethylation landscapes in human cancers
    Nathan A. Damaschke, Clinical Epigenetics - 2020
    Background The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. Results Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. Conclusion These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.
    Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli
    Fabiola Puértolas-Balint, Journal of Antimicrobial Chemotherapy - 2020
    Objectives To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Methods Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Results Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Conclusions Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.
    Microbial community similarity and dissimilarity inside and across full-scale activated sludge processes for simultaneous nitrification and denitrification
    Jianfeng Wen, Water Science and Technology - 2020
    Simultaneous nitrification and denitrification under low dissolved oxygen conditions is an energy-saving modification of the activated sludge process to achieve efficient nitrogen removal. Geographically distinct full-scale treatment plants are excellent platforms to address the links of microbial community with operating parameters. Mixed liquor samples were collected from a sequencing batch reactor plant, oxidation ditch plant, and step-feed activated sludge plant. Next-Generation Sequencing of the samples showed that the microbial communities were similar at the phylum level among the plants, being dominated by Proteobacteria. Microbial composition of functional groups was similar between the react fill and react phases of the sequencing batch reactors, among four sequencing batch reactors, and among four oxidation ditches. Nitrospira was the only identified genus of autotropic nitrifying bacteria with a relative abundance of 2.2–2.5% in the oxidation ditches and 0.4–0.7% at the other plants. Heterotrophic nitrifying–aerobic denitrifying bacteria were dominated by Dechloromonas with a relative abundance of 0.4–1.0%. Microbial community composition and nitrogen removal mechanisms were related to overall level and local zonation of dissolved oxygen, mixed liquor suspended solids concentration, nitrogen and organic loadings, and solids retention time. Low dissolved oxygen and low organic and nitrogen loadings favored growth of Nitrospira.
    Sphingomyelin-Based Nanosystems (SNs) for the Development of Anticancer miRNA Therapeutics
    Surasa Nagachinta, Pharmaceutics - 2020
    Gene replacement therapy with oncosuppressor microRNAs (miRNAs) is a promising alternative to interfere with cancer progression. However, miRNAs are highly inefficient in a biological environment, hampering a successful translation to clinics. Nanotechnology can tackle this drawback by providing delivery systems able to efficiently deliver them to cancer cells. Thus, the objective of this work was to develop biocompatible nanosystems based on sphingomyelin (SM) for the intracellular delivery of miRNAs to colorectal cancer cells. We pursued two different approaches to select the most appropriate composition for miRNA delivery. On the one hand, we prepared sphingomyelin-based nanosystems (SNs) that incorporate the cationic lipid stearylamine (ST) to support the association of miRNA by the establishment of electrostatic interactions (SNs–ST). On the other hand, the cationic surfactant (DOTAP) was used to preform lipidic complexes with miRNA (Lpx), which were further encapsulated into SNs (SNs-Lpx). Restitution of miRNA145 levels after transfection with SNs-Lpx was related to the strongest anticancer effect in terms of tumor proliferation, colony forming, and migration capacity assays. Altogether, our results suggest that SNs have the potential for miRNA delivery to develop innovative anticancer therapies.
    Inhibition of PIM2 in liver cancer decreases tumor cell proliferation in vitro and in vivo primarily through the modulation of cell cycle progression
    Pia Kronschnabl, International Journal of Oncology - 2020
    Liver cancer is the fourth leading cause of cancer‑related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2‑specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST‑1 assays and spheroid assays), 3D‑colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT‑qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh‑7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)‑complexed siRNA. The knockdown of PIM2 resulted in potent anti‑proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh‑7 cells. In vivo, the therapeutic nanoparticle‑mediated delivery of PIM2 siRNA led to profound anti‑tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.
    Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter
    Nohemí Carreras-Villaseñor, Springer Link - 2020
    Background Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. Results Here we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and to a lesser extent to that of Neurospora crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. Conclusions A new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been identified and tested in T. atroviride to express PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.
    The Cutaneous Inflammatory Response to Thermal Burn Injury in a Murine Model
    Zabeen Lateef, International Journal of Molecular Sciences - 2019
    Many burn interventions aim to target the inflammatory response as a means of enhancing healing or limiting hypertrophic scarring. Murine models of human burns have been developed, but the inflammatory response to injury in these models has not been well defined. The aim of this study was to profile inflammatory cell populations and gene expression relative to healing and scarring in a murine model of thermal burns. Cutaneous injuries were created on the dorsal region of C57Bl/6 mice using a heated metal rod. Animals were euthanized at selected time points over ten weeks, with the lesions evaluated using macroscopic measurements, histology, immunofluorescent histochemistry and quantitative PCR. The burn method generated a reproducible, partial-thickness injury that healed within two weeks through both contraction and re-epithelialization, in a manner similar to human burns. The injury caused an immediate increase in pro-inflammatory cytokine and chemokine expression, coinciding with an influx of neutrophils, and the disappearance of Langerhans cells and mast cells. This preceded an influx of dendritic cells and macrophages, a quarter of which displayed an inflammatory (M1) phenotype, with both populations peaking at closure. As with human burns, the residual scar increased in size, epidermal and dermal thickness, and mast cell numbers over 10 weeks, but abnormal collagen I-collagen III ratios, fibre organization and macrophage populations resolved 3–4 weeks after closure. Characterisation of the inflammatory response in this promising murine burn model will assist future studies of burn complications and aid in the preclinical testing of new anti-inflammatory and anti-scarring therapies.
    Fructose-1,6-bisphosphate prevents pulmonary fibrosis by regulating extracellular matrix deposition and inducing phenotype reversal of lung myofibroblasts
    Henrique Bregolin Dias, PLOS ONE - 2019
    Pulmonary fibrosis (PF) is the result of chronic injury where fibroblasts become activated and secrete large amounts of extracellular matrix (ECM), leading to impaired fibroblasts degradation followed by stiffness and loss of lung function. Fructose-1,6-bisphosphate (FBP), an intermediate of glycolytic pathway, decreases PF development, but the underlying mechanism is unknown. To address this issue, PF was induced in vivo using a mouse model, and pulmonary fibroblasts were isolated from healthy and fibrotic animals. In PF model mice, lung function was improved by FBP as revealed by reduced collagen deposition and downregulation of ECM gene expression such as collagens and fibronectin. Fibrotic lung fibroblasts (FLF) treated with FBP for 3 days in vitro showed decreased proliferation, contraction, and migration, which are characteristic of myofibroblast to fibroblast phenotype reversal. ECM-related genes and proteins such as collagens, fibronectin and α-smooth muscle actin, were also downregulated in FBP-treated FLF. Moreover, matrix metalloproteinase (MMP) 1, responsible for ECM degradation, was produced only in fibroblasts obtained from healthy lungs (HLF) and FBP did not alter its expression. On the other hand, tissue inhibitor of metalloproteinase (TIMP)-1, a MMP1 inhibitor, and MMP2, related to fibroblast tissue-invasion, were predominantly produced by FLF and FBP was able to downregulate its expression. These results demonstrate that FBP may prevent bleomycin-induced PF development through reduced expression of collagen and other ECM components mediated by a reduced TIMP-1 and MMP2 expression.
    Glucocorticoid receptor modulation decreases ER-positive breast cancer cell proliferation and suppresses wild-type and mutant ER chromatin association
    Eva Tonsing-Carter, Breast Cancer Research - 2019
    Background: Non-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients. Methods: In vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers. Results: We found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth. Conclusion: These studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression.
    THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress Analysis
    Zaemma Khan, Plants - 2019
    To elucidate dynamic developmental processes in plants, live tissues and organs must be visualised frequently and for extended periods. The development of roots is studied at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualisation and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualisation through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 h. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system’s capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.
    The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2
    Astrid Skjesol, Plos Pathogens - 2019
    Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
    α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron Overload
    Giuseppina Camiolo, International Journal of Molecular Sciences - 2019
    Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma-optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.
    Influence of the concentration of dietary digestible calcium on growth performance, bone mineralization, plasma calcium, and abundance of genes involved in intestinal absorption of calcium in pigs from 11 to 22 kg fed diets with different concentrations o
    L. Vanessa Lagos, Journal of Animal Science and Biotechnology - 2019
    A 21-day experiment was conducted to test the hypothesis that Ca requirements to maximize growth performance expressed as the standardized total tract digestible (STTD) Ca to STTD P ratio is less than 1.40:1. The second hypothesis was that increasing dietary Ca increases plasma Ca concentration and downregulates abundance of genes related to Ca absorption (TRPV6, S100G, and ATP2B1) in the duodenum, and tight junction proteins (OCLN, CLDN1, and ZO1) in the duodenum and ileum. Methods Twenty corn-soybean meal diets were formulated using a 4 × 5 factorial design with diets containing 0.16%, 0.33%, 0.42%, or 0.50% STTD P, and 0.14%, 0.29%, 0.44%, 0.59%, or 0.74% STTD Ca. Six hundred and forty pigs (initial weight: 11.1 ± 1.4 kg) were allotted to 20 diets and 5 blocks in a randomized complete block design. On day 21, weights of pigs and feed left in feeders were recorded and blood, duodenal tissue, ileal mucosa, and the right femur were collected from 1 pig per pen. Abundance of mRNA was determined in duodenal and ileal tissue via quantitative RT-PCR. Data were analyzed using a response surface model. Results The predicted maximum ADG (614 g), G:F (0.65), and bone ash (11.68 g) was obtained at STTD Ca:STTD P ratios of 1.39:1, 1.25:1, and 1.66:1, respectively, when STTD P was provided at the requirement (0.33%). If dietary STTD P was below the requirement, increasing dietary Ca resulted in reduced (P < 0.05) ADG and G:F. However, if dietary STTD P was above the requirement, negative effects (P < 0.05) on ADG and G:F of increasing STTD Ca were observed only if dietary STTD Ca exceeded 0.6%. Plasma Ca concentration was positively affected by STTD Ca over the range studied (quadratic, P < 0.01) and negatively affected by increasing STTD P (linear, P < 0.01). There was a linear negative effect (P < 0.05) of STTD Ca on the abundance of S100G, TRPV6, OCLN, and ZO1 in duodenum, and CLDN and ZO1 in ileum. Conclusions The STTD Ca:STTD P ratio needed to maximize growth performance of 11- to 25-kg pigs is less than 1.40:1, if P is at the estimated requirement. Increasing dietary Ca reduces transcellular absorption of Ca and increases paracellular absorption of Ca.
    Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas
    Dritan Liko, Cell Death and Differentiation - 2019
    RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.
    Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes
    Zhou Fang, Cell Reports - 2019
    Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct β cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.
    No evidence that gut microbiota impose a net cost on their butterfly host
    Alison Ravenscraft, Molecular Ecology - 2019
    Gut microbes are believed to play a critical role in most animal life, yet fitness effects and cost–benefit trade‐offs incurred by the host are poorly understood. Unlike most hosts studied to date, butterflies largely acquire their nutrients from larval feeding, leaving relatively little opportunity for nutritive contributions by the adult's microbiota. This provides an opportunity to measure whether hosting gut microbiota comes at a net nutritional price. Because host and bacteria may compete for sugars, we hypothesized that gut flora would be nutritionally neutral to adult butterflies with plentiful food, but detrimental to semistarved hosts, especially when at high density. We held field‐caught adult Speyeria mormonia under abundant or restricted food conditions. Because antibiotic treatments did not generate consistent variation in their gut microbiota, we used interindividual variability in bacterial loads and operational taxonomic unit abundances to examine correlations between host fitness and the abdominal microbiota present upon natural death. We detected strikingly few relationships between microbial flora and host fitness. Neither total bacterial load nor the abundances of dominant bacterial taxa were related to butterfly fecundity, egg mass or egg chemical content. Increased abundance of a Commensalibacter species did correlate with longer host life span, while increased abundance of a Rhodococcus species correlated with shorter life span. Contrary to our expectations, these relationships were unchanged by food availability to the host and were unrelated to reproductive output. Our results suggest the butterfly microbiota comprises parasitic, commensal and beneficial taxa that together do not impose a net reproductive cost, even under caloric stress.
    Altered Gene Response to Aflatoxin B1 in the Spleens of Susceptible and Resistant Turkeys
    Kent M. Reed, Toxins - 2019
    Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1 exposure
    Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteria
    Nicolas Salcedo-Porras, PLOS ONE - 2019
    The innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosoma cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the “missing” elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions.
    TCO, a Putative Transcriptional Regulator in Arabidopsis, Is a Target of the Protein Kinase CK2
    Laina M. Weinman, International Journal of Molecular Sciences - 2019
    As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.
    Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathways
    Oscar E. Juarez, PLOS ONE - 2019
    White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptorsignaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females’ WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.
    Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice
    Andrew Wang, PNAS - 2019
    Secondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
    Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant women
    Nsoh Godwin Anabire, PLOS ONE - 2019
    Background The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, it is not known if this co-infection could adversely influence clinical and immunological responses. This study investigated these interactions in pregnant women reporting to antenatal clinics in Ghana. Methods Clinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia, and hepatitis B viremia) and cytokine profiles were assayed and compared across four categories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria+CHB group). Results Women with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM = 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin [P<0.001 for all comparisons]. Similarly, the Malaria+CHB group had significantly elevated pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 [P<0.05 for all comparisons]. In women with Malaria+CHB, correlation analysis showed significant negative association of the pro-inflammatory cytokines responses with malaria parasitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394) and IL-12 (P = 0.011; r = -0.49)]. On the other hand, the pro-inflammatory cytokine levels positively correlated with HBV viremia [TNF-α (P = 0.004; r = 0.549), IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r = 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P = 0.008; r = 0.512) and IL-12 (P<0.001; r = 0.655)]. Also, for women in the Malaria+CHB group, parasitemia was observed to diminish HBV viremia [P = 0.003, r = -0.489]. Conclusion Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine responses and increase susceptibility to liver injury among pregnant women in endemic settings.
    Selection for novel metabolic capabilities in Salmonella enterica
    Omar Warsi, Evolution International Journal of Evolution - 2019
    Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.
    Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes
    Pamela Calvo, Journal of Plant Interactions - 2019
    Plant growth-promoting rhizobacteria (PGPR) enhance plant growth under the influence of multigenic processes, including nitrate (NO−3) and ammonium (NH+4) uptake genes, which could potentially explain the improvement in plant nutrition and plant growth promotion. Studies on the effects of PGPR inoculation on regulation of NO−3 and NH+4 plant uptake genes and nutrient accumulation using soil or soil-like substrates are limited. Here, we tested the hypothesis that the application of PGPR Bacillus mixtures increases overall plant growth, nutrient uptake and the transcript levels of nitrate and ammonium uptake genes in Arabidopsis thaliana. All three PGPR mixtures tested in this study significantly increased plant shoot fresh weight, root fresh, chlorophyll content, nutrient uptake and plant diameter. The transcript levels of five nitrate and four ammonium uptake genes were significantly higher in PGPR-treated plants compared to untreated plants. These results demonstrate that plant growth promotion and enhanced nutrient uptake by select PGPR mixtures.
    The effect of maxillary sinus antrostomy size on the sinus microbiome
    Alexander S. Kim BSE, Allergy and Rhinology - 2019
    Background The optimal maxillary antrostomy size to surgically treat sinusitis is not well known. In this study, we examined clinical metrics of disease severity and symptom scores, measured secreted inflammatory markers, and characterized the sinus microbiome to determine if there were significant differences in outcome between different maxillary ostial sizes. Methods Prospective randomized, single‐blinded clinical trial enrolling 12 individuals diagnosed with recurrent acute or chronic rhinosinusitis. Each patient was blinded and randomized to receive minimal maxillary ostial dilation via balloon sinuplasty on 1 side vs a mega‐antrostomy on the contralateral side. Data collected included symptom scores (20‐item Sino‐Nasal Outcome Test [SNOT‐20]), endoscopy, and radiologic Lund‐Mackay scores. During surgery and at their postoperative visit swabs were obtained from each maxillary sinus, and 16S DNA and inflammatory cytokine levels analyzed. The use of each patient as their own control allowed us to minimize confounding variables. Results There was statistically significant improvement in SNOT‐20 symptom scores postoperatively in all patients. There were no significant differences between maxillary ostial size in postoperative endoscopy scores, cytokine profile, or bacterial burden. There were statistically significant differences in relative postoperative abundance of Staphylococcus, Lactococcus, and Cyanobacteria between the mega‐antrostomy and mini‐antrostomy. Conclusions The method used in surgical maxillary antrostomies had no effect on endoscopy scores or cytokine profiles. Microbiome analysis determined significant differences between the different antrostomy sizes in postoperative Staphylococcus, Lactococcus, and Cyanobacteria abundance. The clinical significance of these changes in the sinus microbiome are not known but may be a result of increased access to postoperative sinonasal irrigations.
    An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity Against Colorectal Cancer
    Bastian J. Schmied, Cancers - 2019
    The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E—that reportedly increase the affinity to CD16—with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.
    Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stress
    Ramona Dolling, Heliyon - 2019
    Stress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 μmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 μmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 μmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.
    Epigenetic Changes at the Birc5 Promoter Induced by YM155 in Synovial Sarcoma
    Aleksander Mika, Journal of Clinical Medicine - 2019
    YM155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of survivin (gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is a rare soft-tissue malignancy with high protein expression of survivin. We investigated whether YM155 would be a viable therapeutic option to treat synovial sarcoma. YM155 therapy was applied to human synovial sarcoma cell lines and to a genetically engineered mouse model of synovial sarcoma. We discovered that YM155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM155 by looking at the change of lysine modifications of the histone tails that were within 250 base pairs of the Birc5 promoter. Using chromatin immunoprecipitation (ChIP)-qPCR, we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM155 treatment. H3K27me3 and H3K27ac increased, but the net result was decreased Birc5/survivin expression. Furthermore, the combination of molecular events resulted in caspase 3/7/8 upregulation and death of the sarcoma cells.
    Chemokine Receptor Redundancy and Specificity Are Context Dependent
    Douglas P. Dyer, Immunity - 2019
    Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
    Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice
    Carmen J. Reynolds, The Journal of Steroid Biochemistry and Molecular Biology - 2019
    1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, β-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
    Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cells
    Zejun Zhou, Scientific Reports - 2019
    Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.
    Chronic liver injury drives non‐traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminase
    L. G. Poole, Journal of Thrombosis and Haemostasis - 2019
    Background Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin‐mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit‐deficient (FXIII−/−) mice. Female wild‐type mice, FXIII−/− mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4) twice weekly for 4 weeks or 6 weeks (1 mL kg−1, intraperitoneal). Results Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin‐catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4‐challenged FXIII−/− mice or FibAEK mice as compared with wild‐type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase‐2 (TGM2)‐deficient (TGM2−/−) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2−/− mice did not affect CCl4‐induced liver fibrosis. Conclusions These results indicate that neither traditional fibrin clots, formed by the thrombin–activated FXIII pathway nor atypical TGM2‐crosslinked fibrin(ogen) contribute to experimental CCl4‐induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.
    Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease [version 1; peer review: 1 approved, 1 approved with reservations]
    Serge Dionne, F1000 Research Open for Science - 2019
    Background: Intestinal fibrosis represents a serious complication of inflammatory bowel diseases (IBD), often necessitating surgical resections. Myofibroblasts are primarily responsible for interstitial matrix accumulation in fibrotic diseases. However intestinal myofibroblasts (IMF) remain inadequately characterized. The aim was to examine fibroblast markers and fibrosis-associated gene expression in IMF isolated from resected intestine from IBD and control patients. As well as determining the effect of the fibrogenic cytokine TGFβ. Methods: Intestinal resections were obtained (n =35) from consenting patients undergoing elective surgery (2014-16). Primary cultures of IMF were isolated using DTT and EDTA and cultured. Viability and phenotypic characterization of IMF was carried out by flow cytometry and fluorescence microscopy. IMF (passages 3-8) were treated for 24 hours. Cytokines were quantified in IMF by real time PCR and in supernatants using the human pro-inflammatory cytokine panel Results: All markers and most fibrosis mediators studied were preferentially expressed by IMF compared to mucosal tissue. Metalloproteinases (MMP) 2 and 3, as well as their inhibitor TIMP1, are highly expressed by IMF. They also highly expressed inflammatory mediators, including IL-6, IL-8, CCL2 and PTGS2. Whereas mucosal expression of pro-inflammatory cytokines such as TNFα and IL-17 is increased in IBD, that of fibrosis mediators was not different. Fibrosis-related gene expression in IMF from IBD patients and controls was similar, but IMF from IBD expressed higher levels of several inflammatory genes. IMF from CD and UC had mostly similar expression profiles. TGFβ induced expression of fibrogenic genes αSMA, COL1A1, CTGF, FN1 and LOX. TGFβ-stimulated IMF released increased levels of IL-6, whereas IL-6, IL-8, as well as small amounts of IFN-γ and IL12p70 were produced following stimulation with IL-1β+IL-23. Conclusions: This study extends knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required.
    Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma
    Lemlem Brook, Journal of Cellular Biochemistry - 2019
    Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM‐derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.
    Neuroendocrine Whiplash: Slamming the Breaks on Anabolic-Androgenic Steroids Following Repetitive Mild Traumatic Brain Injury in Rats May Worsen Outcomes
    Jason Tabor, Frontiers in Neurology - 2019
    Sport-related concussion is an increasingly common injury among adolescents, with repetitive mild traumatic brain injuries (RmTBI) being a significant risk factor for long-term neurobiological and psychological consequences. It is not uncommon for younger professional athletes to consume anabolic-androgenic steroids (AAS) in an attempt to enhance their performance, subjecting their hormonally sensitive brains to potential impairment during neurodevelopment. Furthermore, RmTBI produces acute neuroendocrine dysfunction, specifically in the anterior pituitary, disrupting the hypothalamic-pituitary adrenal axis, lowering cortisol secretion that is needed to appropriately respond to injury. Some AAS users exhibit worse symptoms post-RmTBI if they quit their steroid regime. We sought to examine the pathophysiological outcomes associated with the abrupt cessation of the commonly abused AAS, Metandienone (Met) on RmTBI outcomes in rats. Prior to injury, adolescent male rats received either Met or placebo, and exercise. Rats were then administered RmTBIs or sham injuries, followed by steroid and exercise cessation (SEC) or continued treatment. A behavioral battery was conducted to measure outcomes consistent with clinical representations of post-concussion syndrome and chronic AAS exposure, followed by analysis of serum hormone levels, and qRT-PCR for mRNA expression and telomere length. RmTBI increased loss of consciousness and anxiety-like behavior, while also impairing balance and short-term working memory. SEC induced hyperactivity while Met treatment alone increased depressive-like behavior. There were cumulative effects whereby RmTBI and SEC exacerbated anxiety and short-term memory outcomes. mRNA expression in the prefrontal cortex, amygdala, hippocampus, and pituitary were modified in response to Met and SEC. Analysis of telomere length revealed the negative impact of SEC while Met and SEC produced changes in serum levels of testosterone and corticosterone. We identified robust changes in mRNA to serotonergic circuitry, neuroinflammation, and an enhanced stress response. Interestingly, Met treatment promoted glucocorticoid secretion after injury, suggesting that maintained AAS may be more beneficial than abstaining after mTBI.
    MiRNA-27a sensitizes breast cancer cells to treatment with Selective Estrogen Receptor Modulators
    Bojan Ljepoja, The Breast - 2019
    Background MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest. Methods Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment. Results Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression. Conclusion MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.
    Fine-scale spatial and temporal dynamics of kdr haplotypes in Aedes aegypti from Mexico
    Marissa K. Grossman, BMC - 2019
    Background As resistance to insecticides increases in disease vectors, it has become exceedingly important to monitor populations for susceptibility. Most studies of field populations of Aedes aegypti have largely characterized resistance patterns at the spatial scale of the city or country, which may not be completely informative given that insecticide application occurs at the scale of the house or city block. Phenotypic resistance to pyrethroids dominates in Ae. aegypti, and it has been partially explained by mutations in the voltage-gated sodium channel gene. Here, we assess community-level patterns of four knockdown resistance (kdr) haplotypes (C1534/I1016, F1534/I1016, C1534/V1016 and F1534/V1016) in Ae. aegypti in 24 randomly chosen city blocks from a city in Yucatán State, Mexico, during both the dry and wet season and over two years. Results Three of the four haplotypes, C1534/I1016, C1534/V1016 and F1534/V1016 were heterogeneous between city blocks at all four sampling time points, and the double mutant haplotype, C1534/I1016, showed a significant increase following the wet season. The F1534/I1016 haplotype was rarely detected, similar to other studies. However, when haplotype frequencies were aggregated to a coarser spatial scale, the differences in space and time were obscured. Conclusions Our results provide empirical evidence that the selection of kdr alleles is occurring at fine spatial scales, indicating that future studies should include this scale to better understand evolutionary processes of resistance in natural populations.
    Loss-of-function mutations in caspase recruitment domain-containing protein 14 (CARD14) are associated with a severe variant of atopic dermatitis
    Alon Peled BMedSci, Science Direct - 2019
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. Methods Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. Results In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.
    Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
    Vianey Gonzalez-Villasana, Hindawi - 2019
    miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.
    Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis
    Mette Pernille Myklebust, Frontiers in Genetics - 2019
    Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.
    Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes
    Tamara A. Potapova, Journal of Cell Biology - 2019
    The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization
    Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies
    Sarah Irmscher, Nature Communications - 2019
    Persistent inflammation is a hallmark of many human diseases, including anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis. Here, we describe a dominant trigger of inflammation: human serum factor H-related protein FHR1. In vitro, this protein selectively binds to necrotic cells via its N-terminus; in addition, it binds near necrotic glomerular sites of AAV patients and necrotic areas in atherosclerotic plaques. FHR1, but not factor H, FHR2 or FHR3 strongly induces inflammasome NLRP3 in blood-derived human monocytes, which subsequently secrete IL-1β, TNFα, IL-18 and IL-6. FHR1 triggers the phospholipase C-pathway via the G-protein coupled receptor EMR2 independent of complement. Moreover, FHR1 concentrations of AAV patients negatively correlate with glomerular filtration rates and associate with the levels of inflammation and progressive disease. These data highlight an unexpected role for FHR1 during sterile inflammation, may explain why FHR1-deficiency protects against certain diseases, and identifies potential targets for treatment of auto-inflammatory diseases.
    RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes
    Joel Johansson, Cell Stem Cell - 2019
    Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
    A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila
    Alessandro Scopelliti, Cell Metabolism - 2019
    The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. Here, we show that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon α, which signals via its neuronal receptor DLgr2. Bursicon α/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon α/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, our work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis.
    A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing
    Theophilus T. Tettey, Cell Reports - 2019
    FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
    Suppression of UV-B stress induced flavonoids by biotic stress: Is there reciprocal crosstalk?
    Dirk Schenke, Plant Physiology and Biochemistry - 2019
    Plants respond to abiotic UV-B stress with enhanced expression of genes for flavonoid production, especially the key-enzyme chalcone synthase (CHS). Some flavonoids are antioxidative, antimicrobial and/or UV-B protective secondary metabolites. However, when plants are challenged with concomitant biotic stress (simulated e.g. by the bacterial peptide flg22, which induces MAMP triggered immunity, MTI), the production of flavonoids is strongly suppressed in both Arabidopsis thaliana cell cultures and plants. On the other hand, flg22 induces the production of defense related compounds, such as the phytoalexin scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread within the host tissue. Since all these metabolites require the precursor phenylalanine for their production, suppression of the flavonoid production appears to allow the plant to focus its secondary metabolism on the production of pathogen defense related compounds during MTI. Interestingly, several flavonoids have been reported to display anti-microbial activities. For example, the plant flavonoid phloretin targets the Pseudomonas syringae virulence factors flagella and type 3 secretion system. That is, suppression of flavonoid synthesis during MTI might have also negative side-effects on the pathogen defense. To clarify this issue, we deployed an Arabidopsis flavonoid mutant and obtained genetic evidence that flavonoids indeed contribute to ward off the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Finally, we show that UV-B attenuates expression of the flg22 receptor FLS2, indicating that there is negative and reciprocal interaction between this abiotic stress and the plant-pathogen defense responses.
    Diet and diet‐associated bacteria shape early microbiome development in Yellowtail Kingfish (Seriola lalandi)
    Jackson Wilkes Walburn, Microbial Biotechnology - 2019
    The supply of quality juveniles via land‐based larviculture represents a major bottleneck to the growing finfish aquaculture industry. As the microbiome plays a key role in animal health, this study aimed to assess the microbial community associated with early larval development of commercially raised Yellowtail Kingfish (Seriola lalandi). We used qPCR and 16S rRNA gene amplicon sequencing to monitor changes in the microbiome associated with the development of S. lalandi from larvae to juveniles. We observed an increase in the bacterial load during larval development, which consisted of a small but abundant core microbiota including taxa belonging to the families Rhodobacteraceae, Lactobacillaceae and Vibrionaceae. The greatest change in the microbiome occurred as larvae moved from a diet of live feeds to formulated pellets, characterized by a transition from Proteobacteria to Firmicutes as the dominant phylum. A prediction of bacterial gene functions found lipid metabolism and secondary metabolite production were abundant in the early larval stages, with carbohydrate and thiamine metabolism functions increasing in abundance as the larvae age and are fed formulated diets. Together, these results suggest that diet is a major contributor to the early microbiome development of commercially raised S. lalandi.
    Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model
    Benedikt Linder, Cancers - 2019
    Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12–15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability
    On resolving ambiguities in microbial community analysis of partial nitritation anammox reactors
    Laura Orschler, Scientific Reports - 2019
    PCR-based methods have caused a surge for integration of eco-physiological approaches into research on partial nitritation anammox (PNA). However, a lack of rigorous standards for molecular analyses resulted in widespread data misinterpretation and consequently lack of consensus. Data consistency and accuracy strongly depend on the primer selection and data interpretation. An in-silico evaluation of 16S rRNA gene eubacterial primers used in PNA studies from the last ten years unraveled the difficulty of comparing ecological data from different studies due to a variation in the coverage of these primers. Our 16S amplicon sequencing approach, which includes parallel sequencing of six 16S rRNA hypervariable regions, showed that there is no perfect hypervariable region for PNA microbial communities. Using qPCR analysis, we emphasize the significance of primer choice for quantification and caution with data interpretation. We also provide a framework for PCR based analyses that will improve and assist to objectively interpret and compare such results.
    Infection with Toxoplasma gondii (Eucoccidiorida: Sarcocystidae) in bats of Campeche and Yucatán, Mexico
    Marco Torres-Castro, International Journal of Tropical Biology and Conservation - 2019
    Toxoplasma gondii is a protozoan parasite recognized as the causative agent of toxoplasmosis, a zoonotic disease that affects humans and domestic or wild animals. In Mexico, it represents a public and animal health problem, especially in regions with tropical and subtropical climates. Bats have been reported as accidental hosts in the transmission cycle; however, there is no preceding information in Mexico. Therefore, the aim of the present study is to report the T. gondii infection in bats captured in sites of Campeche and Yucatan states, Mexico. Bats were captured in two sites in Yucatan (X’matkuil and Panaba) and one in Campeche (Hampolol), located in the Yucatan Peninsula. Kidneys, spleen, and liver were collected and used in the total DNA extraction. Toxoplasma gondii infection was detected through the amplification of a B1 gene fragment, using nested PCR. The positive PCR products were purified and sent to sequencing for a posterior sequence identity analysis. Additionally, a phylogenetic tree was made. A total of 69 bats belonging to eight different species were processed: 41 (59.4 %, 41/69) Artibeus jamaicensis; six (8.7 %, 6/69) Pteronotus parnellii; six (8.7 %, 6/69) Noctilio leporinus; six (8.7 %, 6/69) Chiroderma villosum; four (5.8 %, 4/69) Glossophaga soricina; two (2.9 %, 2/69) Carollia sowelli; two (2.89 %, 2/69) Artibeus lituratus; and two (2.9 %, 2/69) Rhogeessa aeneus. The nested PCR identified eight (11.6 %, 8/69) infected bats: six (75 %, 6/8) A. jamaicensis, captured in X'matkuil and Panaba, one (12.5 %, 1/8) G. soricina, and one (12.5 %, 1/8) C. villosum, both captured in Panaba. The alignment analysis yielded 99-100 % for cover and 97-99 % for identity to T. gondii sequences. Our results contribute to the understanding of the T. gondii transmission cycle in the region; however, future research is needed to determine circulating genotypes, as previous studies have demonstrated that these animals might be infected with identified genotypes in other domestic or wild animals and even in humans.
    Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST
    Yoko Nakano, Cell Reports - 2019
    A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST.
    Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts
    Zoe E. Gillespie, Scientific Reports - 2019
    Metformin is a widely-used treatment for type 2 diabetes and is reported to extend health and lifespan as a caloric restriction (CR) mimetic. Although the benefits of metformin are well documented, the impact of this compound on the function and organization of the genome in normal tissues is unclear. To explore this impact, primary human fibroblasts were treated in culture with metformin resulting in a significant decrease in cell proliferation without evidence of cell death. Furthermore, metformin induced repositioning of chromosomes 10 and 18 within the nuclear volume indicating altered genome organization. Transcriptome analyses from RNA sequencing datasets revealed that alteration in growth profiles and chromosome positioning occurred concomitantly with changes in gene expression profiles. We further identified that different concentrations of metformin induced different transcript profiles; however, significant enrichment in the activator protein 1 (AP-1) transcription factor network was common between the different treatments. Comparative analyses revealed that metformin induced divergent changes in the transcriptome than that of rapamycin, another proposed mimetic of CR. Promoter analysis and chromatin immunoprecipitation assays of genes that changed expression in response to metformin revealed enrichment of the transcriptional regulator forkhead box O3a (FOXO3a) in normal human fibroblasts, but not of the predicted serum response factor (SRF). Therefore, we have demonstrated that metformin has significant impacts on genome organization and function in normal human fibroblasts, different from those of rapamycin, with FOXO3a likely playing a role in this response.
    The chemokine receptor CXCR2 contributes to murine adipocyte development
    Douglas P. Dyer, Journal of Leukocyte Biology - 2019
    Chemokines are members of a large family of chemotactic cytokines that signal through their receptors to mediate leukocyte recruitment during inflammation and homeostasis. The chemokine receptor CXCR2 has largely been associated with neutrophil recruitment. However, there is emerging evidence of roles for chemokines and their receptors in processes other than leukocyte migration. We have previously demonstrated that CXCR2 knockout (KO) mice have thinner skin compared to wild‐type mice. Herein we demonstrate that this is due to a thinner subcutaneous adipose layer, as a result of fewer and smaller individual adipocytes. We observe a similar phenotype in other fat depots and present data that suggests this may be due to reduced expression of adipogenesis related genes associated with adipocyte specific CXCR2 signaling. Interestingly, this phenotype is evident in female, but not male, CXCR2 KO mice. These findings expand our understanding of nonleukocyte related chemokine receptor functions and help to explain some previously observed adipose‐related phenotypes in CXCR2 KO mice.
    Cytoprotective effects of Avenathramide C against oxidative and inflammatory stress in normal human dermal fibroblasts
    Chenxuan Wang, Scientific Reports - 2019
    Natural polyphenols are promising anti-aging compounds not only for their antioxidant activity, but also their ability to activate specific cellular pathways mediating the aging process. Avenanthramide C (Avn C), found exclusively in oats, is a natural antioxidant associated with free radical scavenging; however, it is how this compound elicits other protective effects. We investigated the intracellular antioxidant activity of Avn C and other cytoprotective potential in normal human skin fibroblasts exposed to extracellular stress. Avn C reduced H2O2-induced oxidative stress by reducing intracellular free radical levels and antioxidant gene transcripts. Avn C also resulted in decreased levels of gene transcripts encoding pro-inflammatory cytokines in response to H2O2 or tumor necrosis factor-α (TNF-α). This reduction in cytokine gene transcription occurred concomitantly with reduced phosphorylated nuclear factor-κB (NF-κB) p65, and decreased NF-κB DNA binding. Avn C further induced heme oxygense-1 (HO-1) expression through increased Nrf2 DNA binding activity, demonstrating a second mechanism by which Avn C attenuates cellular stress. Collectively, our findings indicate that Avn C protects normal human skin fibroblasts against oxidative stress and inflammatory response through NF-κB inhibition and Nrf2/HO-1 activation
    Effects of 50 Hz magnetic fields on circadian rhythm control in mice
    Louise Lundberg, Bio Electro Magnetics - 2019
    Artificial light and power frequency magnetic fields are ubiquitous in the built environment. Light is a potent zeitgeber but it is unclear whether power frequency magnetic fields can influence circadian rhythm control. To study this possibility, 8–12‐week‐old male C57BL/6J mice were exposed for 30 min starting at zeitgeber time 14 (ZT14, 2 h into the dark period of the day) to 50 Hz magnetic fields at 580 μT using a pair of Helmholtz coils and/or a blue LED light at 700 lux or neither. Our experiments revealed an acute adrenal response to blue light, in terms of increased adrenal per1 gene expression, increased serum corticosterone levels, increased time spent sleeping, and decreased locomotor activity (in all cases, P < 0.0001) compared to an unexposed control group. There appeared to be no modulating effect of the magnetic fields on the response to light, and there was also no effect of the magnetic fields alone (in both cases, P > 0.05) except for a decrease in locomotor activity (P < 0.03). Gene expression of the cryptochromes cry1 and cry2 in the adrenals, liver, and hippocampus was also not affected by exposures (in all cases, P > 0.05). In conclusion, these results suggest that 50 Hz magnetic fields do not significantly affect the acute light response to a degree that can be detected in the adrenal response. Bioelectromagnetics. 2019;9999:XX–XX. © 2019 Bioelectromagnetics Society.
    Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface
    Maria Salvany-Celades, Cell Reports - 2019
    During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.
    Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine
    May Levin, Cell Death and Disease - 2019
    Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.
    Transcriptome Response of Female Culicoides sonorensis Biting Midges (Diptera: Ceratopogonidae) to Early Infection with Epizootic Hemorrhagic Disease Virus (EHDV-2)
    Dana Nayduch, Viruses - 2019
    Female Culicoides sonorensis biting midges are vectors of epizootic hemorrhagic disease virus (EHDV), which causes morbidity and mortality in wild and domesticated ruminants. The aims in this study were to identify key changes in female midge transcriptome profiles occurring during early infection with EHDV-2. Midges were fed either negative control bloodmeals or bloodmeals containing EHDV-2 and transcriptomes were acquired at 36 h through deep sequencing. Reads were de novo assembled into a transcriptome comprised of 18,754 unigenes. Overall, there were 2401 differentially expressed unigenes and ~60% were downregulated in response to the virus (953 up; 1448 down). Downstream Gene Ontology enrichment, KEGG pathway mapping, and manual analyses were used to identify the effect of virus ingestion at both the gene and pathway levels. Downregulated unigenes were predominantly assigned to pathways related to cell/tissue structure and integrity (actin cytoskeleton, adherens junction, focal adhesion, hippo signaling), calcium signaling, eye morphogenesis and axon guidance. Unigenes attributed to sensory functions (especially vision), behavior, learning and memory were largely downregulated. Upregulated unigenes included those coding for innate immune processes, olfaction and photoreceptor pigments. Our results suggest that midges respond to virus infection as soon as 36 h post-ingestion, and that EHDV-2 may have a significant phenotypic effect on sensory and neural tissues.
    CYLD Regulates Centriolar Satellites Proteostasis by Counteracting the E3 Ligase MIB1
    Tiphaine Douanne, Cell Reports - 2019
    The tumor suppressor CYLD is a deubiquitinatingenzyme that removes non-degradative ubiquitin link-ages bound to a variety of signal transduction adap-tors. CYLD participates in the formation of primarycilia, a microtubule-based structure that protrudesfrom the cell body to act as a ‘‘sensing antenna.’’Yet, how exactly CYLD regulates ciliogenesis is notfully understood. Here, we conducted an unbiasedproteomic screen of CYLD binding partners andidentified components of the centriolar satellites.These small granular structures, tethered to the scaf-fold protein pericentriolar matrix protein 1 (PCM1),gravitate toward the centrosome and orchestrateciliogenesis. CYLD knockdown promotes PCM1degradation and the subsequent dismantling of thecentriolar satellites. We found that CYLD marshalsthe centriolar satellites by deubiquitinating andpreventing the E3 ligase Mindbomb 1 (MIB1) frommarking PCM1 for proteasomal degradation. Theseresults link CYLD to the regulation of centriolar satel-lites proteostasis and provide insight into howreversible ubiquitination finely tunes ciliogenesis.
    Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune Uveitis
    Cathleen Janowitz, Investigative Ophthalmology & Visual Science - 2019
    Purpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis. Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation. Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis. Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.
    Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing Mice
    Nicholas Moon, Nutrients - 2019
    Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1β and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.
    Insulin-like Growth Factor (IGF)-1 treatment stabilizes the microvascularcytoskeleton under ischemic conditions
    Shameena Bake, Experimental Neurology - 2019
    Our previous studies showed that Insulin-like Growth Factor (IGF)-1 reduced blood brain barrier permeabilityand decreased infarct volume caused by middle cerebral artery occlusion (MCAo) in middle aged female rats.Similarly, cultures of primary brain microvessel endothelial cells from middle-aged female rats and exposed tostroke-like conditions (oxygen glucose deprivation; OGD) confirmed that IGF-1 reduced dye transfer across thiscell monolayer. Surprisingly, IGF-1 did not attenuate endothelial cell death caused by OGD. To reconcile thesefindings, the present study tested the hypothesis that, at the earliest phase of ischemia, IGF-1 promotes barrierfunction by increasing anchorage and stabilizing cell geometry of surviving endothelial cells. Cultures of humanbrain microvessel endothelial cells were subject to oxygen-glucose deprivation (OGD) in the presence of IGF-1,IGF-1 + JB-1 (IGFR inhibitor) or vehicle. OGD disrupted the cell monolayer and reduced cell-cell interactions,which was preserved in IGF-1-treated cultures and reversed by concurrent treatment with JB-1. IGF-1-mediatedpreservation of the endothelial monolayer was reversed with LY294002 treatment, but not by Rapamycin, in-dicating that IGF-1 s actions on cell-cell contacts are likely mediated via the PI3K pathway. In vivo, microvesselmorphology was evaluated in middle-aged female rats that were subjected to ischemia by MCAo, and treated ICVwith IGFeI, IGF-1 + JB-1, or artificial CSF (aCSF; vehicle) after reperfusion. Compared to vehicle controls, IGF-1treated animals displayed larger microvessel diameters in the peri-infarct area and increased staining density forvinculin, an anchorage protein. Both these measures were reversed by concurrent IGF-1 + JB-1 treatment.Moreover these effects were restricted to 24 h after ischemia-reperfusion and no treatment effects were seen at5d post stroke. Collectively, these data suggest that in the earliest hours during ischemia, IGF-1 promotes re-ceptor-mediated anchorage of endothelial cells, and its actions may be accurately characterized as vasculo-protective.
    High diagnostic yield and novel variants in very late-onset spasticity
    Momen Almomen, Journal of Neurogenetics - 2019
    Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to support the pathogenicity of these variants, and provide observations regarding the poor genotypephenotype correlation in these conditions that should be the subject of future study
    Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bonelike Matrix Deposition in Vitro
    Rayan Fairag, ACS Applied Materials & Interfaces - 2019
    Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 μm), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 μm pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 μm pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 μm pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.
    Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening
    Konrad Schwefel, Journal of Cellular and Molecular Medicine - 2019
    CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.
    S-adenosyl methionine prevents ASD like behaviors triggered by earlypostnatal valproic acid exposure in very young mice
    asher Ornoy, Neurotoxicology and Terattology - 2019
    Introduction:A common animal model of ASD is the one induced by valproic acid (VPA), inducing epigeneticchanges and oxidative stress. We studied the possible preventive effect of the methyl donor for epigenetic en-zymatic reactions, S-adenosine methionine (SAM), on ASD like behavioral changes and on redox potential in thebrain and liver in this model.Methods:ICR albino mice were injected on postnatal day 4 with one dose of 300 mg/kg of VPA, with normalsaline (controls) or with VPA and SAM that was given orally for 3 days at the dose of 30 mg/kg body weight.From day 50, we carried out neurobehavioral tests and assessment of the antioxidant status of the prefrontalcerebral cortex, liver assessing SOD and CAT activity, lipid peroxidation and the expression of antioxidant genes.Results:Mice injected with VPA exhibited neurobehavioral deficits typical of ASD that were more prominent inmales. Changes in the activity of SOD and CAT increased lipid peroxidation and changes in the expression ofantioxidant genes were observed in the prefrontal cortex of VPA treated mice, more prominent in females, whileASD like behavior was more prominent in males. There were no changes in the redox potential of the liver. Theco-administration of VPA and SAM alleviated most ASD like neurobehavioral symptoms and normalized theredox potential in the prefrontal cortex.Conclusions:Early postnatal VPA administration induces ASD like behavior that is more severe in males, whilethe redox status changes are more severe in females; SAM corrects both. VPA-induced ASD seems to result fromepigenetic changes, while the redox status changes may be secondary.
    Human-based fibrillar nanocomposite hydrogels as bioinstructive matrices to tune stem cell behavior
    Bárbara B. Mendes, Nanoscale - 2018
    The extracellular matrix (ECM)-biomimetic fibrillar structure of platelet lysate (PL) gels along with their enriched milieu of biomolecules has drawn significant interest in regenerative medicine applications. However, PL-based gels have poor structural stability, which severely limits their performance as a bioinstructive biomaterial. Here, rod-shaped cellulose nanocrystals (CNC) are used as a novel approach to modulate the physical and biochemical microenvironment of PL gels enabling their effective use as injectable human-based cell scaffolds with a level of biomimicry that is difficult to recreate with synthetic biomaterials. The incorporation of CNC (0 to 0.61 wt%) into the PL fibrillar network during the coagulation cascade leads to decreased fiber branching, increased interfiber porosity (from 66 to 83%) and modulates fiber (from 1.4 ± 0.7 to 27 ± 12 kPa) and bulk hydrogel (from 18 ± 4 to 1256 ± 82 Pa) mechanical properties. As a result of these physicochemical alterations, nanocomposite PL hydrogels resist the typical extensive clot retraction (from 76 ± 1 to 24 ± 3 at day 7) and show favored retention of PL bioactive molecules. The feedback of these cues on the fate of human adipose-derived stem cells is evaluated, showing how it can be explored to modulate the commitment of encapsulated stem cells toward different genetic phenotypes without the need for additional external biological stimuli. These fibrillar nanocomposite hydrogels allow therefore the exploration of the outstanding biological properties of human-based PL as an efficient engineered ECM which can be tailored to trigger specific regenerative pathways in minimal invasive strategies.
    Maternal malnourishment induced upregulation of fetuin-B blunts nephrogenesis in the low birth weight neonate
    May M. Rabadi, Developmental Biology - 2018
    Maternal undernutrition during pregnancy (MUN) often leads to low birth weight (LBW) neonates that have a reduced total nephron endowment, leaving these neonates susceptible to kidney disease throughout their lives. For reasons unknown, these LBW neonates have impaired kidney development due to a severe reduction in renal SIX2+ stem cells during nephrogenesis. Using a mouse model of MUN, we investigated SIX2+ stem cell reduction in the LBW neonate. Significant upregulation of the protein fetuin-B (measured by PCR and immunoblotting) in the MUN mother's placenta, organs and circulation yielded a 3-fold increase of this protein in the embryonic kidney. Recombinant fetuin-B, administered to healthy pregnant mothers at the concentration equivalent to that in the MUN mother, crossed the placenta and reduced both SIX2+ stem cells by 50% and nephron formation by 66% in embryonic kidneys (measured by immunofluorescence and the physical dissector/fractionator stereological method). Administration of fetuin-B to kidney explants yielded similar reductions in renal SIX2+ stem cells and nephron formation. Fetuin-B treatment of isolated embryonic renal SIX2+ stem cell primary cultures 1) increased NF-kB activity and apoptosis, 2) reduced cell proliferation due to upregulated p21 nuclear activity and subsequent cell cycle arrest, and 3) enhanced generation of reactive oxygen species (measured by fluorescence microscopy). In conclusion, MUN increases fetuin-B in the developing embryonic kidney. The increase in fetuin-B blunts nephrogenesis by reducing SIX2+ stem cells by promoting their apoptosis (via NF-kB upregulation), blunting their proliferative renewal (via p21 upregulation) and enhancing oxidative stress.
    Polyunsaturated Fatty Acids Induce ROS Synthesis in Microvascular Endothelial Cells
    Simon Trommer, Oxygen Transport to Tissue XL - 2018
    In sepsis, endothelial dysfunction is a crucial driver known to limit the survival rate of affected patients. For this, ROS-mediated signaling plays an important role in endothelial communication and functionality. In the management of sepsis, polyunsaturated fatty acids (PUFA) have received increasing attention regarding their anti-inflammatory potential neglecting the oxidative properties of these substances. Therefore, in the present study we examined the capacity of PUFA to interfere with the expression of major ROS-producing enzymes, as well as endothelial ROS production itself. The human microvascular endothelial cells TIME (ATCC number: CRL-4025) were used. Cells were cultured in medium enriched with LNA (C18:3n3), EPA (C20:5n3), DHA (C22:6n3), LA (C18:2n6), or AA (C20:4n6) in concentrations of 15 μM totaling 144 h. Stimulation of cells was performed in the last 24 h of fatty acid supplementation by addition of the cytokines TNF-α + IL-1β + IFN-γ (5 ng/ml each). Gene expression of eNOS, COX-2, and NOX-4 was evaluated by qPCR. ROS synthesis was analyzed by means of a flow cytometry-based rhodamine 123 assay. Cytokine stimulation was found to differentially affect gene expression of major ROS synthesizing enzymes: eNOS was decreased whereas COX-2 and NOX-4 were increased. As a consequence, cytokine stimulation had no effect on rhodamine accumulation in endothelial cells. PUFA supplementation alone did not affect the gene expression of eNOS, COX-2, and NOX-4. Nevertheless, an increasing action of PUFA on the stimulation-induced reduction in eNOS expression was found. More importantly, the number of rhodamine positive endothelial cells almost doubled following enrichment with the PUFA EPA, DHA or AA. This effect was independent of the stimulation status of the cells but seemed to be related to the number of double bonds of a supplemented fatty acid. Our data warrant further studies to ensure that increased endothelial cell oxidative stress is not boosted by PUFA in septic patients.
    Detection of coliphages and human adenoviruses in a subtropical estuarine lake
    Emily M. Cooksey, Science of The Total Environment - 2018
    Fecal indicator bacteria (FIB) have been used to assess fecal contamination in recreational water. However, enteric viruses have been shown to be more persistent in the environment and resistant to wastewater treatment than bacteria. Recently, U.S Environmental Protection Agency has proposed the use of coliphages as viral indicators to better protect against viral waterborne outbreaks. This study aimed to detect and determine correlation between coliphages (F-specific and somatic), fecal indicator bacteria (enterococci and fecal coliforms), and human enteric viruses (human adenovirus) in a subtropical brackish estuarine lake. Water samples were collected from 9 estuarine recreation sites on Lake Pontchartrain in southeast Louisiana. Water samples (n = 222, collected weekly) were analyzed for coliphages and fecal indicator bacteria using culture-based methods and large volume water samples (n = 54, collected monthly) were analyzed for human adenovirus using quantitative PCR. Somatic coliphage and F-specific coliphage were found in 93.7 and 65.2% of samples with geometric mean concentrations of 30 and 3 plaque forming units (PFU) per 100 mL, respectively. Enterococci, fecal coliforms, and adenovirus were found in all samples with geometric mean concentrations of 27 most probable number (MPN), 77 MPN, and 3.0 × 104 gene copies per 100 mL, respectively. Watersheds in suburban areas exhibited significantly higher concentrations of coliphages and fecal indicator bacteria, indicating potential fecal contamination from septic systems. There was no significant correlation (p > 0.05) observed between the presence of adenoviruses and fecal indicator bacteria and coliphages. The presence of human adenovirus in Lake Pontchartrain poses a significant public health problem for both recreational use and seafood harvesting as it increases exposure risks. This study demonstrated the lack of relationship between fecal indicators and human viral pathogen in Lake Pontchartrain supporting an alternative microbial surveillance system such as direct pathogen detection.
    Restoring Endothelial Function by Targeting Desert Hedgehog Downstream of Klf2 Improves Critical Limb Ischemia in Adults
    Caroline Caradu, Circulation Research - 2018
    Rationale: Klf2 is critical to establish and maintain endothelial integrity. Objective:Therefore, determining upstream and downstream mediators of Klf2 would lead to alternative therapeutic targets in cardiovascular disease management. Methods and Results: Here we identify Desert Hedgehog (Dhh) as a downstream effector of Klf2, whose expression in endothelial cells (ECs) is upregulated by shear stress and decreased by inflammatory cytokines. Consequently, we show that Dhh knock down in ECs promotes endothelial permeability and EC activation and that Dhh agonist prevents TNFα or glucose-induced EC dysfunction. Moreover, we demonstrate that human critical limb ischemia (CLI), a pathological condition linked to diabetes and inflammation, is associated to major EC dysfunction. By recreating a complex model of CLI in diabetic mice, we found that Dhh-signaling agonist significantly improved EC function without promoting angiogenesis, which subsequently improved muscle perfusion. Conclusions: Restoring EC functi...
    AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice
    Lemlem Brook, Journal of Cellular Biochemistry - 2018
    Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.
    AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice
    Aimee E. Vozenilek, Atherosclerosis - 2018
    Abstrat Background and aims The recombinant adeno-associated viral vector serotype 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8- PCSK9) is a new model for the induction of hypercholesterolemia. AAV8 preferentially infects hepatocytes and the incorporated liver-specific promoter should ensure expression of PCSK9 in the liver. Since tissue distribution of AAVs can differ between male and female mice, we investigated the differences in PCSK9 expression and hypercholesterolemia development between male and female mice using the AAV8-PCSK9 model. Methods Male and female C57BL/6 mice were injected with either a low-dose or high-dose of AAV8-PCSK9 and fed a high-fat diet. Plasma lipid levels were evaluated as a measure of the induction of hypercholesterolemia. Results Injection of mice with low dose AAV8-PCSK9 dramatically elevated both serum PCSK9 and cholesterol levels in male but not female mice. Increasing the dose of AAV8-PCSK9 threefold in female mice rescued the hypercholesterolemia phenotype but did not result in full restoration of AAV8-PCSK9 transduction of livers in female mice compared to the low-dose male mice. Our data demonstrate female mice respond differently to AAV8-PCSK9 injection compared to male mice. Conclusions These differences do not hinder the use of female mice when AAV8-PCSK9 doses are taken into consideration. However, localization to and production of AAV8-PCSK9 in organs besides the liver in mice may introduce confounding factors into studies and should be considered during experimental design.
    Genetic diversity of Hepatitis C Virus in Pakistan using Next Generation Sequencing
    Sana Saleem, Journal of Clinical Virology - 2018
    Background In Pakistan, HCV disease is considered a major public health issue with about 10–17 million people suffering with this infection and rate is increasing every day without any hindrance. The currently available Pyrosequencing approach used to analyze complex viral genomes as it can determine minor variants. It is crucial to understand viral evolution and quasispecies diversity in complex viral strains. Objectives To assess genetic diversity in patients with HCV using Next Generation Sequencing (NGS) and compare nucleotide diversity of genotype 3a with respect to other genotypes. Study design Intra-host viral diversity of HCV was determined using NGS from 13 chronically HCV infected individuals. NGS of three different regions (E2 (HVR1), NS3 and NS5B) of HCV-3a allowed for a comprehensive analysis of the viral population. Result Phylogenetic analysis of different HCV genes revealed great variability within the Pakistani population. The average nucleotide diversity for HVR1, NS3 and NS5B was 0.029, 0.011 and 0.010 respectively. Conclusion Our findings clearly indicate that patient-2 greater quasispecies heterogeneity than other patients of same genotype-3a using phylogenetic and one step network analyses. Initially phylogenetic analysis of these three genes showed that genotype 3a samples have greater genetic diversity. However, no significant difference was determined when nucleotide variability of genotype 3a compared with other genotypes (1a, 1b, 2a & 4a).
    Sperm capacitation is associated with phosphorylation of the testis-specific radial spoke protein Rsph6a
    Bidur Paudel, Biology of Reproduction - 2018
    A sequence corresponding to the N-terminal domain of the radial spoke protein Rsph6 was found phosphorylated in capacitated sperm. Rsph6 expression is a testis-
    Localization of the 1,25-dihydroxyvitamin D-mediated response in the intestines of mice
    Carmen J. Reynolds, The Journal of Steroid Biochemistry and Molecular Biology - 2018
    1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, β-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
    Loss-of-function mutations in CARD14 are associated with a severe variant of atopic dermatitis
    Alon Peled, Journal of Allergy and Clinical Immunology - 2018
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease which is known to be, at least in part, genetically determined. Mutations in CARD14 have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe atopic dermatitis. Methods Whole exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA and functional assays in human keratinocytes were used. Results In a cohort of individuals referred with severe atopic dermatitis, DNA sequencing revealed in 4 patients two rare heterozygous missense mutations in CARD14 encoding the Caspase Recruitment Domain-Containing Protein 14, a major regulator of NF-κB. A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patient skin as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions While dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of atopic dermatitis.
    Tissue-specific gene regulation corresponds with seasonal plasticity in female testosterone
    Alexandra B. Bentz, General and Comparative Endocrinology - 2018
    Testosterone (T) is a sex steroid hormone that often varies seasonally and mediates trade-offs between territorial aggression and parental care. Prior work has provided key insights into the ‘top-down’ hypothalamic control of this seasonal plasticity in T, yet mechanisms acting outside of the brain may also influence circulating T levels. We hypothesized that peripheral mechanisms may be especially critical for females, because peripheral regulation may mitigate the costs of systemically elevated T. Here, we begin to test this hypothesis using a seasonal comparative approach, measuring gene expression in peripheral tissues in tree swallows (Tachycineta bicolor), a songbird with intense female-female competition and T-mediated aggression. We focused on the gonad and liver for their role in T production and metabolism, respectively, and we contrasted females captured during territory establishment versus incubation. During territory establishment, when T levels are highest, we found elevated gene expression of the hepatic steroid metabolizing enzyme CYP2C19 along with several ovarian steroidogenic enzymes, including the androgenic 5α-reductase. Despite these seasonal changes in gene expression along the steroidogenic pathway, we did not observe seasonal changes in sensitivity to upstream signals, measured as ovarian mRNA abundance of luteinizing hormone receptor. Together, these data suggest that differential regulation of steroidogenic gene expression in the ovary is a potentially major contributor to seasonal changes in T levels in females. Furthermore, these data provide a unique and organismal glimpse into tissue-specific gene regulation and its potential role in hormonal plasticity in females.
    Potent in vivo lung cancer Wnt signaling inhibition via cyclodextrin-LGK974 inclusion complexes
    Pedro P. G. Guimaraes, Journal of Controlled Release - 2018
    Activation of the Wnt signaling pathway promotes lung cancer progression and contributes to poor patient prognosis. The porcupine inhibitor LGK974, a novel orally bioavailable cancer therapeutic in Phase I clinical trials, induces potent Wnt inhibition leading to suppressed growth and progression of multiple types of cancers. The clinical use of LGK974, however, is limited in part due to its low solubility and high toxicity in tissues that rely on Wnt signaling for normal homeostasis. Here, we report the use of host-guest chemistry to enhance solubility and bioavailability of LGK974 in mice through complexation with cyclodextrins (CD). We assessed the effects of these complexes to inhibit Wnt signaling in lung adenocarcinomas that are typically driven by overactive Wnt signaling. 2D H1 NMR confirmed host-guest complexation of CDs with LGK974. CD:LGK974 complexes significantly decreased the expression of Wnt target genes both in vitro and in vivo. Further, CD:LGK974 complexes increased the bioavailability upon oral administration in mice compared to free LGK974. In a mouse lung cancer allograft model, CD:LGK974 complexes induced potent Wnt signaling inhibition with reduced intestinal toxicity compared to administration of free drug. Collectively, the development of these complexes enables safer and repeated oral or parenteral administration of porcupine inhibitors, which hold promise for the treatment of multiple types of malignancies.
    Alterations of EDEM1 functions enhance ATF6 pro-survival signaling
    Alexandra Papaioannou, The FEBS Journal - 2018
    Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the 3 sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions. This article is protected by copyright. All rights reserved.
    Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility
    Yao Xiao, Biotechnology for Biofuels - 2018
    Expression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates.
    Endocrine-immune signaling as a predictor of survival: A prospective study in developing songbird chicks
    Emily E. Virgin, General and Comparative Endocrinology - 2018
    Immune function varies with an animal’s endocrine physiology and energy reserves, as well as its abiotic and biotic environment. This context-dependency is thought to relate to adaptive trade-off resolution that varies from one context to the next; however, it is less clear how state- and environmentally-dependent differences in endocrine-immune signaling relate to survival in natural populations. We begin to address this question in a prospective study on a free-living passerine bird, the tree swallow (Tachycineta bicolor), by capitalizing upon naturally-occurring variation in ectoparasitism in 12-day old chicks. We measured body mass, hematological gene expression of the pro-inflammatory cytokine interleukin-6 (IL-6) as well as corticosterone (CORT) secretion at baseline and in response to 30 min of handling. We found that chicks with ectoparasites had smaller body mass and higher levels of IL-6 gene expression at this critical stage of post-natal growth and development. Mass and IL-6 were positively correlated, but only among parasitized chicks, suggesting that larger chicks mount stronger immune responses when necessary, i.e. in the presence of ectoparasites that are known to induce inflammation. IL-6 mRNA expression was negatively correlated with stress-induced CORT levels, suggesting that this proxy of inflammation may be co-regulated with or coordinated by glucocorticoids. More importantly, these endocrine-immune parameters predicted survival to fledging, which was positively associated with IL-6 mRNA abundance and, to a lesser degree, CORT reactivity. These results suggest a link between endocrine-immune interactions and performance in nature, and as a consequence, they shed light on the potentially adaptive, context-dependent interplay between body mass, immunity, and endocrine physiology during development.
    time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance
    Wojciech Siwek, Histone Variants - 2018
    Understanding chromatin dynamics is essential to define the contribution of chromatin to heritable gene silencing and the long-term maintenance of gene expression. Here we present a detailed protocol for time-ChIP, a novel method to measure histone turnover at high resolution across long timescales. This method is based on the SNAP-tag, a self-labeling enzyme that can be pulse labeled with small molecules in cells. Upon pulse biotinylation of a cohort of SNAP-tagged histones we can determine their abundance and fate across a chase period using a biotin-specific chromatin pulldown followed by DNA sequencing or quantitative PCR. This method is unique in its ability to trace the long-term fate of a chromatin bound histone pool, genome wide. In addition to a step by step protocol, we outline advantages and limitations of the method in relation to other existing techniques. time-ChIP can define regions of high and low histone turnover and identify the location of pools of long lived histones.
    Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth
    Rita C. Assunção-Silva, Biochimie - 2018
    Cell transplantation free-based therapies using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro, using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth.
    Bifidobacterium pseudolongum in the Ceca of Rats Fed Hi-Maize Starch Has Characteristics of a Keystone Species in Bifidobacterial Blooms
    Manuela Centanni, Applied and Environmental Microbiology - 2018
    Starches resistant to mammalian digestion are present in foods and pass to the large bowel, where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis. However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a type 1 pullulanase, alpha-amylase, and glycogen debranching enzyme by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium, and B. animalis cultures had shorter doubling times in maltose medium than did B. pseudolongum. Thus, B. pseudolongum, which was present at a consistently low abundance in the microbiota, but which has extensive enzymatic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom. IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community of the large bowel of animals, including humans, has been studied extensively through the use of high-throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that the increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch (Bifidobacterium animalis) but cohabits with a species that can (Bifidobacterium pseudolongum). B. pseudolongum has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function, the hydrolysis of resistant starch.
    Increased Alternative Splicing as a Host Response to Edwardsiella ictaluri Infection in Catfish
    Suxu Tan, Marine Biotechnology - 2018
    Alternative splicing is the process of generating multiple transcripts from a single pre-mRNA used by eukaryotes to regulate gene expression and increase proteomic complexity. Although alternative splicing profiles have been well studied in mammalian species, they have not been well studied in aquatic species, especially after biotic stresses. In the present study, genomic information and RNA-Seq datasets were utilized to characterize alternative splicing profiles and their induced changes after bacterial infection with Edwardsiella ictaluri in channel catfish (Ictalurus punctatus). A total of 27,476 alternative splicing events, derived from 9694 genes, were identified in channel catfish. Exon skipping was the most abundant while mutually exclusive exon was the least abundant type of alternative splicing. Alternative splicing was greatly induced by E. ictaluri infection with 21.9% increase in alternative splicing events. Interestingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced genes after infection. Sequence analyses of splice variants of a representative alternatively spliced gene, splicing factor srsf2, revealed that certain spliced transcripts may undergo nonsense-mediated decay (NMD), suggesting functional significance of the induced alternative splicing. Although statistical analysis was not possible with such large datasets, results from quantitative real-time PCR from representative differential alternative splicing events provided general validation of the bacterial infection-induced alternative splicing. This is the first comprehensive study of alternative splicing and its changes in response to bacterial infection in fish species, providing insights into the molecular mechanisms of host responses to biotic stresses.
    Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells
    Guillaume M. Fortin, The FASEB Journal - 2018
    Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.—Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.
    Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle stimulating hormone receptor signaling
    Dongwon Kim, Molecular Reproduction and Development - 2018
    Recruitment of a single follicle into the preovulatory hierarchy of the domestic hen ovary occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. We have previously reported that granulosa cells (GC) collected from prehierarchal follicles express highest levels of membrane-localized follicle-stimulating hormone receptor (FSHR) during follicle development, yet fail to initiate signaling via cAMP following short-term incubation with FSH. Consequently, GC from prehierarchal follicles remain in an undifferentiated state and lack the capacity for steroidogenesis due to a deficiency of cAMP-dependent STAR protein and CYP11A1 gene expression. The present studies investigate FSH responsiveness in GC before and after the transition from undifferentiated to a differentiated state at follicle recruitment. Prior to recruitment focus is directed towards the inhibition of FSHR signaling by β-ARRESTIN (βARR). Specifically, knockdown of βARR mRNA in cultured, undifferentiated GC using small interfering RNA (siRNA) facilitated FSH-induced cAMP formation, STAR expression and progesterone production. Furthermore, over-expression of bovine βARR1 and G PROTEIN-COUPLED RECEPTOR KINASE2 in actively differentiating GC significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. We propose that a βARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment. This article is protected by copyright. All rights reserved.
    Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer
    Nashwa Kabil, Breast Cancer Research and Treatment - 2018
    Background/purposeTriple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target.Methods/resultsIn the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice.ConclusionOur study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.
    Exercise Preconditioning Diminishes Skeletal Muscle Atrophy after Hindlimb Suspension in Mice*
    Nicholas T. Theilen, Journal of Applied Physiology - 2018
    The aim of the present study was to investigate if short-term, concurrent exercise training prior to hindlimb suspension (HLS) prevents or diminishes both soleus and gastrocnemius atrophy and to analyze if changes in mitochondrial molecular markers were associated. Male C57BL/6 (WT) mice (12-14 weeks old) were assigned to control, 7-day HLS (HLS), 2-weeks exercise training prior to 7-day HLS (Ex+HLS), and 2-weeks exercise training (Ex) groups. HLS resulted in a 27.1% and 21.5% decrease in soleus and gastrocnemius muscle weight to bodyweight ratio, respectively, in WT mice. Exercise training prior to HLS resulted in a 5.6% and 8.1% decrease in soleus and gastrocnemius weight to bodyweight ratio, respectively. Exercise increased mitochondrial biogenesis and function associated markers, slow myosin heavy chain (SMHC) expression, and reduced the fiber-type transitioning marker myosin heavy chain 4 (Myh4). Ex+HLS revealed decreased reactive oxygen species (ROS) and oxidative stress compared to HLS. Our data indicated the time prior to an atrophic setting, particularly caused by muscle unloading, may be a useful period to intervene short-term, progressive exercise training to prevent skeletal muscle atrophy and is associated with mitochondrial biogenesis, function, and redox balance.
    In Silico Analyses of Rice Thionin genes and the Antimicrobial Activity of OsTHION15 against Phytopathogens
    Krissana Boonpa, Phytopathology - 2018
    Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa L.) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa L. ssp. indica ‘KDML 105’ was expressed in all of the tissues/organs examined, including germinating seeds, leaves and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice pathogenic bacteria, such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed towards Fusarium oxysporum ssp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against Xanthomonas campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.
    Pax-5 Inhibits NF-κB Activity in Breast Cancer Cells Through IKKε and miRNA-155 Effectors
    Jason Harquail, Journal of Mammary Gland Biology and Neoplasia - 2018
    Pax-5, an essential transcription factor in B cell development, is aberrantly expressed in various B cell cancer lesions and solid tumors such as breast carcinoma. We have recently shown that Pax-5 regulates NF-κB activity which lead to the modulation of breast cancer phenotypic features (EMT-MET). NF-κB is known as a central mediator in inflammation, stress response as well as being a gatekeeper of pro-tumorigenic activity. However, little is known as to how Pax-5 affects this modulation. We thus turned our attention to microRNAs as potential regulatory effectors. In this study, we set out to elucidate the regulatory network between differential Pax-5 expression and NF-κB activity which dictate breast cancer malignancy. Through next-generation sequencing (NGS) of breast cancer cells conditionally expressing Pax-5, we profile significantly upregulated microRNAs; including microRNA-155, a known regulator of pathological processes and suppressor of malignant growth. Through the conditional expression of microRNA-155 in breast cancer models, we identify and validate IKKε (IKBKE) as a downstream target and an essential effector of Pax-5-mediated suppression of NF-κB signaling. Using rescue experiments, we also confirm that Pax-5 modulates NF-κB activity via IKKε downregulation. Interestingly, we also show that microRNA-155, in turn, supresses Pax-5 expression, indicative of an auto-regulatory feedback loop. Altogether, we demonstrate that Pax-5 inhibits NF-κB signalling through the regulation of microRNA-155 and its downstream target IKKε. The elucidation of this signaling network is relevant as Pax-5 and NF-κB are potent transcriptional regulators of breast cancer aggressivity. In addition, IKKε is relevant oncogene aberrantly expressed in 30% of breast carcinomas. Further insight into the regulatory pathways of breast cancer progression will eventually identify strategic therapeutic and prognostic targets to improve cancer patient outcome.
    The effects of electronic cigarette vapor on placental trophoblast cell function: Short title : E-cigarette and trophoblast function
    Sergio Raez-Villanueva, Reproductive Toxicology - 2018
    Despite evidence that maternal smoking is associated with numerous adverse outcomes, 10-35% of women still smoke during pregnancy. Recently, many smokers have turned to electronic cigarettes (e-cigarettes) as a smoking cessation tool. However, there is considerable uncertainty regarding their safety for use during pregnancy. The goal of this study was to examine the effects of e-cigarette vapor on placental trophoblast function. HTR-8/SVneo cells were exposed to unflavored e-cigarette vapor-conditioned media with and without nicotine to assess cell viability, proliferation, migration (wound healing assay), invasion (transwell extracellular matrix invasion assay), and tube formation, a surrogate for angiogenesis. While there was no effect on cell viability, proliferation or migration (all p > 0.05), e-cigarette conditioned media significantly reduced trophoblast invasion and tube formation; these effects could not be solely attributed to the presence of nicotine. These results suggest that an evaluation of the safety of e-cigarette use during pregnancy is urgently required.
    Post-exposure effects of the piscicide 3-trifluoromethyl-4-nitrophenol (TFM) on the stress response and liver metabolic capacity in rainbow trout (Oncorhynchus mykiss)
    Oana Birceanu, PLOS ONE - 2018
    The piscicide 3-trifluoromethyl-4-nitrophenol (TFM) has been used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes for almost 60 years. Applied to rivers and streams containing larval lampreys, TFM seldom harms non-target fishes, but the effects of sub-lethal treatments on fish physiology are not well understood. We examined the effects of 9 h exposure to TFM on the stress axis and liver metabolic capacity of rainbow trout (Oncorhynchus mykiss) using in vivo and in vitro approaches. The fish that had been acutely exposed to TFM in vivo had increased plasma cortisol levels at 12 h post-treatment, but TFM exposure did not interfere with in vitro cortisol production in head kidney preparations. Subjecting trout to an acute handling stressor 12 h post-TFM exposure resulted in a relative attenuation of the plasma cortisol and glucose response compared to pre-stress levels. We conclude that routine TFM treatments can lead to elevations of plasma cortisol following exposure, plus a relative dampening of the stress response in rainbow trout, with high cortisol levels lasting at least 12 h post-treatment. Since the ability of the fish to produce cortisol and the liver metabolic capacity were not compromised following TFM exposure, it is likely that their ability to cope with other stressors is not altered in the long-term.
    Antimicrobial peptide expression in swine granulosa cells in response to lipopolysaccharide
    Xiaofeng Sun, Theriogenology - 2018
    Antimicrobial peptides (AMP) are host defense peptides present in all species examined. The objective of the current study was to characterize the expression of a group of antimicrobial peptides in ovarian cells, and to investigate their expression response to pathogen ligands. It was found that while PG1 transcript was not detected in the ovary, the expression of BD2 is the highest in small follicle derived granulosa cells (SGC), and its expression decreases during follicular development to large follicle stage (LGC; p < 0.05). The expression of BD2 in cumulus cells also decreased from GV to MII stage of oocyte maturation. ANG4 expression increased in granulosa cells during follicular development from SGC to LGC stage (p < 0.05), although no significant difference was observed in cumulus cells from different stages of oocyte maturation. We further examined AMP expression in follicle cells treated with different toll-like receptor (TLR) ligands which mimic pathogen exposure in the ovary. Of the four TLR ligands examined, lipopolysaccharide (LPS) exposure resulted in a 11.5 fold increase of BD2 expression, and a significant decrease of LYZ in LGC. A similar response pattern in BD2 and LYZ expression was also observed in SGC. These responses of AMP expression to LPS are associated with increased TLR4 signaling pathway component in mRNA and protein level, such as MyD88 and NFkB, and pro-inflammatory cytokines/chemokines, such as IL-6, TNFα and IL-8 (p < 0.05). Our data suggest that AMPs may play a role in innate defense as well as other physiological functions during ovarian follicular development and oocyte maturation.
    Effect of Tim23 Knockdown in vivo on Mitochondrial Protein Import and Retrograde Signaling to the UPRmt in Muscle
    Ashley N Oliveira, American Journal of Physiology-Cell Physiology - 2018
    The mitochondrial unfolded protein response (UPRmt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Due to the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mito-nuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPRmt. Thus, we sought to perturb this genomic coordination by using a systemic anti-sense oligonucleotide (in-vivo Morpholino) targeted to Tim23, the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate ROS emission under maximal respiration conditions. This import defect activated the CHOP-branch of the UPRmt, as evident from increases in ClpP and cpn10, but not the ATF5 arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically-derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.
    HISTONE DEACETYLASE 19 and the flowering time gene FD maintain reproductive meristem identity in an age-dependent manner
    Sasha R. Gorham, Journal of Experimental Botany - 2018
    The shoot apical meristem (SAM) undergoes developmental transitions that include a shift from vegetative to reproductive growth. This transition is triggered by flowering time genes, which up-regulate floral meristem (FM) identity genes that, in turn, control flower development by activating floral organ identity genes. This cascade of transcriptional activation is refined by repression mechanisms that temporally and spatially restrict gene expression to ensure proper development. Here, we demonstrate that HISTONE DEACETYLASE 19 (HDA19) maintains the identity of the reproductive SAM, or inflorescence meristem (IM), late in Arabidopsis thaliana development. At late stages of growth, hda19 IMs display a striking patterning defect characterized by ectopic expression of floral organ identity genes and the replacement of flowers with individual stamenoid organs. We further show that the flowering time gene FD has a specific function in this regulatory process, as fd hastens the emergence of these patterning defects in hda19 growth. Our work therefore identifies a new role for FD in reproductive patterning, as FD regulates IM function together with HDA19 in an age-dependent fashion. To effect these abnormalities, hda19 and fd may accentuate the weakening of transcriptional repression that occurs naturally with reproductive meristem proliferation.
    Rnd3/RhoE expression is regulated by G-actin through MKL1-SRF signaling pathway
    Léo Piquet, Experimental Cell Research - 2018
    Rnd3/RhoE is an atypical member of the Rho family of small GTPases, devoid of intrinsic GTP hydrolytic activity and a general modulator of important cellular processes such as migration and proliferation. Here, we show that Rnd3 is a target of the transcription factor SRF and its co-activator MKL1. The MKL1-SRF pathway assures the translation of physical forces into a transcriptional response. Rho GTPases can modulate the activity of this mechanotransduction pathway through actin cytoskeleton regulation, and many MKL1-SRF targets are involved in the regulation of actin. We found that Rnd3 expression is altered by G-actin signaling and sensitive to actin-targeting drugs and MKL1 mutants. We further characterized a consensus SRF binding site in the Rnd3 promoter. We found that MKL1-SRF modulation regulates Rnd3 promoter activity and Rnd3 expression can affect MKL1-SRF pathway activity in return. We demonstrated that this novel MKL1-SRF target is required in mechanosensitive mechanisms such as cell spreading and spheroid formation. Thus, Rnd3 is a MKL1-SRF target that plays a key role in the feedback loop described between the MKL1-SRF pathway and the organization of the actin cytoskeleton.
    Improved fatty acid profiles in seeds of Camelina sativa by artificial microRNA mediated FATB gene suppression
    Mehmet E. Ozseyhan, Biochemical and Biophysical Research Communications - 2018
    The fatty acid profile of plant oils determines their quality and uses. Saturated fatty acids are often not desirable from the standpoints of nutrition and some industrial applications. Camelina sativa is a re-emerged oilseed crop, however its oil needs to be improved to meet different application requirements. In this study, saturated fatty acids were greatly reduced by down-regulating genes encoding the fatty acyl-ACP thioesterases (FATB). An artificial microRNA (amiFATB) was created by replacing a microRNA sequence in the camelina Csa-miR159a gene with a FATB gene specific sequence. Seed-specific expression of amiFATB caused a 45% reduction of palmitic acid (16:0) and a 38% reduction of stearic acid (18:0) compared to wildtype seeds. The total saturated fatty acid content was decreased by 35% from 14.6% to 9.4% of total fatty acids. When amiFATB was expressed in a high-oleic acid transgenic line, it caused further increased oleic acid content. This work demonstrates that the FATB genes in camelina can be effectively knocked down by an artificial microRNA targeting gene-specific sequences, thus provides an additional tool to improve seed oils for desired properties.
    Mechanical load increase–induced changes in cytoskeletal structure and cellular barrier function in human cerebral endothelial cells
    Dongjoo Kim, Biotechnology and Bioengineering - 2018
    Globally, approximately a billion patients are estimated to suffer from neurological disorders. Although there are many therapeutic candidates for the central nervous system, treatment of brain disorders is restricted by the blood–brain barrier (BBB), which is a highly selective membrane that protects the brain from exogenous substances. This study was undertaken to develop a novel strategy to overcome the BBB and improve the efficiency of drug delivery to the brain by mechanical load increase using hypergravity. Human cerebral microvascular endothelial cells were exposed three times to 20 min hypergravity (10g), with a 20-min rest period between each exposure. The applied hypergravity reversibly decreased the cellular metabolic activity and increased the permeation rate of fluorescein sodium salt, fluorescein isothiocyanate–labeled dextran (FD-4), and fluorescein-labeled jacalin. Following the exposure to hypergravity, we also observed structural changes of the cytoskeleton and tight junctions, and an alteration in the expression levels of related genes. These results indicate that increased mechanical load due to the applied hypergravity affects the cytoskeletal arrangement and tight junctions, thereby weakening the cell barrier function and enhancing the permeability of the paracellular pathway. Thus, the mechanical load increase by hypergravity has the potential of being used as a novel strategy to overcome the BBB for brain drug delivery.
    The ATP-stimulated translocation promoter (ASTP) activity of glycerol kinase plays central role in adipogenesis
    Lilly S. Parr, Molecular Genetics and Metabolism - 2018
    Glycerol kinase (GK) is a multifunctional enzyme located at the interface of carbohydrate and fat metabolism. It contributes to both central carbon metabolism and adipogenesis; specifically, through its role as the ATP-stimulated translocation promoter (ASTP). GK overexpression leads to increased ASTP activity and increased fat storage in H4IIE cells. We performed metabolic flux analysis in human GK-overexpressing H4IIE cells and found that overexpressing cells had significantly altered fluxes through central carbon and lipid metabolism including increased flux through the pentose phosphate pathway and increased production of lipids. We also observed an equal contribution of glycerol to carbohydrate metabolism in all cell lines, suggesting that GK's alternate functions rather than its enzymatic function are important for these processes. To further elucidate the contributions of the enzymatic (phosphorylation) and alternative (ASTP) functions of GK in adipogenesis, we performed experiments on mammalian GK and E. coli GK. We determined that the ASTP function of GK (which is absent in E. coli GK) plays a greater role than the enzymatic activity in these processes. These studies further emphasize GK's diverse functionality and provides fundamental insights into the multiple protein functions of glycerol kinase.
    IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms
    Akash H. Verma, The Journal of Immunology - 2018
    Protection against microbial infection by the induction of inflammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the key players in driving these responses remain poorly defined. Recent work has identified a central role played by IL-1 in inducing innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1α/β, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R−/− mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17–dependent genes, indicating that this protection occurs via an alternative pathway to IL-1–driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell–driven protective pathways in immunity to oral C. albicans infection.
    Molecular cloning and characterization of a sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from Y-organs of the blue crab (Callinectes sapidus)
    Megan E. Roegner, Gene - 2018
    Existing data indicate that a Ca2+ signal stimulates ecdysteroid hormone production by crustacean molting glands (Y-organs). Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, with intracellular free Ca2+ maintained at a low basal level (typically sub-micromolar). This is achieved through the action of proteins intrinsic to the plasma membrane and the membranes of organelles. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the endoplasmic reticulum. As a step toward understanding Ca2+-mediated regulation of ecdysteroidogenesis, we have begun investigating Ca2+ transport proteins in Y-organs. In studies reported here, we used a PCR-based strategy to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative SERCA protein. The cloned Cas-SERCA cDNA (3806 bp) includes a 3057-bp open reading frame that encodes a 1019-residue protein (Cas-SERCA). The conceptually translated protein has a predicted molecular mass of 111.42 × 103 and contains all signature domains of an authentic SERCA, including ten transmembrane domains and a phosphorylation site at aspartate 351. A homology model of Cas-SERCA closely resembles models of related SERCA proteins. Phylogenetic analysis shows Cas-SERCA clusters with SERCA proteins from other arthropods. An assessment of tissue distribution indicates the Cas-SERCA transcript is widely distributed across tissues. Studies using quantitative PCR showed Cas-SERCA transcript abundance increased significantly in Y-organs activated by eyestalk ablation, a pattern consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs.
    Accelerated neural differentiation of mouse embryonic stem cells on aligned GYIGSR-functionalized nanofibers
    Elena A. Silantyeva, Acta Biomaterialia - 2018
    Substrates for embryonic stem cell culture are typified by poorly defined xenogenic, whole proteins or cellular components that are difficult and expensive to generate, characterize, and recapitulate. Herein, the generation of well-defined scaffolds of Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide-functionalized poly(ε-caprolactone) (PCL) aligned nanofibers are used to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells (mESCs). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation progression than D3 mESCs on laminin. Further, GYIGSR-functionalized nanofiber substrates yielded an increased gene expression of Sox1, a neural progenitor cell marker, and Tubb3, Cdh2, Syp, neuronal cell markers, at early time points. In addition, guidance of neurites was found to parallel the fiber direction. We demonstrate the fabrication of a well-defined, xeno-free functional nanofiber scaffold and demonstrates its use as a surrogate for xenogenic and complex matrixes currently used for the neural differentiation of stem cells ex vivo. Statement of Significance In this paper, we report the use of GYIGSR-functionalized poly(ε-caprolactone) aligned nanofibers as a tool to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells. The results indicate that functional nanofiber substrates promote faster differentiation than laminin coated substrates. The data suggest that aligned nanofibers and post-electrospinning surface modification with bioactive species can be combined to produce translationally relevant xeno-free substrates for stem cell therapy. Future development efforts are focused on additional bioactive species that are able to function as surrogates for other xenogenic factors found in differentiation media.
    Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria
    A. Bruce Cahoon, Current Genetics - 2018
    The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3′ untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3′ UTR upstream of the 5′ start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3′–5′ junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5′ ends (the start AUG) and a variable 3′ terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3′ ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3′ termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3′ termini of these mRNAs—adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.
    The Yeast Three-Hybrid System for Screening RNA-Binding Proteins in Plants
    Sung Ki Cho, Two-Hybrid Systems - 2018
    Yeast-hybrid methods have been successfully applied for screening interacting partners of DNAs or proteins. A yeast-based method, the yeast three-hybrid system, using a chimeric protein of a DNA-binding domain (LexA or GAL4BD) with a protein (MS2 coat protein or HIV Rev. M10) having a hybrid RNA at the 3′ end of a target RNA sequence, has been developed for screening RNA-binding proteins. When the target RNA interacts with RNA-binding proteins fused with an activation domain (AD), yeast cells having all the interacting components can survive on selection media, and interacting reporters, HIS3 and LacZ, are activated. Based on this selection, interaction can be easily monitored and detected by simple biochemical assays. The in vivo screening strategy has been widely applied for characterizing and evaluating specific interactions between target RNAs and RNA-binding proteins. Here, we describe a library screening strategy for isolating RNA-binding proteins of select target RNAs using the yeast three-hybrid method. We also describe strategies to verify binding specificity using both a yeast-dependent reporter system and a yeast-independent method, in vivo RNA immunoprecipitation (RIP).
    PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1
    Vitaliy V. Bondar, Human Molecular Genetics - 2018
    Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein’s stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.
    The distribution and detection of grapevine red blotch virus in its host depends on time of sampling and tissue type
    Felicia J Setiono, Plant Disease - 2018
    Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative qPCR method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a similar pattern to that found for the greenhouse-grown plants, but most strikingly close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.
    Porcine circovirus 2 infection induces IFNβ expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways
    Cheryl M. T.Dvorak, Virus Research - 2018
    Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNβ, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.
    Molecular network, pathway, and functional analysis of time-dependent gene changes related to cathepsin G exposure in neonatal rat cardiomyocytes
    Sanket Kumar Shukla, Gene - 2018
    The molecular pathways activated in response to acute cathepsin G (CG) exposure, as well as the mechanisms involved in activation of signaling pathways that culminate in myocyte detachment and apoptosis remain unclear. This study aimed to determine the changes in gene expression patterns associated with time dependent CG exposure to neonatal rat cardiomyocytes (NRCMs). Microarray analysis revealed a total of 451, 572 and 1127 differentially expressed genes after CG exposure at 1, 4 and 8 h respectively. A total of 54 overlapped genes at each time point were mapped by Ingenuity Pathway Analysis (IPA). The top up-regulated genes included Hamp, SMAD6, NR4A1, FOSL2, ID3 and SLAMF7, and down-regulated genes included CYR61, GDF6, Olr640, Vom2r36, DUSP6 and MMP20. Our data suggest that there are multiple deregulated pathways associated with cardiomyocyte death after CG exposure, including JAK/Stat signaling, IL-9 signaling and Nur77 signaling. In addition, we also generated the molecular network of expressed gene and found most of the molecules were connected to ERK1/2, caspase, BCR (complex) and Cyclins. Our study reveals the ability to assess time-dependent changes in gene expression patterns in NRCMs associated with CG exposure. The global gene expression profiles may provide insight into the cellular mechanism that regulates CG dependent myocyte apoptosis. In future, the pathways important in CG response, as well as the genes found to be differentially expressed might represent the therapeutic targets for myocyte survival in heart failure.
    Treatment of limb wounds of horses with orf virus IL-10 and VEGF-E accelerates resolution of exuberant granulation tissue, but does not prevent its development
    Lyn M. Wise, PLOS ONE - 2018
    Bandaging of limb wounds in horses leads to formation of exuberant granulation tissue (EGT) that retards healing due to protracted inflammation, aberrant vascularisation and delayed epithelialisation. EGT is not observed if wounds are left undressed or when wounds are on the body. A previous study showed that short-term administration of proteins derived from orf virus dampened inflammation and promoted epithelialisation of open wounds in horses. Here, we investigated the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on the development and resolution of EGT. Excisional wounds were created on the forelimb of four horses, and bandages were maintained until full healing to induce EGT formation. Matching body wounds were created to ensure EGT was limited to the limb, and to differentiate the effects of the viral proteins on normal healing and on EGT formation. Viral proteins or the hydrogel vehicle control were administered topically to site-matched wounds at day 1, with repeat administration at day 8. Wound healing and EGT formation were monitored macroscopically. Wound margin samples were harvested at 2, 7 and 14 days, and at full healing, with histology used to observe epithelialisation, immunofluorescence used to detect inflammatory cells, angiogenesis and cell death, and qPCR to measure expression of genes regulating inflammation and angiogenesis. Limb wounds developed EGT, and exhibited slower healing than body wounds. Viral protein treatment did not accelerate healing at either location nor limit EGT formation in limb wounds. Treatment of limb wounds did however increase epithelialisation and angiogenesis, without dampening inflammatory cell infiltration or gene expression. The healed wounds also had less occlusion and death of blood vessels and fewer epidermal rete ridges following viral protein treatment. These findings indicate that the viral protein treatment does not suppress wound inflammation or EGT formation, but does promote vascular and epidermal repair and EGT resolution.
    Association of residual feed intake with abundance of ruminal bacteria and biopolymer hydrolyzing enzyme activities during the peripartal period and early lactation in Holstein dairy cows
    Ahmed A. Elolimy, Journal of Animal Science and Biotechnology - 2018
    Residual feed intake (RFI) in dairy cattle typically calculated at peak lactation is a measure of feed efficiency independent of milk production level. The objective of this study was to evaluate differences in ruminal bacteria, biopolymer hydrolyzing enzyme activities, and overall performance between the most- and the least-efficient dairy cows during the peripartal period. Twenty multiparous Holstein dairy cows with daily ad libitum access to a total mixed ration from d − 10 to d 60 relative to the calving date were used. Cows were classified into most-efficient (i.e. with low RFI, n = 10) and least-efficient (i.e. with high RFI, n = 10) based on a linear regression model involving dry matter intake (DMI), fat-corrected milk (FCM), changes in body weight (BW), and metabolic BW.
    Different adaptive strategies in E. coli populations evolving under macronutrient limitation and metal ion limitation
    Omar M. Warsi, BMC Evolutionary Biology - 2018
    Adaptive responses to nutrient limitation involve mutations that increase the efficiency of usage or uptake of the limiting nutrient. However, starvation of different nutrients has contrasting effects on physiology, resulting in different evolutionary responses. Most studies performed to understand these evolutionary responses have focused only on macronutrient limitation. Hence our understanding of adaptation under limitation of other forms of nutrients is limited. In this study, we compared the evolutionary response in populations evolving under growth-limiting conditions for a macronutrient and a major cation.
    Cocoa procyanidins modulate transcriptional pathways linked to inflammation and metabolism in human dendritic cells
    Helene L. E. Midttun, Food & Function - 2018
    Foods rich in polyphenols such as procyanidins (PC) have been proposed to have anti-inflammatory properties, and we have previously reported inhibition of lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in human dendritic cells (DCs) by PC derived from cocoa. To explore the mechanistic basis of this inhibition, here we conducted transcriptomic analysis on DCs cultured with either LPS or LPS combined with oligomeric cocoa PC. Procyanidins suppressed a number of genes encoding cytokines and chemokines such as CXCL1, but also genes involved in the cGMP pathway such as GUCY1A3 (encoding guanylate cyclase soluble subunit alpha-3). Upregulated genes were involved in diverse metabolic pathways, but notably two of the four most upregulated genes (NMB, encoding neuromedin B and ADCY3, encoding adenyl cyclase type 3) were involved in the cAMP signalling pathway. Gene-set enrichment analysis demonstrated that upregulated gene pathways were primarily involved in nutrient transport, carbohydrate metabolism and lysosome function, whereas down-regulated gene pathways involved cell cycle, signal transduction and gene transcription, as well as immune function. qPCR analysis verified differential expression of GUCY1A3, ADCY3, NMB as well as a number of other genes, and marked suppression of LPS-induced CXCL1 and IL-23 protein secretion was also observed. Thus, our results confirm a marked anti-inflammatory effect of PC in human DCs, which may be related mechanistically to second-messenger function and metabolic activity. Our results provide a foundation to further investigate metabolic pathways altered by PC during intestinal inflammation, and further encourage investigation of the health-promoting potential of PC-rich functional foods.
    The Phenotypic Effects of Exosomes Secreted from Distinct Cellular Sources: a Comparative Study Based on miRNA Composition
    Scott Ferguson, The AAPS Journal - 2018
    Exosomes are nano-sized vesicles composed of lipids, proteins, and nucleic acids. Their molecular landscape is diverse, and exosomes derived from different cell types have distinct biological activities. Since exosomes are now being utilized as delivery vehicles for exogenous therapeutic cargoes, their intrinsic properties and biological effects must be understood. We performed miRNA profiling and found substantial differences in the miRNA landscape of prostate cancer (PC3) and human embryonic kidney (HEK) 293 exosomes with little correlation in abundance of common miRNAs (R2 = 0.16). Using a systems-level bioinformatics approach, the most abundant miRNAs in PC3 exosomes but not HEK exosomes were predicted to significantly modulate integrin signaling, with integrin-β3 loss inducing macrophage M2 polarization. PC3 but not HEK exosomes downregulated integrin-β3 expression levels by 70%. There was a dose-dependent polarization of RAW 264.7 macrophages toward an M2 phenotype when treated with PC3-derived exosomes but not HEK-derived exosomes. Conversely, HEK exosomes, widely utilized as delivery vehicles, were predicted to target cadherin signaling, with experimental validation showing a significant increase in the migratory potential of MCF7 breast cancer cells treated with HEK exosomes. Even widely utilized exosomes are unlikely to be inert, and their intrinsic activity ought to be assessed before therapeutic deployment.
    Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon-gamma-mediated Tryptophan Limitation
    Scot P. Ouellette, Infection and Immunity - 2018
    As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon-γ (IFNγ), the tryptophan (trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during trp starvation with transcripts for trp-codon containing genes disproportionately increased. Yet, the presence of trp codons destabilized 3′ ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on trp codons rendered the 3′ ends sensitive to ribonuclease activity. The half-life of chlamydial transcripts containing different numbers of trp codons was thus measured in untreated and IFNγ-treated infected cells to determine whether trp codons influenced the stability of transcripts. However, no effect of trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a mid-cycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, as well as overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3′ to trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.
    Probiotics Strains Modulate Gut Microbiota and Lipid Metabolism in Mule Ducks
    Maxime Even, The Open Microbiology Journal - 2018
    Probiotics Strains Modulate Gut Microbiota and Lipid Metabolism in Mule Ducks
    The influence of oxygen and methane on nitrogen fixation in subarctic
    Martine A. R. Kox, AMB Express - 2018
    Biological nitrogen fixation is an important source of bioavailable nitrogen in Sphagnum dominated peatlands. Sphagnum mosses harbor a diverse microbiome including nitrogen-fixing and methane (CH4) oxidizing bacteria. The inhibitory effect of oxygen on microbial nitrogen fixation is documented for many bacteria. However, the role of nitrogen-fixing methanotrophs in nitrogen supply to Sphagnum peat mosses is not well explored. Here, we investigated the role of both oxygen and methane on nitrogen fixation in subarctic Sphagnum peat mosses. Five species of Sphagnum mosses were sampled from two mesotrophic and three oligotrophic sites within the Lakkasuo peatland in Orivesi, central Finland. Mosses were incubated under either ambient or low oxygen conditions in the presence or absence of methane. Stable isotope activity assays revealed considerable nitrogen-fixing and methane-assimilating rates at all sites (1.4 ± 0.2 µmol 15N–N2 g−1 DW day−1 and 12.0 ± 1.1 µmol 13C–CH4 g−1 DW day−1, respectively). Addition of methane did not stimulate incorporation of 15N-nitrogen into biomass, whereas oxygen depletion increased the activity of the nitrogen-fixing community. Analysis of the 16S rRNA genes at the bacterial community level showed a very diverse microbiome that was dominated by Alphaproteobacteria in all sites. Bona fide methane-oxidizing taxa were not very abundant (relative abundance less than 0.1%). Based on our results we conclude that methanotrophs did not contribute significantly to nitrogen fixation in the investigated peatlands.
    Orsay δ protein is required for non-lytic viral egress
    Wang Yuan, Journal of Virology - 2018
    Non-enveloped gastrointestinal viruses such as human rotavirus can exit infected cells from the apical surface without cell lysis. The mechanism of such non-lytic exit is poorly understood. The non-enveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans. Dye staining results suggested that Orsay exits from the intestine of infected worms in a non-lytic manner. Therefore, the Orsay-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had normal CP-δ fusion but could not produce free δ due to lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells in C. elegans and co-localized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay infection rearranged ACT-5 apical localization. Reduction of ACT-5 level via RNAi significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay exit. Together, these data support a model that the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate polarized, non-lytic egress of the Orsay virus. Importance An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain non-enveloped viruses can exit cultured cells in non-lytic ways, however, such non-lytic exit has not been demonstrated in vivo. In addition, it is not clear how such non-lytic exit is achieved mechanistically in vivo. Orsay is a non-enveloped RNA virus that infects the intestine cells of the nematode C. elegans. It is currently the only virus known to naturally infect C. elegans. Using this in vivo model, we show that the δ protein encoded by Orsay facilitates the non-lytic exit of the virus, possibly by interacting with host actin on the apical side of the worm intestine cells.
    The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function
    Yenkel Grinberg-Bleyer, The Journal of Immunology - 2018
    CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-κB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-κB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-κB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity.
    LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as Ca. Liberibacter asiaticus Infection in Nicotiana benthamiana
    Marco Pitino, Frontiers in Plant Science - 2018
    Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. HLB is associated with a phloem-limited α-proteobacterium, Candidatus Liberibacter asiaticus (Las). Las infection causes yellow shoots and blotchy mottle on leaves and is associated with excessive starch accumulation. However, the mechanisms underlying the starch accumulation remain unknown. We previously showed that the Las5315mp effector induced callose deposition and cell death in Nicotiana benthamiana. In this study, we demonstrated that Las can experimentally infect N. benthamiana via dodder transmission. Furthermore, we revealed another key function of the Las5315 effector by demonstrating that transient expression of the truncated form of the effector, LasΔ5315, induced excessive starch accumulation by 6 fold after 8 dpi in N. benthamiana after removal of the chloroplast transit peptide from the Las5315mp. The induction mechanisms of LasΔ5315 in N. benthamiana were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant N. benthamiana. Using this model plant, we demonstrated that the LasΔ5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in N. benthamiana. Altogether the Las 5315 effector is critical for Las pathogenesis, and therefore, an important target for interference.
    Development-related aberrations in Kv1.1 α-subunit exert disruptive effects on bioelectrical activities of neurons in a mouse model of fragile X syndrome
    Pingping Zhu, Progress in Neuro-Psychopharmacology and Biological Psychiatry - 2018
    Kv1.1, a Shaker homologue potassium channel, plays a critical role in homeostatic regulation of neuronal excitability. Aberrations in the functional properties of Kv1.1 have been implicated in several neurological disorders featured by neuronal hyperexcitability. Fragile X syndrome (FXS), the most common form of inherited mental retardation, is characterized by hyperexcitability in neural network and intrinsic membrane properties. The Kv1.1 channel provides an intriguing mechanistic candidate for FXS. We investigated the development-related expression pattern of the Kv1.1 α-subunit by using a Fmr1 knockout (KO) mouse model of FXS. Markedly decreased protein expression of Kv1.1 was found in neonatal and adult stages when compared to age-matched wild-type (WT) mice. Immunohistochemical investigations supported the delayed development-related increases in Kv1.1 expression, especially in CA3 pyramidal neurons. By applying a Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ), we isolated the Kv1.1-mediated currents in the CA3 pyramidal neurons. The isolated DTX-κ-sensitive current of neurons from KO mice exhibited decreased amplitude, lower threshold of activation, and faster recovery from inactivation. The equivalent reduction in potassium current in the WT neurons following application of the appropriate amount of DTX-κ reproduced the enhanced firing abilities of KO neurons, suggesting the Kv1.1 channel as a critical contributor to the hyperexcitability of KO neurons. The role of Kv1.1 in controlling neuronal discharges was further supported by the parallel developmental trajectories of Kv1.1 expression, current amplitude, and discharge impacts, with a significant correlation between the amplitude of Kv1.1-mediated currents and Kv1.1-blocking-induced firing enhancement. These data suggest that the expression of the Kv1.1 α-subunit has a profound pathological relevance to hyperexcitability in FXS, as well as implications for normal development, maintenance, and control of neuronal activities.
    The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows
    Y. Qu, Journal of Dairy Science - 2018
    The 4 major tocopherol isoforms differ in their biochemical reactivity and cellular effects due to basic chemical structural differences. Alpha-tocopherol has been well studied regarding effects on bovine polymorphonuclear leukocyte (PMN) function and its involvement in respiratory burst. However, no studies to date have identified the effects of supplementing a mixed tocopherol oil (Tmix) particularly enriched in non-α tocopherol isoforms (i.e., γ- and δ-isoforms) on fundamental immunometabolic changes in dairy cows. Therefore, the objectives of this study were to determine whether short-term feeding of vegetable oil–derived Tmix alters specific biomarkers of metabolism, whole-blood leukocyte populations, respiratory burst, immunometabolic-related gene expression of PMN, or gene expression of isolated PMN when challenged with lipopolysaccharides (LPS). Clinically healthy multiparous lactating Holstein cows (n = 12; 179 ± 17 d in milk, 40.65 ± 3.68 kg of milk yield) were fed Tmix (620 g/d) for 7 consecutive days. Jugular blood (EDTA anticoagulant) was collected from all cows on d 0 before treatment initiation and again on d 7 after Tmix feeding. Total stimulated respiratory burst activity (RBA) and leukocyte populations were assessed in whole blood, and tocopherol isoform concentrations, metabolites, and hormones were measured in plasma. For gene expression analysis, isolated PMN from cows before and after Tmix feeding were incubated with LPS at a final concentration of either 0.0 or 1.5 µg/mL. Feeding of Tmix for 7 d increased the concentrations of α- and γ-tocopherol. The Tmix did not alter plasma insulin but decreased cholesterol. The Tmix did not alter whole-blood RBA or the leukocyte populations. The LPS challenge increased the expression of proinflammatory genes TNFA and IL6. However, Tmix treatment did not alter the patterns of LPS-affected expression of genes (e.g., TNFA, ITGB2, PPARA, and RXRA) associated with the immune or metabolic response. In conclusion, short-term feeding of Tmix may have no negative effect on animal health as Tmix increased α- and γ-tocopherol concentrations in blood and did not impair whole-blood RBA or alter leukocyte populations. The data provide further support that the α- and γ-tocopherol isoforms do not interfere with normal immune or metabolic function.
    Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiac Hypertrophy Through p90 Ribosomal S6 Kinase
    Nabeel Abdulrahman, Physiological Genomics - 2018
    Cardiovascular diseases are the leading cause of death worldwide. One in three cases of heart failure is due to dilated cardiomyopathy. The Na+/H+ exchanger isoform 1 (NHE1), a multifunctional protein and the key pH regulator in the heart, has been demonstrated to be increased in this condition. We have previously demonstrated that elevated NHE1 activity induced cardiac hypertrophy in vivo. Furthermore, the overexpression of active NHE1 elicited modulation of gene expression in cardiomyocytes including an upregulation of myocardial osteopontin (OPN) expression. To determine the role of OPN in inducing NHE1 mediated cardiomyocyte hypertrophy, a double transgenic mouse expressing active NHE1 and OPN knock-out were generated and assessed by echocardiography and the cardiac phenotype. Our studies showed that hearts expressing active NHE1 exhibited cardiac remodeling indicated by increased systolic and diastolic left ventricular internal diameter and increased ventricular volume. Moreover, these hearts demonstrated impaired function with decreased fractional shortening and ejection fraction. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA was upregulated, and there was an increase in heart cell cross sectional area confirming the cardiac hypertrophic effect. Moreover, NHE1 transgenic mice also showed increased collagen deposition, upregulation of CD44 and phosphorylation of p90 ribosomal s6 kinase (RSK), effects that were regressed in OPN knock-out mice. In conclusion, we developed an interesting comparative model of active NHE1 transgenic mouse lines which express a dilated hypertrophic phenotype expressing CD44 and phosphorylated RSK, effects which are regressed in absence of OPN.
    Inhibition of tomato fruit ripening by 1-MCP, wortmannin and hexanal is associated with a decrease in transcript levels of phospholipase D and other ripening related genes
    Mohd Sabri Pak Dek, Postharvest Biology and Technology - 2018
    Membrane deterioration is an inherent aspect of the advancement in senescence and loss in fruit quality during storage. Postharvest technologies used for extending shelf life and quality are targeted to reduce membrane damage through downregulating or blocking ethylene action. In this study, mature green tomato fruit were treated with inhibitors of ethylene receptor (ETR), phosphatidylinositol 3-kinase (PI3K) and phospholipase D (PLD), all recognized to be targets of regulation of fruit ripening. The inhibitors used included 1-methylcyclopropene (1-MCP, an ethylene receptor blocker), wortmannin (an inhibitor of PI3K), and hexanal (a PLD inhibitor). Fruit were treated at optimal levels of the inhibitors and were stored at 21 °C for 10 days. Color development was strongly delayed in wortmannin treated tomatoes just as in 1-MCP treated fruit; while, changes in respiration, firmness and ethylene evolution were very similar to that of control fruit. Hexanal delayed the initiation of these changes; while 1-MCP and wortmannin blocked the ripening process. Changes in expression levels of key genes involved in ethylene signalling, phosphoinositide metabolism, and lycopene synthesis that occurred in response to inhibitors, suggested potential roles for PI3K and PLD in ethylene signalling. Furthermore, fruit treated with all the three inhibitors showed a marked reduction in PLD transcript levels; suggesting that, regulation of PLD gene expression is a common critical regulatory point that regulates ripening. Lowered PLD levels may reduce membrane lipid catabolism and the generation of phosphatidic acid (PA), an intermediate in ethylene signalling regulation through downstream components.
    Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia
    Rafiou Agoro, The FASEB Journal - 2018
    Severe anemia and iron deficiency are common complications in chronic kidney disease. The cause of renal anemia is multifactorial and includes decreased erythropoietin (Epo) production, iron deficiency, and inflammation, and it is currently treated with injections of synthetic Epo. However, the use of recombinant Epo has several adverse effects. We previously reported that high fibroblast growth factor 23 (FGF23) levels in mice are associated with decreased red blood cell production, whereas genetic inactivation of Fgf23 results in expansion of the erythroid lineage. The present study is the first to show that high FGF23 levels in a mouse model of renal failure contribute to renal anemia, and inhibiting FGF23 signaling stimulates erythropoiesis and abolishes anemia and iron deficiency. Moreover, we show that inhibition of FGF23 signaling significantly decreases erythroid cell apoptosis and influences the commitment of hematopoietic stem cells toward the erythroid linage. Furthermore, we show that blocking FGF23 signaling attenuates inflammation, resulting in increased serum iron and ferritin levels. Our data clearly demonstrate that elevated FGF23 is a causative factor in the development of renal anemia and iron deficiency, and importantly, blocking FGF23 signaling represents a novel approach to stimulate erythropoiesis and possibly improve survival for millions of chronic kidney disease patients worldwide.—Agoro, R., Montagna, A., Goetz, R., Aligbe, O., Singh, G., Coe, L. M., Mohammadi, M., Rivella, S., Sitara, D. Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia.
    Blood Glutamate Scavenger as a novel neuroprotective treatment in spinal cord injury
    Yona Goldshmit, Journal of Neurotrauma - 2018
    Neurotrauma causes immediate elevation of extracellular glutamate levels, which creates excitotoxicity and facilitates inflammation, glial scar formation and consequently neuronal death. Finding factors that reduce the inflammatory response, glial scar formation and increase neuronal survival and neurite outgrowth, are of major importance for improving the outcome after spinal cord injury (SCI). In the present study, we evaluated a new treatment aiming to remove CNS glutamate into the systemic blood circulation by intravenous administration of blood glutamate scavengers (BGS) such as recombinant enzyme glutamate-oxaloacetate transaminase (rGOT1) and its co-substrate. In this study we induced in mice a spinal cord injury (hemisection), and one-hour post injury started administering BGS treatment for five consecutive days. The treatment reduced the expression levels of p-p38, which regulates apoptosis and increased the expression of p-Akt, which mediates cell survival. Moreover, this treatment decreased pro-inflammatory cytokine expression and microglia activation, reduced astrocytes’ reactivity and facilitated expression of radial glia markers such as Pax6 and nestin. BGS treatment increased the survival of neurons at lesion site and enabled axonal regeneration into the injury site. These effects were correlated with improved functional recovery of the left paretic hindlimb. Thus, early pharmacological intervention with BGS following SCI may be neuroprotective and create a pro-regenerative environment by modulating glia cell response. In light of our results, the availability of the method to remove excess glutamate from CNS without the need to deliver drugs across the blood-brain barrier (BBB) and with minimal or no adverse effects may provide a major therapeutic asset.
    Safety Analysis of Leishmania Vaccine Used in a Randomized Canine Vaccine/Immunotherapy Trial
    Mandy Larson, ASTMH - 2018
    In Leishmania infantum–endemic countries, controlling infection within dogs, the domestic reservoir, is critical to public health. There is a need for safe vaccines that prevent canine progression with disease and transmission to others. Protective vaccination against Leishmania requires mounting a strong, inflammatory, Type 1 response. Three commercially available canine vaccines on the global veterinary market use saponin or inflammatory antigen components (Letifend) as a strong pro-inflammatory adjuvant. There is very little information detailing safety of saponin as an adjuvant in field trials. Safety analyses for the use of vaccine as an immunotherapeutic in asymptomatically infected animals are completely lacking. Leishmania infantum, the causative agent of canine leishmaniasis, is enzootic within U.S. hunting hounds. We assessed the safety of LeishTec® after use in dogs from two different clinical states: 1) without clinical signs and tested negative on polymerase chain reaction and serology or 2) without clinical signs and positive for at least one Leishmania diagnostic test. Vaccine safety was assessed after all three vaccinations to quantify the number and severity of adverse events. Vaccinated animals had an adverse event rate of 3.09%, whereas placebo animals had 0.68%. Receiving vaccine was correlated with the occurrence of mild, site-specific, reactions. Occurrence of severe adverse events was not associated with having received vaccine. Infected, asymptomatic animals did not have a higher rate of adverse events. Use of vaccination is, therefore, likely to be safe in infected, asymptomatic animals.
    Altered DNA methylation is associated with aberrant gene expression in parenchymal but not airway fibroblasts isolated from individuals with COPD
    Rachel L. Clifford, Clinical Epigenetics - 2018
    Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms, including DNA methylation, have the potential to mediate the interactions between an individual’s genetics and environmental exposure. DNA methylation is highly cell type-specific, and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in the airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD.
    Polycyclic aromatic hydrocarbon exposure in seaside sparrows (Ammodramus maritimus) following the 2010 Deepwater Horizon oil spill
    Anna A. Perez-Umphrey, Science of The Total Environment - 2018
    The seaside sparrow (Ammodramus maritimus) is an abundant and permanent resident of coastal salt marshes impacted by the 2010 BP Deepwater Horizon oil spill. Such terrestrial species are often overlooked in the aftermath of marine spills, despite the potential for long-term oil exposure. We sampled the livers of seaside sparrows residing in oiled and unoiled sites from 2011 to 2014 and quantified expression of cytochrome p450 1A (CYP1A), a gene involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). In August 2011, CYP1A expression was markedly higher in birds from an oiled site compared to an unoiled site, but differences had disappeared by June 2012. In June 2013, CYP1A expression was elevated compared to 2012 levels on all sites, including those collected from sites that had not been directly oiled during the spill. This rise in CYP1A expression was possibly due to Hurricane Isaac, which made landfall near our sites between the 2012 and 2013 sampling periods. CYP1A expression was significantly attenuated again in June 2014. We also collected sediment samples from the same marshes for a total concentration analysis of PAHs. The PAH concentrations in sediment samples exhibited a similar pattern to the CYP1A data, supporting the link between marsh PAHs and bird CYP1A expression. These results indicate that contamination from marine oil spills can immediately extend to terrestrial ecosystems, and that storms, weather, or other factors may influence subsequent spatial and temporal oil exposure for several additional years.
    Steroid Receptor Coactivator-1 Knockdown Decreases Synaptic Plasticity and Impairs Spatial Memory in the Hippocampus of Mice
    Chen Bian, Neuroscience - 2018
    Steroids have been demonstrated to play profound roles in the regulation of hippocampal function by acting on their receptors, which need coactivators for their transcriptional activities. Previous studies have shown that steroid receptor coactivator-1 (SRC-1) is the predominant coactivator in the hippocampus, but its exact role and the underlying mechanisms remain unclear. In this study, we constructed SRC-1 RNA interference (RNAi) lentiviruses, injected them into the hippocampus of male mice, and then examined the changes in the expression of selected synaptic proteins, CA1 synapse density, postsynaptic density (PSD) thickness, and in vivo long-term potentiation (LTP). Spatial learning and memory behavior changes were investigated using the Morris water maze. We then transfected the lentiviruses into cultured hippocampal cells and examined the changes in synaptic protein and phospho-cyclic AMP response element-binding protein (pCREB) expression. The in vivo results showed that SRC-1 knockdown significantly decreased the expression of synaptic proteins and CA1 synapse density as well as PSD thickness; SRC-1 knockdown also significantly impaired in vivo LTP and disrupted spatial learning and memory. The in vitro results showed that while the expression of synaptic proteins was significantly decreased by SRC-1 knockdown, pCREB expression was also significantly decreased. The above results suggest a pivotal role of SRC-1 in the regulation of hippocampal synaptic plasticity and spatial learning and memory, strongly indicating SRC-1 may serve as a novel therapeutic target for hippocampus-dependent memory disorders.
    Inhibition of pMAPK14 Overcomes Resistance to Sorafenib in Hepatoma Cells with Hepatitis B Virus
    Dvora Witt-Kehati, Translational Oncology - 2018
    Hepatitis B virus (HBV) targets the liver and is a major driver for liver cancer. Clinical data suggest that HBV infection is associated with reduced response to treatment with the multi-kinase inhibitor sorafenib, the first available molecularly targeted anti-hepatocellular carcinoma (HCC) drug. Given that Raf is one of the major targets of sorafenib, we investigated the activation state of the Raf-Mek-Erk pathway in the presence of HBV and in response to sorafenib. Here we show that hepatoma cells with replicating HBV are less susceptible to sorafenib inhibitory effect as compared to cells in which HBV expression is suppressed. However, although HBV replication is associated with increased level of pErk, its blockade only modestly augments sorafenib effect. In contrast, the phosphorylated form of the pro-oncogenic Mitogen-Activated Protein Kinase 14 (pMAPK14), a protein kinase that was recently linked to sorafenib resistance, is induced in sorafenib-treated hepatoma cells in association with HBV X protein expression. Knocking down pMAPK14 results in augmentation of the therapeutic efficacy of sorafenib and largely alleviates resistance to sorafenib in the presence of HBV. Thus, this study suggests that HBV promotes HCC resistance to sorafenib. Combining pMAPK14 inhibitors with sorafenib may be beneficial in patients with HBV-associated HCC.
    Implant delivering hydroxychloroquine attenuates vaginal T lymphocyte activation and inflammation
    Yufei Chen, Journal of Controlled Release - 2018
    Evidence suggests that women who are naturally resistant to HIV infection exhibit low baseline immune activation at the female genital tract (FGT). This “immune quiescent” state is associated with lower expression of T-cell activation markers, reduced levels of gene transcription and pro-inflammatory cytokine or chemokine production involved in HIV infection while maintaining an intact immune response against pathogens. Therefore, if this unique immune quiescent state can be pharmacologically induced locally, it will provide an excellent women-oriented strategy against HIV infection To our knowledge, this is the first research article evaluating in vivo, an innovative trackable implant that can provide controlled delivery of hydroxychloroquine (HCQ) to successfully attenuate vaginal T lymphocyte activation and inflammation in a rabbit model as a potential strategy to induce an “immune quiescent” state within the FGT for the prevention of HIV infection. This biocompatible implant can deliver HCQ above therapeutic concentrations in a controlled manner, reduce submucosal immune cell recruitment, improve mucosal epithelium integrity, decrease protein and gene expression of T-cell activation markers, and attenuate the induction of key pro-inflammatory mediators. Our results suggest that microbicides designed to maintain a low level of immune activation at the FGT may offer a promising new strategy for reducing HIV infection.
    Effect of contractile activity on PGC-1α transcription in young and aged skeletal muscle
    Heather N. Carter, Journal of Applied Physiology - 2018
    Mitochondrial impairments are often noted in aged skeletal muscle. The transcriptional coactivator PGC-1α is integral to maintaining mitochondria, and its expression declines in aged muscle. It remains unknown whether this is due to a transcriptional deficit during aging. Our study examined PGC-1α transcription in muscle from young and old F344BN rats. Using a rat PGC-1α promoter-reporter construct, we found that PGC-1α transcription was reduced by ~65% in aged TA muscle, accompanied by decreases in PGC-1α mRNA and transcript stability. Altered expression patterns in PGC-1α transcription regulatory factors, including Nrf2, USF1, ATF2 and YY1, were noted in aged muscle. Acute contractile activity (CA) followed by recovery was employed to examine whether PGC-1α transcription could be activated in aged muscle similar to that observed in young muscle. AMPK and p38 signaling was attenuated in aged muscle. CA evoked an upregulation of PGC-1α transcription in both young and aged groups, while mRNAs encoding PGC-1α and COX IV were induced during the recovery period. Global DNA methylation, an inhibitory event for transcription, was enhanced in aged muscle, likely a result of elevated methyltransferase enzyme Dnmt3b in aged muscle. Successive bouts of CA for 7 days to evaluate longer-term consequences resulted in a rescue of PGC-1α and downstream mRNAs in aged muscle. Our data indicate that diminished mitochondria in aged muscle is partly due to a deficit in PGC-1α transcription, a result of attenuated upstream signaling. Contractile activity is an appropriate countermeasure to restore PGC-1α expression and mitochondrial content in aged muscle.
    Overexpression of miR169o, an Overlapping MicroRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice
    Chao Yu, Plant and Cell Physiology - 2018
    Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs (miRNAs) in this phenomenon, 63 differentially expressed overlapping miRNAs in response to Xoo infection and N limitation stress in rice were identified through deep RNA sequencing and stem–loop quantitative real-time PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than the wild type (WT). Transcript level assays showed that under different N supply conditions, miR169o oppositely regulated NRT2, and this is reduced under normal N supply conditions but remarkably induced under N-limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with the WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to BB in rice. Consistently, transient expression of NF-YA genes in rice protoplasts promoted the transcripts of PR genes and NRT2 genes, while it reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the co ordinated regulatory mechanisms of cross-talk between Xoo infection and N deficiency responses in rice.
    Continuous Exposure to Simulated Hypergravity-Induced Changes in Proliferation, Morphology, and Gene Expression of Human Tendon Cells
    Raquel Costa-Almeida, Stem Cells and Development - 2018
    Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention toward a better understanding of microgravity-, hypogravity (<1 g)-, or hypergravity (>1 g)-induced alterations. In this work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15, and 20 g for 4 or 16 h, using a large diameter centrifuge. Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10, and 15 g for 16 h, in comparison to 1 g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10 g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency toward an upregulation of tenogenic markers was observed; scleraxis (SCX), tenascin C (TNC), collagen type III (COL3A1), and decorin (DCN) were significantly upregulated in hTDCs cultured at 15 g and COL3A1 and DCN were significantly upregulated in hTDCs cultured at 20 g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation.
    High-fat diet modifies expression of hepatic cellular senescence gene p16(INK4a) through chromatin modifications in adult male rats
    Xiyuan Zhang, Genes & Nutrition - 2018
    Liver is the crucial organ as a hub for metabolic reactions. p16(INK4a) is a well-established cyclin-dependent kinase (CDK) inhibitor that plays important role in the molecular pathways of senescence, which lead to irreversible cell cycle arrest with secretion of proinflammatory cytokines and mitochondrial dysfunction. This study tested the hypothesis that cellular senescence regulated by p16(INK4a) is associated with high-fat diet in adult male rats.
    High calcium, phosphate and calcitriol supplementation leads to an osteocyte-like phenotype in calcified vessels and bone mineralisation defect in uremic rats
    Sarah-Kim Bisson, Journal of Bone and Mineral Metabolism - 2018
    A link between vascular calcification and bone anomalies has been suggested in chronic kidney disease (CKD) patients with low bone turnover disease. We investigated the vascular expression of osteocyte markers in relation to bone microarchitecture and mineralization defects in a model of low bone turnover CKD rats with vascular calcification. CKD with vascular calcification was induced by 5/6 nephrectomy followed by high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). CKD + Ca/P/VitD group (n = 12) was compared to CKD + normal diet (n = 12), control + normal diet (n = 8) and control + Ca/P/VitD supplementation (n = 8). At week 6, tibia, femurs and the thoracic aorta were analysed by Micro-Ct, histomorphometry and for expression of osteocyte markers. High Ca/P/VitD treatment induced vascular calcification only in CKD rats, suppressed serum parathyroid hormone levels and led to higher sclerostin, DKK1 and FGF23 serum levels. Expression of sclerostin, DKK1 and DMP1 but not FGF23 were increased in calcified vessels from CKD + Ca/P/VitD rats. Despite low parathyroid hormone levels, tibia bone cortical thickness was significantly lower in CKD + Ca/P/VitD rats as compared to control rats fed a normal diet, which is likely the result of radial growth impairment. Finally, Ca/P/VitD treatment in CKD rats induced a bone mineralization defect, which is likely explained by the high calcitriol dose. In conclusion, Ca/P/VitD supplementation in CKD rats induces expression of osteocyte markers in vessels and bone mineralisation anomalies. Further studies should evaluate the mechanisms of high dose calcitriol-induced bone mineralisation defects in CKD.
    The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis
    Jianqiang Bao, Nucleic Acids Research - 2018
    CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development. Loss of Carm1 led to a low sperm count and deformed sperm heads that can be attributed to defective elongation of round spermatids. RNA-seq analysis of Carm1-null spermatids revealed that the deregulated genes fell into similar categories as those impacted by p300-loss, thus providing a link between Carm1 and p300. Importantly, p300 has long been known to be a major Carm1 substrate. We found that CREMτ, a key testis-specific transcription factor, associates with p300 through its activator, ACT, and that this interaction is negatively regulated by the methylation of p300 by Carm1. Thus, high nuclear Carm1 levels negatively impact the p300•ACT•CREMτ axis during late stages of spermiogenesis.
    In vitro bioassessment of the immunomodulatory activity of Saccharomyces cerevisiae components using bovine macrophages and Mycobacterium avium ssp. paratuberculosis
    Z. Li, Journal of Dairy Science - 2018
    The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor β, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection.
    Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide
    Robert M. Immormino, American Journal of Physiology-Lung Cellular and Molecular Physiology - 2018
    Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AM) in the steady-state, but is dramatically up-regulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AM with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AM was dependent upon the MyD88 signaling pathway and the transcription factor nuclear factor kappa B (NFκB). In addition to up-regulating display of NRP2 on the cell membrane, inhaled LPS also triggered AM to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine Ccl2 in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AM regulates LPS-induced inflammatory cell recruitment to the airways, and reveal a novel role for NRP2 during innate immune responses in the lungs.
    Genome-wide expression analysis suggests a role for jasmonates in the resistance to blue mold in apple
    Masoud Ahmadi-Afzadi, Plant Growth Regulation - 2018
    Blue mold, caused by the necrotrophic fungal pathogen Penicillium expansum, causes serious postharvest losses in apple, and threatens human health through production of the potent mycotoxin patulin. Recent studies indicate a quantitative control of resistance against this disease in apple cultivars. A whole genome apple microarray covering 60k transcripts was used to identify gene(s) that appear to be differentially regulated between resistant and susceptible cultivars in P. expansum-infected fruits. A number of potential candidates was encountered among defense- and oxidative stress-related genes, cell wall modification and lignification genes, and genes related to localization and transport. Induction of one cell wall-related gene and three genes involved in the ‘down-stream’ flavonoid biosynthesis pathway, demonstrates the fundamental role of the cell wall as an important barrier, and suggests that fruit flavonoids are involved in the resistance to blue mold. Moreover, exogenous application of the plant hormone methyl jasmonate (MeJA) reduced the symptoms resulting from inoculating apples with P. expansum. This is the first report linking MeJA and activation of cell wall and flavonoid pathway genes to resistance against blue mold in a study comparing different cultivars of domesticated apple. Our results provide an initial categorization of genes that are potentially involved in the resistance mechanism, and should be useful for developing tools for gene marker-assisted breeding of apple cultivars with an improved resistance to blue mold.
    Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks
    L. Q. Han, Journal of Dairy Science - 2018
    Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at −30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from −30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined.
    Combined analytical approaches to define biodistribution and biological activity of semi-synthetic berberrubine, the active metabolite of natural berberine
    Emanuele Porru, Analytical and Bioanalytical Chemistry - 2018
    Berberine (BBR) is a natural alkaloid obtained from Berberis species plants, known for its protective effects against several diseases. Among the primary BBR metabolites, berberrubine (M1) showed the highest plasma concentration but few and conflicting data are available regarding its concentration in biological fluids related to its new potential activity on vascular cells. A combined analytical approach was applied to study biodistribution of M1 in comparison with BBR. The optimization of sample clean-up combined with a fully validated HPLC-ESI-MS/MS tailored for M1 allows sufficient detectability and accuracy to be reached in the different studied organs even when administered at low dose, comparable to that assumed by human. A predictive human vascular endothelial cell-based assay to measure intracellular xanthine oxidase has been developed and applied to study unexplored activities of M1 alongside other common activities. Results showed that oral M1 treatment exhibits higher plasma levels than BBR, reaching maximum concentration 400-fold higher than BBR (204 vs 0.5 ng/mL); moreover, M1 exhibits higher concentrations than BBR also in all the biological compartments analyzed. Noteworthy, the two compounds follow two different excretion routes: M1 through urine, while BBR through feces. In vitro studies demonstrated that M1 inhibited intracellular xanthine oxidase activity, one of the major sources of reactive oxygen species in vasculature, with an IC50 = 9.90 ± 0.01 μg/mL and reduced the expression of the inflammatory marker ICAM-1. These peculiar characteristics allow new perspectives to be opened up for the direct use of M1 instead of BBR in endothelial dysfunction treatment.
    Teriparatide (human PTH1–34) compensates for impaired fracture healing in COX-2 deficient mice
    Kiminori Yukata, Bone - 2018
    Genetic ablation of cyclooxygenase-2 (COX-2) in mice is known to impair fracture healing. To determine if teriparatide (human PTH1–34) can promote healing of Cox-2-deficient fractures, we performed detailed in vivo analyses using a murine stabilized tibia fracture model. Periosteal progenitor cell proliferation as well as bony callus formation was markedly reduced in Cox-2−/− mice at day 10 post-fracture. Remarkably, intermittent PTH1–34 administration increased proliferation of periosteal progenitor cells, restored callus formation on day 7, and enhanced bone formation on days 10, 14 and 21 in Cox-2-deficient mice. PTH1–34 also increased biomechanical torsional properties at days 10 or 14 in all genotypes, consistent with enhanced bony callus formation by radiologic examinations. To determine the effects of intermittent PTH1–34 for callus remodeling, TRAP staining was performed. Intermittent PTH1–34 treatment increased the number of TRAP positive cells per total callus area on day 21 in Cox-2−/− fractures. Taken together, the present findings indicate that intermittent PTH1–34 treatment could compensate for COX-2 deficiency and improve impaired fracture healing in Cox-2-deficient mice.
    Histamine H3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons
    Rafael Koerich Varaschin, Neuroscience - 2018
    Histamine H3 receptors are widely distributed Gi-coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H3 agonist α-methylhistamine significantly reduced electrically- evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H3-modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H3 receptors by α-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by α-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by α-methylhistamine. Together, these results indicate that histamine H3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons.
    Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice
    Caroline Caradu, Cardiovascular Research - 2018
    AimsHedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated.The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis.Methods and resultsTo this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes.ConclusionAlthough ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis.
    Pituitary adenylate-cyclase activating-polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats
    Adam J. Kirry, Neuropharmacology - 2018
    A genetic polymorphism within the gene encoding the pituitary adenylate-cyclase activating polypeptide (PACAP) receptor type I (PAC1R) has recently been associated with hyper-reactivity to threat-related cues in women, but not men, with post-traumatic stress disorder (PTSD). PACAP is a highly conserved peptide, whose role in mediating adaptive physiological stress responses is well established. Far less is understood about the contribution of PACAP signaling in emotional learning and memory, particularly the encoding of fear to discrete cues. Moreover, a neurobiological substrate that may account for the observed link between PAC1R and PTSD in women, but not men, has yet to be identified. Sex differences in PACAP signaling during emotional learning could provide novel targets for the treatment of PTSD. Here we investigated the contribution of PAC1R signaling within the prefrontal cortex to the acquisition of cued fear in female and male rats. We used a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. We found that cued fear learning, but not spatial working memory, was impaired by administration of a PAC1R antagonist directly into the prelimbic area of the prefrontal cortex. This effect was specific to females. We also found that levels of mRNA for the PAC1R receptor in the prelimbic cortex were greater in females compared with males, and were highest during and immediately following the proestrus stage of the estrous cycle. Together, these results demonstrate a sex-specific role of PAC1R signaling in learning about threat-related cues.
    Matrix Metalloproteinase 12 (MMP-12) Promotes Tumor Propagation in the Lung
    Ezra Ella, The Journal of Thoracic and Cardiovascular Surgery - 2018
    Objective Past studies are inconsistent with regard to the role of MMP-12 in lung tumorigenesis. This is due, in part, to differential tumorigenesis based on tumor- versus immune-derived MMP-12 expression. Our study aims to thoroughly dissect the role of MMP-12 in lung tumorigenesis. Methods We tested MMP-12 expression, and association with prognosis using a tissue-array and a published NSCLC gene expression database. In addition, we characterized the contribution of MMP-12 to tumor propagation in the lung using a series of in vitro and in vivo studies. Results Tumor cells of a diverse set of human lung-cancers stained positive for MMP-12 and high MMP-12 mRNA levels in the tumor were associated with reduced survival. The lung microenvironment stimulated endogenous production of MMP-12 in lung-cancer cells (H460, LLC). In-vitro MMP-12 KO LLC and LLC cells had the same proliferation rate but LLC showed increased invasiveness. In-vivo, deficiency of MMP-12 in LLC cells -- but not in the host -- reduced tumor growth and invasiveness. Conclusion We suggest that tumor-cell-derived MMP-12 promotes tumor propagation in the lung and that in the context of pulmonary malignancies MMP-12 should further be tested as a potential novel therapeutic target.
    Fibroblast Growth Factor-2 and Transforming Growth Factor-beta1 Oppositely Regulate miR-221 that Targets Thrombospondin-1 in Bovine Luteal Endothelial Cells
    Svetlana Farberov, Biology of Reproduction - 2018
    Thrombospondin-1 (THBS1) is an important mediator of corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. In this study we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced SERPINE1 in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL.
    Crosstalk between adipose stem cells and tendon cells reveals a temporal regulation of tenogenesis by matrix deposition and remodeling
    Raquel Costa-Almeida, Journal of Cellular Physiology - 2018
    Tendon injuries constitute an unmet clinical challenge owing to the limited intrinsic regenerative ability of this tissue. Cell-based therapies aim at improving tendon healing through the delicate orchestration of tissue rebuilding and regain of function. Hence, human adipose-derived stem cells (hASCs) have been proposed as a promising cell source for boosting tendon regeneration. In this work, we investigated the influence of hASCs on native human tendon-derived cells (hTDCs) through the establishment of a direct contact co-culture system. Results demonstrated that direct interactions between these cell types resulted in controlled proliferation and spontaneous cell elongation. ECM-related genes, particularly COL1A1 and TNC, and genes involved in ECM remodeling, such as MMP1, MMP2, MMP3 and TIMP1, were expressed in co-cultures in a temporally regulated manner. In addition, deposition of collagen type I was accelerated in co-cultures systems and favored over the production of collagen type III, resulting in an enhanced COL1/COL3 ratio as soon as 7 days. In conclusion, hASCs seem to be good candidates in modulating the behavior of native tendon cells, particularly through a balanced process of ECM synthesis and degradation. This article is protected by copyright. All rights reserved
    Identification of Estrogen-Related Receptor Alpha Agonists in the Tox21 Compound Library
    Caitlin Lynch, Endocrinology - 2018
    The estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has also recently been shown to regulate an enzyme in the β-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8,311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., siRNA knockdown) we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. This study is the first comprehensive analysis in discovering potential endocrine disrupters which affect the ERRα signaling pathway.
    Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph
    R. Kyle Bennett, Metabolic Engineering - 2018
    Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. Introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.
    The effect of within-instar development on tracheal diameter and hypoxia-inducible factors α and β in the tobacco hornworm, Manduca sexta
    Taylor A. Lundquist, Journal of Insect Physiology - 2018
    As insects grow within an instar, body mass increases, often more than doubling. The increase in mass causes an increase in metabolic rate and hence oxygen demand. However, the insect tracheal system is hypothesized to increase only after molting and may be compressed as tissues grow within an instar. The increase in oxygen demand in the face of a potentially fixed or decreasing supply could result in hypoxia as insects near the end of an instar. To test these hypotheses, we first used synchrotron X-ray imaging to determine how diameters of large tracheae change within an instar and after molting to the next instar in the tobacco hornworm, Manduca sexta. Large tracheae did not increase in diameter within the first, second, third, and fourth instars, but increased upon molting. To determine if insects are hypoxic at the end of instars, we used the presence of hypoxia-inducible factors (HIFs) as an index. HIF-α and HIF-β dimerize in hypoxia and act as a transcription factor that turns on genes that will increase oxygen delivery. We sequenced both of these genes and measured their mRNA levels at the beginning and end of each larval instar. Finally, we obtained an antibody to HIF-α and measured protein expression during the same time. Both mRNA and protein levels of HIFs were increased at the end of most instars. These data support the hypothesis that some insects may experience hypoxia at the end of an instar, which could be a signal for molting. Summary Statement As caterpillars grow within an instar, major tracheae do not increase in size, while metabolic demand increases. At the same life stages, caterpillars increased expression of hypoxia inducible factors, suggesting that they become hypoxic near the end of an instar.
    Effects of a novel microtubule-depolymerizer on pro-inflammatory signaling in RAW264.7 macrophages
    Samuel P. Gilmore, Chemico-Biological Interactions - 2018
    The Nuclear Factor-kappa B (NF-κB) pathway is vital for immune system regulation and pro-inflammatory signaling. Many inflammatory disorders and diseases, including cancer, are linked to dysregulation of NF-κB signaling. When macrophages recognize the presence of a pathogen, the signaling pathway is activated, resulting in the nuclear translocation of the transcription factor, NF-κB, to turn on pro-inflammatory genes. Here, we demonstrate the effects of a novel microtubule depolymerizer, NT-07-16, a polysubstituted pyrrole compound, on this process. Treatment with NT-07-16 decreased the production of pro-inflammatory cytokines in RAW264.7 mouse macrophages. It appears that the reduction in pro-inflammatory mediators produced by the macrophages after exposure to NT-07-16 may be due to activities upstream of the translocation of NF-κB into the nucleus. NF-κB translocation occurs after its inhibitory protein, IκB-α is phosphorylated which signals for its degradation releasing NF-κB so it is free to move into the nucleus. Previous studies from other laboratories indicate that these processes are associated with the microtubule network. Our results show that exposure to the microtubule-depolymerizer, NT-07-16 reduces the phosphorylation of IκB-α and also decreases the association of NF-κB with tubulin which may affect the ability of NF-κB to translocate into the nucleus. Therefore, the anti-inflammatory activity of NT-07-16 may be explained, at least in part, by alterations in these steps in the NF-κB signaling pathway leading to less NF-κB entering the nucleus and reducing the production of pro-inflammatory mediators by the activated macrophages.
    Nlrp12 Mediates Adverse Neutrophil Recruitment during Influenza Virus Infection
    Emma E. Hornick, The Journal of Immunology - 2018
    Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12−/− mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12−/− neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12−/− dendritic cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses.
    BDNF Function in Long-Term Synaptic Plasticity in the Dentate Gyrus In Vivo: Methods for Local Drug Delivery and Biochemical Analysis of Translation
    Debabrata Panja, SpringerLink - 2018
    Neurotrophins are essential for multiple aspects of neuronal development and to important functions like synaptic plasticity. Brain-derived neurotrophic factor (BDNF) is a critical activity-dependent modulator of gene expression which regulates both transcription and translation. BDNF is crucial in the maintenance of long-term potentiation (LTP) at synapses and regulates protein synthesis at the dendritic and synaptic level. To elucidate the mechanisms operating in the hippocampal dentate gyrus region, in vivo electrophysiology and pharmacology is combined with analysis of signaling pathways and protein synthesis. Here, we present methods for the analysis of translation initiation, polysome formation, and translational efficiency in the context of LTP consolidation in live rodents.
    Allelic variants of the aryl hydrocarbon receptor differentially influence UVB-mediated skin inflammatory responses in SKH1 mice
    Kayla J. Smith, Toxicology - 2017
    The mouse strain SKH1 is widely used in skin research due to its hairless phenotype and intact immune system. Due to the complex nature of aryl hydrocarbon receptor (AHR) function in the skin, the development of additional in vivo models is necessary to study its role in cutaneous homeostasis and pathology. Variants of the Ah allele, exist among different mouse strains. The Ahb−1 and Ahd alleles express high and low affinity ligand binding forms of the AHR, respectively. The outbred SKH1 mice express the Ahb−2 and/or Ahd alleles. SKH1 mice were crossed with C57BL/6J mice, which harbor the Ahb−1 allele, to create useful models for studying endogenous AHR function. SKH1 mice were bred to be homozygous for either the Ahb−1 or Ahd allele to establish strains for use in comparative studies of the effects of differential ligand-mediated activation through gene expression changes upon UVB exposure. Ahb−1 or Ahd allelic status was confirmed by DNA sequence analysis. We tested the hypothesis that SKH1-Ahb−1 mice would display enhanced inflammatory signaling upon UVB exposure compared to SKH1-Ahd mice. Differential basal AHR activation between the strains was determined by assessing Cyp1a1 expression levels in the small intestine, liver, and skin of the SKH1-Ahb−1 mice compared to SKH1-Ahd mice. To determine whether SKH1-Ahb−1 mice are more prone to a pro-inflammatory phenotype in response to UVB, gene expression of inflammatory mediators was analyzed. SKH1-Ahb−1 mice expressed enhanced gene expression of the chemotactic factors Cxcl5, Cxcl1, and Ccl20, as well as the inflammatory signaling factors S100a9 and Ptgs2, compared to SKH1-Ahd mice in skin. These data supports a role for AHR activation and enhanced inflammatory signaling in skin.
    Uptake and biological responses in land snail Cornu aspersum exposed to vaporized CdCl2
    L. Sturba, Ecotoxicology and Environmental Safety - 2017
    The uptake of Cd and some biomarkers of exposure and effects have been investigated in specimens of land snail Cornu aspersum exposed to vaporized CdCl2 (10mg/L) for 7 days. The Cd levels quantified in snail's whole bodies confirmed Cd bioavailability trough vaporization and an higher accumulation in the midgut gland compared to the foot. Biological responses investigated showed a reduction of destabilization time of lysosomal membranes (NRRT) in hemocytes and an induction of catalase activities (CAT) in midgut gland. A further evidence of CdCl2 vaporized exposure was given by an increase in MT protein content as well as induction of Cd-MT gene expression, highlighting the central role of the midgut gland in Cd detoxification. These biomarkers can thus be considered as sensitive tools for the assessment of Cd contamination in the air using land snails as bioindicators. No changes in of GST activity and MDA were observed. From the overall results, the land snail, C. aspersum, could be used as good bioindicator of air quality for pollution monitoring purposes having shown clear signs of exposure and effects due Cd exposure by air.
    Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results
    Paulo Refinetti, BMC Research Notes - 2017
    Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The $$\Delta C_T$$ Δ C T method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitting a robust regression to the rtPCR signal. This new data analysis tool reduces potential biases and does not require all of the compared DNA fragments to have the same PCR efficiency.
    Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion of Ctcf in Camk2a-Cre-Expressing Neurons. | Journal of Neuroscience
    Bryan E.McGill, Journal of Neuroscience - 2017
    CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying three dimensional chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor co-ordination, and decreased sociability by 4 months of age. Ctcf CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from Ctcf CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from Ctcf CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in Ctcf CKO hippocampus. Finally, we found that microglia in Ctcf CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that Ctcf knockout in postnatal neurons causes a neurobehavioral phenotype in mice, and we provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction. SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in CTCF cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking Ctcf in Camk2a-expressing neurons (Ctcf CKO mice) have: spatial learning/memory deficits, impaired fine-motor skills, subtly altered social interactions, and decreased dendritic spine density. We uniquely demonstrate that Ctcf CKO mice overexpress inflammation-related genes in the brain, and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.
    Effects of EPSPS Copy Number Variation (CNV) and Glyphosate Application on the Aromatic and Branched Chain Amino Acid Synthesis Pathways in Amaranthus palmeri
    Manuel Fernández-Escalada, Frontiers in Plant Science - 2017
    A key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC, is the known target of the widely used herbicide glyphosate. Glyphosate resistance in Amaranthus palmeri, one of the most troublesome weeds in agriculture, has evolved through increased EPSPS gene copy number. The aim of this work was to study the pleiotropic effects of (i) EPSPS increased transcript abundance due to gene copy number variation (CNV) and of (ii) glyphosate application on the aromatic amino acid (AAA) and branched chain amino acid (BCAA) synthesis pathways. Hydroponically grown glyphosate sensitive (GS) and glyphosate resistant (GR) plants were treated with glyphosate three days after treatment. In absence of glyphosate treatment, high EPSPS gene copy number had only a subtle effect on transcriptional regulation of AAA and BCAA pathway genes. In contrast, glyphosate treatment provoked a general accumulation of the transcripts corresponding to genes of the AAA pathway leading to synthesis of chorismate in both GS and GR. After chorismate, anthranilate synthase transcript abundance was higher while chorismate mutase transcription showed a small decrease in GR and remained stable in GS, suggesting a regulatory branch point in the pathway that favors synthesis towards tryptophan over phenylalanine and tyrosine after glyphosate treatment. This was confirmed by studying enzyme activities in vitro and amino acid analysis. Importantly, this upregulation was glyphosate dose dependent and was observed similarly in both GS and GR populations. Glyphosate treatment also had a slight effect on the expression of BCAA genes but no general effect on the pathway could be observed. Taken together, our observations suggest that the high copy number variation of EPSPS in A. palmeri GR populations has no major pleiotropic effect on the expression of AAA biosynthetic genes, even in response to glyphosate treatment. This finding supports the idea that the fitness cost associated with EPSPS CNV in A. palmeri may be limited.
    miR-25 Tough Decoy enhances cardiac function in heart failure
    Dongtak Jeong, Molecular Therapy - 2017
    microRNAs are promising therapeutic targets since their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomiRs against miR-25 enhances cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 Tough Decoy inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 Tough Decoy showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR25 Tough Decoy delivered to the murine pressure-overload heart failure model selectively decreased expression of miR- 25, increased levels of SERCA2a protein and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 Tough Decoy is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.
    Leishmania-derived trimannose modulates inflammatory response to significantly reduce Leishmania (L.) major-induced lesions
    Tara L. Grinnage-Pulley, Infection and Immunity - 2017
    Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection is poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads demonstrated that C57BL/6 mice co-inoculated with L. major and trimannose-coated beads produced significantly higher levels of IL-12 p40 and other pro-inflammatory, type 1 cytokines compared L. major infection alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose or carrier (uncoated) beads, infected with L. major alone, co-inoculated with carrier beads and L. major, or co-inoculated with trimannose beads and L. major. Trimannose treatment of L. major-infected mice decreased parasite load and significantly decreased lesion size at 14 days post infection (pi) compared to non-treated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased IFN-γ at 14 days pi. Mice lacking the ability to detect trimannose, mannose-receptor deleted mice (MR-/-), when treated with trimannose beads and infected with L. major did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control parasite load and alter the course of cutaneous leishmaniasis.
    Mark Tarnopolsky, EXERKINE CORPORATION (Hamilton, CA) - 2017
    An exosome pellet or physiological solution comprising resuspended exosomes is provided. The exosomes are essentially free from undesirable particles having a diameter less than 20 nm or greater than 140 nm, and the exosomes comprise one or more metabolic products. The exosomes may be used to induce mitochondrial biogenesis, increase thermogenesis (browning) of subcutaneous white adipose tissue, and/or mediate other systemic effects of exercise in a mammal.
    Exercise induces TFEB expression and activity in skeletal muscle in a PGC-1α-dependent manner
    Avigail T. Erlich, American Journal of Physiology - Cell Physiology - 2017
    The mitochondrial network in muscle is controlled by the opposing processes of mitochondrial biogenesis and mitophagy. The coactivator PGC-1α regulates biogenesis, while the transcription of mitophagy-related genes is controlled by transcription factor EB (TFEB). PGC-1α activation is induced with exercise, however the effect of exercise on TFEB is not fully known. We investigated the interplay between PGC-1α and TFEB on mitochondria in response to acute contractile activity in C2C12 myotubes, and following exercise in WT and PGC-1α KO mice. TFEB nuclear localization was increased by 1.6-fold following 2 hours of acute myotube contractile activity. TFEB transcription and LC3 localization to mitochondria were also simultaneously increased by 2-3-fold. Viral overexpression of TFEB increased PGC-1α and COXIV gene expression. In WT mice, TFEB translocation to the nucleus increased 2.4-fold in response to acute exercise, while TFEB transcription, assessed through the electroporation of a TFEB promoter construct, was elevated by 4-fold. These exercise effects were dependent on the presence of PGC-1α. Our data suggest that acute exercise provokes TFEB expression and activation both in vitro and in vivo, in a PGC-1α-dependent manner. Our results indicate that TFEB, along with PGC-1α, are important regulators of mitochondrial biogenesis in muscle as a result of exercise.
    TIEG and estrogen modulate SOST expression in the murine skeleton
    Malayannan Subramaniam, Journal of Cellular Physiology - 2017
    TIEG knockout (KO) mice exhibit a female-specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up-regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG-SW3 osteocyte cells using shRNA and CRISPR-Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and since changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen-regulated miRNAs in modulating SOST expression in bone. This article is protected by copyright. All rights reserved
    A comparative evaluation of crowding stress on muscle HSP90 and myostatin expression in salmonids
    Nicholas J. Galt, Aquaculture - 2017
    Stress is a major factor that contributes to poor production and animal welfare concerns in aquaculture. As such, a thorough understanding of mechanisms involved in the stress response is imperative to developing strategies to mitigate the negative side effects of stressors, including the impact of high stocking densities on growth. The purpose of this study was to determine how the muscle growth inhibitor, myostatin, and the stress-responsive gene HSP90 are regulated in response to crowding stress in rainbow trout (Oncorhynchus mykiss), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar). All species exhibited higher cortisol and glucose levels following the handling stress, indicating physiological response to the treatment. Additionally, all species, except rainbow trout, exhibited higher HSP90 levels in muscle after a 48h crowding stress. Crowding stress resulted in a decrease of myostatin-1a in brook trout white muscle but not red muscle, while, myostatin-1a and -2a levels increased in white muscle and myostatin-1b levels increased in red muscle in Atlantic salmon. In rainbow trout, no significant changes were detected in either muscle type, but myostatin-1a was upregulated in both white and red skeletal muscle in the closely related cutthroat trout. The variation in response to crowding suggests a complex and species-specific interaction between stress and the muscle gene regulation in these salmonids. Only Atlantic salmon and cutthroat trout exhibited increased muscle myostatin transcription, and also exhibited the largest increase in circulating glucose in response to crowding. These results suggest that species-specific farming practices should be carefully examined in order to optimize low stress culture conditions.
    T regulatory cell induced Foxp3 binds the IL2, IFNγ, and TNFα promoters in virus-specific CD8+ T cells from feline immunodeficiency virus infected cats.
    Yan Annie Wang, AIDS Research and Human Retroviruses - 2017
    Polyfunctional CD8+ T cells play a critical role in controlling viremia during AIDS lentiviral infections. However, for most HIV infected individuals, virus-specific CD8+ T cells exhibit loss of polyfunctionality including loss of IL2, TNFα, and IFNγ. Using the feline immunodeficiency virus (FIV) model for AIDS lentiviral persistence, our laboratory has demonstrated that FIV-activated Treg cells target CD8+ T cells, leading to a reduction in IL2 and IFNγ production. Further, we have demonstrated that Treg cells induce expression of the repressive transcription factor, Foxp3 in CD8+ T cells. Based upon these findings, we asked if Treg-induced Foxp3 could bind to the IL2, TNFα, and IFNγ promoter regions in virus-specific CD8+ T cells. Following coculture with autologous Treg cells, we demonstrated decreased mRNA levels of IL2 and IFNγ at weeks 4 and 8 post-infection and decreased TNFα at week 4 post infection in virus-specific CD8+ T cells. We also clearly demonstrated Treg cell induced Foxp3 expression in virus-specific CD8+ T cells at weeks 1, 4, and 8 post-infection. Finally, we documented Foxp3 binding to the IL2, TNFα and IFNγ promoters at 8 weeks and 6 months post-infection in virus-specific CD8+ T cells following Treg cell coculture. In summary, the results here clearly demonstrate that Foxp3 inhibits IL2, TNFα and IFNγ transcription by binding to their promoter regions in lentivirus-specific CD8+ T cells. We believe this is the first description of this process during the course of AIDS lentiviral infection.
    Genome-wide binding analysis of AtGNC and AtCGA1 demonstrates their cross-regulation and common and specific functions
    Zhenhua Xu, Plant Direct - 2017
    GATA transcription factors are involved in multiple processes in plant growth and development. Two GATA factors, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1, also named GNL), are important regulators in greening, flowering, senescence, and hormone signaling. However, their direct target genes related to these biological processes are poorly characterized. Here, GNC and CGA1 are shown to be transcription activators and by using chromatin immunoprecipitation sequencing (ChIP-seq), 1475 and 638 genes are identified to be associated with GNC and CGA1 binding, respectively. Enrichment of diverse motifs in the peak binding regions for GNC and CGA1 suggests the possibility that these two transcription factors also interact with other transcription factors and in addition genes coding for DNA-binding proteins are highly enriched among GNC- and CGA1-associated genes. Despite the fact that these two GATA factors are known to share a large portion of co-expressed genes, our analysis revealed a low percentage of overlapping binding-associated genes for these two homologues. This suggests a possible cross-regulation between these, which is verified using ChIP-qPCR. The common and specific biological processes regulated by GNC and CGA1 also support this notion. Functional analysis of the binding-associated genes revealed that those encoding transcription factors, E3 ligase, as well as genes with roles in plant development are highly enriched, indicating that GNC and CGA1 mediate complex genetic networks in regulating different aspects of plant growth and development.
    A newly distal hereditary motor neuropathy caused by a rare AIFM1 mutation
    Paula Sancho, neurogenetics - 2017
    In two siblings, who suffer from an early childhood-onset axonal polyneuropathy with exclusive involvement of motor fibers, the c.629T>C (p.F210S) mutation was identified in the X-linked AIFM1 gene, which encodes for the apoptosis-inducing factor (AIF). The mutation was predicted as deleterious, according to in silico analysis. A decreased expression of the AIF protein, altered cellular morphology, and a fragmented mitochondrial network were observed in the proband’s fibroblasts. This new form of motor neuropathy expands the phenotypic spectrum of AIFM1 mutations and therefore, the AIFM1 gene should be considered in the diagnosis of hereditary motor neuropathies.
    Loop-mediated isothermal DNA amplification for asymptomatic malaria detection in challenging field settings: Technical performance and pilot implementation in the Peruvian Amazon
    Elisa Serra-Casas, PLOS ONE - 2017
    Background Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. Methods Overall, we recruited 1167 individuals from terrestrial (‘road’) and hydric (‘riverine’) communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. Results LAMP had a sensitivity of 91.8% (87.7–94.9) and specificity of 91.9% (87.8–95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12–24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. Conclusions LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.
    Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription
    Nicolás Cifuentes-Muñoz, Journal of Virology - 2017
    Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease to all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 Kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and genomic RNA (vRNA). Time-course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times post-infection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with translation of viral proteins closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time-course RT-qPCR analysis suggested that coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatio-temporal analysis of HMPV replication and transcription in bronchial epithelial-derived immortal cells was performed. HMPV was shown to induce formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures such as stress granules and P-bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though nuanced, inclusion body formation corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, understanding inclusion body formation is crucial for the discovery of innovative therapeutic targets.
    Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants
    Melissa M Doud, Horticulture Research - 2017
    Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with ‘Candidatus Liberibacter asiaticus’ (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3–6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18–36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.
    Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells
    Devi Rajan, Virology - 2017
    RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection.
    High environmental ammonia exposure has developmental-stage specific and long-term consequences on the cortisol stress response in zebrafish
    Tegan A. Williams, General and Comparative Endocrinology - 2017
    The capacity for early life environmental stressors to induce programming effects on the endocrine stress response in fish is largely unknown. In this study we determined the effects of high environmental ammonia (HEA) exposure on the stress response in larval zebrafish, assessed the tolerance of embryonic and larval stages to HEA, and evaluated whether early life HEA exposure has long-term consequences on the cortisol response to a novel stressor. Exposure to 500–2000μM NH4Cl for 16h did not affect the gene expression of corticotropin-releasing factor (CRF) system components in 1day post-fertilization (dpf) embryos, but differentially increased crfa, crfb and CRF binding protein (crfbp) expression and stimulated both dose- and time-dependent increases in the whole body cortisol of 5dpf larvae. Pre-acclimation to HEA at 1dpf did not affect the cortisol response to a subsequent NH4Cl exposure at 5dpf. In contrast, pre-acclimation to HEA at 5dpf caused a small but significant reduction in the cortisol response to a second NH4Cl exposure at 10dpf. While continuous exposure to 500–2000μM NH4Cl between 0 and 5dpf had a modest effect on mean survival time, exposure to 400–1000μM NH4Cl between 10 and 14dpf decreased mean survival time in a dose-dependent manner. Moreover, pre-acclimation to HEA at 5dpf significantly decreased the risk of mortality to continuous NH4Cl exposure between 10 and 14dpf. Finally, while HEA at 1dpf did not affect the cortisol stress response to a novel vortex stressor at 5dpf, the same HEA treatment at 5dpf abolished vortex stressor-induced increases in whole body cortisol at 10 and 60dpf. Together these results show that the impact of HEA on the cortisol stress response during development is life-stage specific and closely linked to ammonia tolerance. Further, we demonstrate that HEA exposure at the larval stage can have persistent effects on the capacity to respond to stressors in later life.
    H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3
    Sarah A. LaMere, The Journal of Immunology - 2017
    The changes to the epigenetic landscape in response to Ag during CD4 T cell activation have not been well characterized. Although CD4 T cell subsets have been mapped globally for numerous epigenetic marks, little has been done to study their dynamics early after activation. We have studied changes to promoter H3K27me3 during activation of human naive and memory CD4 T cells. Our results show that these changes occur relatively early (1 d) after activation of naive and memory cells and that demethylation is the predominant change to H3K27me3 at this time point, reinforcing high expression of target genes. Additionally, inhibition of the H3K27 demethylase JMJD3 in naive CD4 T cells demonstrates how critically important molecules required for T cell differentiation, such as JAK2 and IL12RB2, are regulated by H3K27me3. Our results show that H3K27me3 is a dynamic and important epigenetic modification during CD4 T cell activation and that JMJD3-driven H3K27 demethylation is critical for CD4 T cell function.
    Sphingosine Kinase 1 expression in Peritoneal Macrophages is required for Colon Carcinogenesis
    Hideki Furuya, Carcinogenesis - 2017
    Accumulating evidence suggests that the sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) pathway plays a pivotal role in colon carcinogenesis. Our previous studies indicate that the SphK1/S1P pathway mediates colon carcinogenesis at least by regulating COX-2 expression and prostaglandin E2 (PGE2) production. However, the mechanisms by which this pathway regulates colon carcinogenesis are still unclear. First, we show that SphK1 deficient mice significantly attenuated azoxymethane (AOM)-induced colon carcinogenesis as measured by colon tumor incidence, multiplicity, and volume. We found that AOM activates peritoneal macrophages to induce SphK1, COX-2, and TNF-α expression in WT mice. Interestingly, SphK1 KO mice revealed significant reduction of COX-2 and TNF-α expression from AOM-activated peritoneal macrophages, suggesting that SphK1 regulates COX-2 and TNF-α expression in peritoneal macrophages. We found that inoculation of WT peritoneal macrophages restored the carcinogenic effect of AOM in Sphk1 KO mice as measured by aberrant crypt foci (ACF) formation, preneoplastic lesions of colon cancer. In addition, downregulation of SphK1 only in peritoneal macrophage by shRNA reduced the number of ACF per colon induced by AOM. Intraperitoneal injection of sphingolipids demonstrates that S1P enhanced AOM-induced ACF formation, while ceramide inhibited. Finally, we show that SphK inhibitor SKI-II significantly reduced the number of ACF per colon. These results suggest that SphK1 expression plays a pivotal role in the early stages of colon carcinogenesis through regulating COX-2 and TNF-α expression from activated peritoneal macrophages.
    Short chain fatty acids ameliorate immune-mediated uveitis partially by altering migration of lymphocytes from the intestine
    Yukiko K. Nakamura, Scientific Reports - 2017
    Short chain fatty acids (SCFA) are metabolites of intestinal bacteria resulting from fermentation of dietary fiber. SCFA are protective in various animal models of inflammatory disease. We investigated the effects of exogenous administration of SFCAs, particularly propionate, on uveitis using an inducible model of experimental autoimmune uveitis (EAU). Oral SCFA administration attenuated uveitis severity in a mouse strain-dependent manner through regulatory T cell induction among lymphocytes in the intestinal lamina propria (LPL) and cervical lymph nodes (CLN). SCFA also suppressed effector T cell induction in the CLN and mesenteric lymph nodes (MLN). Alterations in intestinal morphology and gene expression demonstrated in the EAU model prior to the onset of uveitis were blunted by oral SCFA administration. Using a Kaede transgenic mouse, we demonstrated enhanced leukocyte trafficking between the intestine and the eye in EAU. Propionate suppressed T effector cell migration between the intestine and the spleen in EAU Kaede mice. In conclusion, our findings support exogenous administration of SCFAs as a potential treatment strategy for uveitis through the stabilization of subclinical intestinal alterations that occur in inflammatory diseases including uveitis, as well as prevention of trafficking of leukocytes between the gastrointestinal tract and extra-intestinal tissues.
    Maternal provision of transformer-2 is required for female development and embryo viability in the wasp Nasonia vitripennis
    Elzemiek Geuverink, Insect Biochemistry and Molecular Biology - 2017
    In insect sex determination a primary signal starts the genetic sex determination cascade that, in most insect orders, is subsequently transduced down the cascade by a transformer (tra) ortholog. Only a female-specifically spliced tra mRNA yields a functional TRA-protein that forms a complex with TRA2, encoded by a transformer-2 (tra2) ortholog, to act as a sex specific splicing regulator of the downstream transcription factors doublesex (dsx) and fruitless (fru). Here, we identify the tra2 ortholog of the haplodiploid parasitoid wasp N. vitripennis (Nv-tra2) and confirm its function in N. vitripennis sex determination. Knock down of Nv-tra2 by parental RNA interference (pRNAi) results in complete sex reversal of diploid offspring from female to male, indicating the requirement of Nv-tra2 for female sex determination. As Nv-tra2 pRNAi leads to frequent lethality in early developmental stages, maternal provision of Nv-tra2 transcripts is apparently also required for another, non-sex determining function during embryogenesis. In addition, lethality following Nv-tra2 pRNAi appears more pronounced in diploid than in haploid offspring. This diploid lethal effect was also observed following Nv-tra pRNAi, which served as a positive control in our experiments. As diploid embryos from fertilized eggs have a paternal chromosome set in addition to the maternal one, this suggests that either the presence of this paternal chromosome set or the dosage effect resulting from the diploid state is incompatible with the induced male development in N. vitripennis caused by either Nv-tra2 or Nv-tra pRNAi. The role of Nv-tra2 in activating the female sex determination pathway yields more insight into the sex determination mechanism of Nasonia.
    Cooperative regulation of Gja1 expression by members of the AP-1 family cJun and cFos in TM3 Leydig and TM4 Sertoli cells
    Firas Ghouili, Gene - 2017
    Within the testis, connexin43 encoded by Gja1 plays an important role in cell-to-cell communication between Leydig cells as well as between Sertoli cells and spermatogonia. In the adult male, Leydig cells are the principal producers of testosterone sustaining spermatogenesis, while Sertoli cells nourish, protect and support the differentiating germ cells. It has been shown previously that members of the AP-1 family regulate Gja1 expression in myometrial cells, suggesting that such regulatory mechanism may also be relevant within the testis. Thus, we performed cotransfections of AP-1 expression plasmids with different mouse Gja1 promoter/luciferase reporter constructs within TM3 Leydig and TM4 Sertoli cells. We showed that a functional cooperation between cJun and cFos activates Gja1 expression and requires an AP-1 DNA regulatory element located between − 132 and − 26 bp. In addition, such synergy relies on the recruitment of cFos to this region of the mouse Gja1 promoter. Hence, our data indicate that AP-1 members are important for optimal expression of Gja1 within Sertoli and Leydig cells from the testis.
    Identification of novel biomarkers for MLL translocated acute myeloid leukemia
    Karine Lagacé, Experimental Hematology - 2017
    Acute myeloid leukemias (AML) with translocations of the Mixed Lineage Leukemia (MLL/KMT2A) gene are common in young patients and are generally associated with poor clinical outcomes. The molecular biology of MLL fusion genes remains incompletely characterized and is complicated by the fact that over 100 different partner genes have been identified in fusions with MLL. The continually growing list of MLL fusions also represents a clinical challenge with respect to identification of novel fusions and tracking the fusions to monitor progression of the disease after treatment. We have recently developed a novel single donor model leukemia system that permits the development of human AML from normal cord blood cells. Gene expression analysis of this model and MLL-AML patient samples has identified a number of candidate biomarker genes with highly biased expression on leukemic cells. Here we present the data demonstrating the potential clinical utility of several of these candidate genes for identifying known and novel MLL fusions.
    An LC-MS Approach to Quantitative Measurement of Ammonia Isotopologues
    Jessica B. Spinelli, Scientific Reports - 2017
    Ammonia is a fundamental aspect of metabolism spanning all of phylogeny. Metabolomics, including metabolic tracing studies, are an integral part of elucidating the role of ammonia in these systems. However, current methods for measurement of ammonia are spectrophotometric, and cannot distinguish isotopologues of ammonia, significantly limiting metabolic tracing studies. Here, we describe a novel LC-MS-based method that quantitatively assesses both 14N-and 15N-isotopologues of ammonia in polar metabolite extracts. This assay (1) quantitatively measures the concentration of ammonia in polar metabolite isolates used for metabolomic studies, and (2) accurately determines the percent isotope abundance of 15N-ammonia in a cell lysate for 15N-isotope tracing studies. We apply this assay to quantitatively measure glutamine-derived ammonia in lung cancer cell lines with differential expression of glutaminase.
    Acute blood loss stimulates fibroblast growth factor 23 production
    Seham Rabadi, American Journal of Physiology - Renal Physiology - 2017
    Fibroblast growth factor 23 (FGF23) production is upregulated by iron deficiency and hypoxia. However, the influence of acute blood loss, and the resulting increases in circulating erythropoietin, on FGF23 production is unknown. Using wild-type C57BL/6 mice, we show that acute loss of 10% total blood volume leads to an increase in plasma C-terminal FGF23 (cFGF23) levels within six hours, while plasma levels of intact FGF23, phosphate, calcium, parathyroid hormone, iron, and ferritin remain similar to control mice without acute blood loss. Volume resuscitation with PBS did not significantly alter these findings. The increase in plasma cFGF23 levels in bled animals was accompanied by increased plasma erythropoietin levels at 6 hours. Administration of erythropoietin led to an acute increase in plasma cFGF23 levels similar to that observed in acute blood loss. Fgf23 mRNA expression was increased 20-fold in bone marrow, but not in bone, of bled versus control mice, suggesting bone marrow as a key source of elevated plasma FGF23 levels following acute blood loss. To extend these findings to humans, we measured plasma cFGF23 levels in 131 critically ill patients admitted to the intensive care unit. In univariate and multivariate models, we found a positive association between number of red blood cell transfusions, an indirect indicator of acute blood loss, and plasma cFGF23 levels. We conclude that FGF23 production is rapidly increased after acute blood loss, and that erythropoietin may be the mediator of this increase. Thus, erythropoietin may represent a novel physiologic regulator of FGF23 production.
    Expression of the chemokine receptors CCR1 and CCR2B is up-regulated in peripheral blood B cells upon EBV infection and in established lymphoblastoid cell lines
    Irina Kholodnyuk, Virology - 2017
    In immunocompetent individuals, EBV establishes in B cells an asymptomatic lifelong latent infection controlled by the immune system. Chemokine receptors regulate immune system function. CCR1 and CCR2 share protein sequence similarity and exert responses to multiple chemokines. The role of these receptors in B cells is largely unknown. We show that the mRNA and functional protein expression of CCR1 and CCR2 is induced in ex vivo B cells upon EBV infection and in established lymphoblastoid cell lines (LCLs). The CCR1 and CCR2B ORF transcripts were determined in LCLs. In contrast, in both the EBV-negative and EBV-positive Burkitt lymphoma cell lines, neither the CCR1, CCR2A, and CCR2B ORF transcripts nor their corresponding proteins were detected. Our data suggest that CCR1/CCR2B could be involved in clearing EBV-infected latency III B cells in immunocompetent individuals via directing the migration of these cells and attracting the chemokines-expressing immune cells.
    Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent manner
    Troy D. Hubbard, Journal of Functional Foods - 2017
    Consumption of broccoli mediates numerous chemo-protective benefits through the intake of phytochemicals, some of which modulate aryl hydrocarbon receptor (AHR) activity. Whether AHR activation is a critical aspect of the therapeutic potential of dietary broccoli is not known. Here we administered isocaloric diets, with or without supplementation of whole broccoli (15% w/w), to congenic mice expressing the high-affinity Ahrb/b or low-affinity Ahrd/d alleles, for 24 days and examined the effects on AHR activity, intestinal microbial community structure, inflammatory status, and response to chemically induced colitis. Cecal microbial community structure and metabolic potential were segregated according to host dietary and AHR status. Dietary broccoli associated with heightened intestinal AHR activity, decreased microbial abundance of the family Erysipelotrichaceae, and attenuation of colitis. In summary, broccoli consumption elicited an enhanced response in ligand-sensitive Ahrb/b mice, demonstrating that in part the beneficial aspects of dietary broccoli upon intestinal health are associated with heightened AHR activity.
    Reptin regulates insulin-stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP-1/PTPN6
    Anne-Aurélie Raymond, Cell Biochemistry and Function - 2017
    Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.
    Long-term correction of diabetic hyperglycemia through glucose-responsive hepatic insulin production using lentivirus
    Handorf Am, Journal of Diabetology and Endocrinolog - 2017
    Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of the insulin-producing β cells of the pancreas. Insulin gene therapy is a promising strategy capable of overcoming the limitations of current treatments, but to become a viable option, it must provide long-term, glucose-responsive control of insulin production. We have previously achieved glucose-responsivity by incorporating glucose-inducible response elements (GIREs) upstream of a liver-specific insulin expression cassette (3xGIRE.ALB.Ins1-2xfur). In this study, 3xGIRE.ALB.Ins1-2xfur was delivered into streptozotocin-induced diabetic rats using lentivirus, resulting in remission of diabetic hyperglycemia for at least 482 days while restoring rate of weight gain in a dose-dependent fashion. Insulin immunostaining showed abundant insulin production in the liver, and qPCR showed 13-20 lentiviral integrations per cell in the liver of rats treated with high dose lentivirus. Negligible integration was found in the pancreas, kidney, spleen and muscle of LV-treated rats, confirming liver specificity. In vitro, LV.3xGIRE.ALB.Ins1-2xfur produced a 4.5-fold increase in insulin production in high glucose conditions, and in vivo, a 1.7-fold increase in insulin levels was found during an intraperitoneal glucose tolerance test. Unfortunately, limitations in large-scale lentivirus production and use of a tissue-specific promoter prevented treatment of more than one rat per batch of lentivirus. Thus, two of the LV-treated diabetic rats were undertreated, while another two rats were over treated, becoming hypoglycemic in the fed state. Nonetheless, we have established the framework for a long-term, glucose-responsive treatment for T1DM from which further improvements can be made.
    Sakacin G is the main responsible bacteriocin for the anti-listerial activity of meat-borne Lactobacillus curvatus ACU-1
    Mónica Adriana Mechoud, Annals of Microbiology - 2017
    The present study was conducted to quantify the expression of the sakacins produced by Lactobacillus curvatus ACU-1, a strain isolated from artisanal dry fermented sausages of Argentina. Polymerase chain reaction (PCR) screening indicated the presence of sakacin G, P, and Q genes in L. curvatus ACU-1. Purification and activity assays determined that anti-Listeria activity was mainly associated to sakacin G, as mass spectrometry analysis revealed a single peak of 3832.60 Da. Further characterization by quantitative PCR demonstrated that L. curvatus ACU-1 transcription of the sakacin G structural gene was three orders of magnitude higher than the others. Interestingly, L. curvatus ACU-1 had skgA1/skgA2 as well as sppQ genes encoded in a plasmid, while the sppA gene that encodes for sakacin P was present in the bacterial chromosome. These results point out that sakacin G is the main peptide responsible for the anti-listerial activity of L. curvatus ACU-1, with little or no contribution of sakacin P and sakacin Q. The high level of expression of sakacin G demonstrated in the present work would facilitate its potential use in food preservation, improving the food quality, safety, and shelf life. In addition, the sakacin G promoter may serve as an interesting tool for the expression of other bacteriocins at higher levels.
    PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans
    Alex Keshet, Molecular Genetics and Genomics - 2017
    The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.
    Blood and Nasal Epigenetics Correlate to Allergic Rhinitis Symptom Development in the Environmental Exposure Unit
    Michelle L. North, Allergy - 2017
    Background Epigenetic alterations may represent new therapeutic targets and/or biomarkers of allergic rhinitis (AR). Our aim was to examine genome-wide epigenetic changes induced by controlled pollen exposure in the Environmental Exposure Unit (EEU). Methods 38 AR-sufferers and 8 non-allergic controls were exposed to grass pollen for 3h on two consecutive days. We interrogated DNA methylation at baseline and 3h in peripheral blood mononuclear cells (PBMCs) using the Infinium Methylation 450K array. We corrected for demographics, cell composition, and multiple testing (Benjamini-Hochberg), and verified hits using bisulfite PCR-pyrosequencing and qPCR. To extend these findings to a clinically relevant tissue, we investigated DNA methylation and gene expression of mucin 4 (MUC4), in nasal brushings from a separate validation cohort exposed to birch pollen. Results In PBMCs of allergic rhinitis participants, 42 sites showed significant DNA methylation changes of 2% or greater. DNA methylation changes in tryptase gamma 1 (TPSG1), schlafen 12 (SLFN12) and MUC4 in response to exposure were validated by pyrosequencing. SLFN12 DNA methylation significantly correlated with symptoms (p<0.05), and baseline DNA methylation pattern was found to be predictive of symptom severity upon grass allergen exposure (p<0.05). Changes in MUC4 DNA methylation in nasal brushings in the validation cohort correlated with drop in peak nasal inspiratory flow (Spearman r = 0.314, p = 0.034), and MUC4 gene expression was significantly increased (p<0.0001). Conclusion This study revealed novel and rapid epigenetic changes upon exposure in a controlled allergen challenge facility, identified baseline epigenetic status as a predictor of symptom severity. This article is protected by copyright. All rights reserved.
    Selective Inhibition of Janus Kinase 3 Has No Impact on Infarct Size or Neurobehavioral Outcomes in Permanent Ischemic Stroke in Mice
    Kelly M. DeMars, Frontiers in Neurology - 2017
    Janus kinase 3 (JAK3) is associated with the common gamma chain of several interleukin receptors essential to inflammatory signaling. To study the potential role of JAK3 in stroke-induced neuroinflammation, we subjected mice to permanent middle cerebral artery occlusion, and investigated the effects of JAK3 inhibition with decernotinib (VX-509) on infarct size, behavior, and levels of several inflammatory mediators. Results from our double immunofluorescence staining showed JAK3 expression on neurons, endothelial cells, and microglia/macrophages in the ischemic mouse brain (n=3). We found for the first time that total as well as phosphorylated/activated JAK3 are dramatically increased after stroke in the ipsilateral hemisphere (**P<0.01; n=5-13/group) in addition to increased IL-21 expression after stroke (**P<0.01; n=5-7/group). However, inhibition of JAK3 confirmed by reduced phosphorylation of its activation loop at tyrosine residues 980/981 does not reduce infarct volume measured at 48 h after stroke (n=6-10/group) nor does it alter behavioral outcomes sensitive to neurological deficits or stroke-induced neuroinflammatory response (n=9-10/group). These results do not support a detrimental role for JAK3 in acute neuroinflammation following permanent focal cerebral ischemia. The functional role of increased JAK3 activation after stroke remains to be further investigated.
    Treatment with antioxidants ameliorates oxidative damage in a mouse model of propionic acidemia
    Ana Rivera-Barahona, Molecular Genetics and Metabolism - 2017
    Oxidative stress contributes to the pathogenesis of propionic acidemia (PA), a life threatening disease caused by the deficiency of propionyl CoA-carboxylase, in the catabolic pathway of branched-chain amino acids, odd-number chain fatty acids and cholesterol. Patients develop multisystemic complications including seizures, extrapyramidal symptoms, basal ganglia deterioration, pancreatitis and cardiomyopathy. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species and oxidative damage, all of which have been documented in patients' samples and in a hypomorphic mouse model. Here we set out to investigate whether treatment with a mitochondria-targeted antioxidant, MitoQ, or with the natural polyphenol resveratrol, which is reported to have antioxidant and mitochondrial activation properties, could ameliorate the altered redox status and its functional consequences in the PA mouse model. The results show that oral treatment with MitoQ or resveratrol decreases lipid peroxidation and the expression levels of DNA repair enzyme OGG1 in PA mouse liver, as well as inducing tissue-specific changes in the expression of antioxidant enzymes. Notably, treatment decreased the cardiac hypertrophy marker BNP that is found upregulated in the PA mouse heart. Overall, the results provide in vivo evidence to justify more in depth investigations of antioxidants as adjuvant therapy in PA.
    Loss of Calreticulin Uncovers a Critical Role for Calcium in Regulating Cellular Lipid Homeostasis
    Wen-An Wang, Scientific Reports - 2017
    A direct link between Ca2+ and lipid homeostasis has not been definitively demonstrated. In this study, we show that manipulation of ER Ca2+ causes the re-distribution of a portion of the intracellular unesterified cholesterol to a pool that is not available to the SCAP-SREBP complex. The SREBP processing pathway in ER Ca2+ depleted cells remained fully functional and responsive to changes in cellular cholesterol status but differed unexpectedly in basal activity. These findings establish the role of Ca2+ in determining the reference set-point for controlling cellular lipid homeostasis. We propose that ER Ca2+ status is an important determinant of the basal sensitivity of the sterol sensing mechanism inherent to the SREBP processing pathway.
    BITC and S-Carvone Restrain High-Fat Diet-Induced Obesity and Ameliorate Hepatic Steatosis and Insulin Resistance
    Sary Alsanea, Pharmaceutical Research - 2017
    PurposeTo investigate the preventative activity of benzyl isothiocyante and S-carvone against high-fat diet-induced obesity and metabolic complications.MethodsTen-week-old C57BL/6 male mice were fed a high-fat diet and injected intraperitoneally twice per week with benzyl isothiocyante, S-carvone, or vehicle for 8 weeks. The body weight, food intake, and body composition were monitored, and glucose tolerance and insulin tolerance tests were performed at the end of the experiment. Serum and tissue samples were studied using serum biochemistry, histological, and gene expression analysis to define the effects of benzyl isothiocyante and S-carvone treatments on lipid and glucose metabolism and inflammatory responses.ResultsBenzyl isothiocyante and S-carvone blocked high-fat diet-induced weight gain, fat accumulation in the liver, and insulin resistance. The beneficial effects were found to be associated with an improvement of expression of macrophage marker genes in white adipose tissue, including F4/80, Cd11b, Cd11c, Cd206, and Tnf-α, and reduced expression of genes (Pparγ2, Scd1, Cd36) responsible for lipid synthesis and transport in the liver.ConclusionBenzyl isothiocyante and S-carvone block high-fat diet-induced obesity and metabolism disorders and can be considered for management of the obesity epidemic that affects approximately 36% of adults and 17% of children in the USA.
    Impact of environmental microbiota on human microbiota of workers in academic mouse research facilities: An observational study
    Peggy S. Lai, PLOS ONE - 2017
    Objectives To characterize the microbial environment of workers in academic mouse research facilities using endotoxin, 16S qPCR, and 16S amplicon sequencing. To determine whether the work microbiome contributes to the human microbiome of workers. Methods We performed area air sampling from the animal rooms, dirty, middle, and setup cage wash locations in four academic mouse research facilities. 10 workers in the dirty cage wash area underwent personal air sampling as well as repeated collection of nasal, oral, and skin samples before and after the work shift. Environmental samples underwent measurement of endotoxin, mouse allergen, bacteria copy number via 16S qPCR, and microbial identification via 16S rDNA sequencing. 16S rDNA sequencing was also performed on human samples before and after the work shift. SourceTracker was used to identify the contribution of the work microbiome to the human microbiome. Results Median endotoxin levels ranged from undetectable to 1.0 EU/m3. Significant differences in mouse allergen levels, bacterial copy number, microbial richness, and microbial community structure were identified between animal, dirty, middle, and setup cage wash locations. Endotoxin levels had only a moderate correlation with microbial composition. Location within a facility was a stronger predictor of microbial community composition (R2 = 0.41, p = 0.002) than facility. The contribution of the work microbiome to the pre-shift human microbiome of workers was estimated to be 0.1 ± 0.1% for the oral microbiome; 3.1 ± 1.9% for the nasal microbiome; and 3.0 ± 1.5% for the skin microbiome. Conclusions The microbial environment of academic animal care facilities varies significantly by location rather than facility. Endotoxin is not a proxy for assessment of environmental microbial exposures using 16S qPCR or 16S rDNA sequencing. The work microbiome contributes to the composition of the nasal and skin microbiome of workers; the clinical implications of this observation should be further studied.
    Numerical Relationships Between Archaeal and Bacterial amoA Genes Vary by Icelandic Andosol Classes
    Hendrikus J. Laanbroek, Microbial Ecology - 2017
    Bacterial amoA genes had not been detectable by qPCR in freshly sampled Icelandic Andosols thus far. Hence, a new primer set yielding shorter gene fragments has been designed to verify the absence of ammonia-oxidizing bacteria in different Icelandic Andosol classes. At the same time, a new primer set was also constructed for archaeal amoA genes that should improve the quality of PCR products. Although a large part of the soil samples were found to be amoA-negative, bacterial amoA genes were detectable with new as well as old primer sets. The same results were obtained for the archaeal amoA genes. The relative distribution of archaeal and bacterial amoA genes varied between Andosol classes. Archaeal amoA genes were significantly more abundant in Brown than in Histic Andosols, while the opposite was observed for bacterial amoA genes. The numbers of archaeal and bacterial amoA genes in Gleyic Andosols were not significantly different from those in Histic and Brown Andosols. The numbers of bacterial amoA genes, but not the numbers of archaeal amoA genes, correlated significantly and positively with potential ammonia oxidation activities. The presence of the bacterial nitrification inhibitor allylthiourea inhibited the potential ammonia oxidation activities during the first 12 h of incubation. Hence, it was concluded that ammonia-oxidizing bacteria profited most from the conditions during the measurements of potential ammonia oxidation activities.
    Bioaccessibility, bioavailability and anti-inflammatory effects of anthocyanins from purple root vegetables using mono- and co-culture cell models
    Hua Zhang, Molecular Nutrition & Food Research - 2017
    Scope Immune-inflammatory, signalling and metabolic effects are the main pillars for bioactivity of anthocyanins derived from highly pigmented root vegetables. This study aims to assess the bioaccessibility and bioavailability of purple carrot and potato derived anthocyanins and the molecular mechanisms of their ability to ameliorate cellular inflammation in a mono- and co-culture cell models. Methods and Results An in vitro gastrointestinal model was used and demonstrated bioaccessibility of 44.62% and 71.8% for anthocyanins of purple carrot and potato, respectively. These accessible anthocyanins significantly inhibited cellular inflammation in Caco-2 cells. Intact cyanidin glycoside or petunidin glycoside (respectively from carrots and potatoes) were transported across a transmembrane cell model and detected by LC-MS/MS. Computational docking and glucose uptake analyses suggested uptake of anthocyanins was mediated by hexose transporters. Subsequent experiment using an inflamed Caco-2 BBe1/THP-1 co-culture cell model showed these transported anthocyanins inhibited IL-8 and TNF-α secretion, and expression of pro-inflammatory cytokines by blocking NF-κB, and MAPK mediated inflammatory cellular signalling cascades, but with varying degrees due to structural features. Conclusion Anthocyanins from purple carrots and potatoes possess a promising anti-inflammatory effect in model gut system. They can be absorbed and act differently but are in general beneficial for inflammation-mediated diseases. This article is protected by copyright. All rights reserved
    Use of a real-time PCR to explore the intensity of Plasmodium spp. infections in native, endemic and introduced New Zealand birds
    D. C. Sijbranda, Cambridge Core - 2017
    SUMMARY Avian malaria, caused by Plasmodium spp., is an emerging disease in New Zealand (NZ). To detect Plasmodium spp. infection and quantify parasite load in NZ birds, a real-time polymerase chain reaction (PCR) (qPCR) protocol was used and compared with a nested PCR (nPCR) assay. A total of 202 blood samples from 14 bird species with known nPCR results were tested. The qPCR prevalences for introduced, native and endemic species groups were 70, 11 and 21%, respectively, with a sensitivity and specificity of 96·7 and 98%, respectively, for the qPCR, while a sensitivity and specificity of 80·9 and 85·4% were determined for the nPCR. The qPCR appeared to be more sensitive in detecting lower levels of parasitaemia. The mean parasite load was significantly higher in introduced bird species (2245 parasites per 10 000 erythrocytes) compared with endemic species (31·5 parasites per 10 000 erythrocytes). In NZ robins (Petroica longipes), a significantly lower packed cell volume was found in birds that were positive for Plasmodium spp. compared with birds that were negative. Our data suggest that introduced bird species, such as blackbirds (Turdus merula), have a higher tolerance for circulating parasite stages of Plasmodium spp., indicating that introduced species are an important reservoir of avian malaria due to a high infection prevalence and parasite load.
    Pigment epithelium derived factor play a positive role in bone mineralization of osteoblasts derived from diabetic patients
    Na Song, Gene - 2017
    Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein which plays important role in anti-angiogenic, anti-tumorigenic, as well as involves in the metabolism and regeneration of bone. In this study, our aim is to investigate the role of PEDF in regulating mineralization of osteoblasts from diabetic patients (DP). We isolated and cultured osteoblasts derived from DP and non-diabetic patients (NDP), in order to analyze the variable differences via gene expression and calcification assay in vitro. Gene expression analysis and alizarin red S staining revealed that osteoblasts from DP exhibited defective mineralization. PEDF and vascular endothelial growth factor (VEGF) levels were lower in osteoblasts from DP than those from NDP. Interestingly, exogenous PEDF could upregulate the gene expression levels of VEGF and osteoblast-related genes, further to restore mineralization ability in osteoblasts from DP. Our results demonstrated that PEDF played a positive role in maintaining bone development in diabetic osteoblasts, therefore, we confidently believe that PEDF may be a promising cytokine to consider in development of treatments for diabetic bone diseases.
    Simple and fast quantification of DNA damage by real-time PCR, and its application to nuclear and mitochondrial DNA from multiple tissues of aging zebrafish
    Shusen Zhu, BMC Research Notes - 2017
    We describe a real-time (rt) PCR-based method of quantifying DNA damage, adapted from the long-run rtPCR method of DNA damage quantification (LORD-Q) developed by Lehle et al. (Nucleic Acids Res 42(6):e41, 2014). We show that semi-long run rtPCR, which generates amplicons half the length of those generated in LORD-Q, provides equivalent sensitivity for detecting low lesion frequencies, and better sensitivity for detecting high frequencies. The smaller amplicon size greatly facilitates PCR optimization and allows greater flexibility in the use of detection dyes, and a modified data analysis method simplifies the calculation of lesion frequency. The method was used to measure DNA damage in the nuclear and mitochondrial genomes of different tissues in zebrafish of different ages. We find that nuclear DNA damage generally increases with age, and that the amount of mitochondrial DNA damage varies substantially between tissues, increasing with age in liver and brain but not in heart or skeletal muscle, the latter having the highest levels of damage irrespective of age.
    Curcumin Protects Skin against UVB-Induced Cytotoxicity via the
    Maya Ben Yehuda Greenwald, Hindawi Oxidative Medicine and Cellular Longevity - 2017
    Curcumin was found to be beneficial in treating several skin pathologies and diseases, providing antioxidant protection due to its reducing properties and its electrophilic properties (the ability to activate the Nrf2 pathway and induce phase II cytoprotective enzymes). Nevertheless, clinical applications of curcumin are being hampered by its insufficient solubility, chemical instability,and poor absorption, leading to low efficacy in preventing skin pathologies. These limitations can be overcome by using a nanotechnology-based delivery system. Here, we elucidated the possibility of using curcumin encapsulated in a microemulsion preserving its unique chemical structure. We also examined whether curcumin microemulsion would reduce UVB-induced toxicity in skin. A significant curcumin concentration was found in the human skin dermis following topical application of a curcumin microemulsion. Moreover, curcumin microemulsion enhanced the reduction of UV-induced cytotoxicity in epidermal cells, paving the way for other incorporated electrophiles in encapsulated form protecting skin against stress-related diseases.
    Increased incidence of non-alcoholic fatty liver disease in male rat offspring exposed to fluoxetine during fetal and neonatal life involves the NLRP3 inflammasome and augmented de novo hepatic lipogenesis
    Nicole E.De Long, Journal of Applied Toxicology - 2017
    Up to 10% of women take selective serotonin reuptake inhibitors (SSRI) during pregnancy. Children exposed to SSRIs in utero have an increased risk of being overweight suggesting that fetal exposure to SSRIs can cause permanent metabolic changes. We have previously shown in rats that fetal and neonatal exposure to the SSRI antidepressant fluoxetine results in metabolic perturbations including increased hepatic triglyceride content; a hallmark of non-alcoholic fatty liver disease (NAFLD). Therefore, the aim of this study was to identify the mechanism(s) underlying the fluoxetine-induced increase in intrahepatic triglyceride content. Female nulliparous Wistar rats were given vehicle or fluoxetine (10 mg/kg/day) orally for 2 weeks prior to mating until weaning. At 6 months of age, we assessed whether SSRI exposure altered components of the hepatic triglyceride biosynthesis pathway in the offspring and examined the molecular mechanisms underlying these changes. Male SSRI-exposed offspring had a significant increase in the steady-state mRNA levels of Elovl6 and Dgat1 and core components of the NLRP3 inflammasome (apoptosis-associated speck-like protein containing a caspase activation recruitment domain [ASC] and caspase-1). Augmented expression of Asc in the SSRI-exposed offspring coincided with increased histone acetylation in the proximal promoter region. Given that we have previously demonstrated that antenatal exposure to SSRIs can lead to fatty liver in the offspring, this raises concerns regarding the long-term metabolic sequelae of fetal SSRI exposure. Moreover, this study suggests that elevated hepatic triglyceride levels observed in the SSRI-exposed offspring may be due, in part, to activation of the NLRP3 inflammasome and augmentation of de novo lipogenesis.
    YY1 Is Required for Posttranscriptional Stability of SOX2 and OCT4 Proteins
    Mary C.Wallingford, Cellular Reprogramming - 2017
    Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein–protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.
    Increasing corn distillers solubles alters the liquid fraction of the ruminal microbiome
    J. C.McCann, Journal of Animal Science - 2017
    Five ruminally fistulated steers were used in a 5 × 5 Latin square design to determine the effects of increasing dietary fat and sulfur from condensed distiller’s solubles (CDS) on the ruminal microbiome. Treatments included a corn-based control (CON) and 4 levels of CDS (0, 10, 19, and 27%) in a coproduct-based (corn gluten feed and soybean hulls) diet. Fat concentrations were 1.79, 4.43, 6.80, and 8.91% for diets containing 0, 10, 19, and 27% CDS, respectively. Steers were fed for ad libitum intake once daily. After feeding each diet for 18 d, ruminal samples were collected 3 h after feeding on d 19. Samples were separated into solid and liquid fractions. Microbial DNA was extracted for bacterial analysis using paired-end sequencing of the V3 through V4 region of the 16S rRNA gene on the MiSeq Illumina platform and quantitative PCR of selected species. Orthogonal contrasts were used to determine linear and quadratic effects of CDS inclusion. Increasing CDS inclusion decreased (linear, P < 0.05) α-diversity and species richness in the liquid fraction. Analysis of Bray–Curtis similarity indicated a treatment effect (P = 0.01) in the liquid fraction. At the phyla level, relative abundance of Bacteroidetes decreased in steers fed increasing dietary inclusion of CDS as Firmicutes increased to 82% of sequences for the 27% CDS treatment. Family Ruminococcaceae increased (linear, P < 0.01) 2-fold in the liquid fraction when feeding CDS increased from 0 to 27% CDS, yet genera Ruminococcus tended (P = 0.09) to decrease in steers fed greater CDS. The most abundant family of sulfate-reducing bacteria, Desulfovibrionaceae, increased (P < 0.03) in the solid and liquid fraction in steers fed additional dietary CDS and sulfur. Relative abundance of family Veillonellaceae and Selenomonas ruminantium were increased (linear, P ≤ 0.02) in the solid fraction as steers were fed increasing CDS. There were no effects (P > 0.10) of feeding increasing dietary fat from CDS on fibroylytic genus Fibrobacter in either fraction. Results demonstrate increasing fat and sulfur from CDS in a coproduct-based diet markedly alters the liquid fraction ruminal microbiome but does not elicit negative effects on relative abundance of identified fiber-fermenting bacteria.
    Pancreatic Beta Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes
    Tehila Dahan, Diabetes - 2016
    Beta-cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of beta-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. While in mice gastrin is expressed insulin+ cells, in humans with T2D gastrin expression occurs in both insulin+ and somatostatin+ cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated beta-cells following exposure to severe hyperglycemia. Gastrin expression in adult beta-cells does not involve the endocrine progenitor cell regulator NeuroG3 but requires membrane depolarization, calcium influx and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of beta cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human beta-cell reprogramming in diabetes.
    Contribution of asparagine catabolism to Salmonella virulence
    Patrick A. McLaughlin, Infection and Immunity - 2016
    Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that L-Asparaginase II produced by Salmonella enterica serovar Typhimurium (S. Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by L-Asparaginase II of S. Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for L-Asparaginase II stability and function. Furthermore, we report that L-Asparaginase II localizes primarily to the periplasm and acts together with L-Asparaginase I to provide S. Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S. Typhimurium lacking both L-Asparaginase I and II genes compete poorly with wild-type S. Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S. Typhimurium virulence, providing new insights into the competition for nutrients at the host pathogen interface.
    Physiological characterization of drought stress response and expression of two transcription factors and two LEA genes in three Prunus genotypes
    Beatriz Bielsa, Scientia Horticulturae - 2016
    Global warming has led to a progressive decrease in rainfall, which is reflected by a reduction of water resources in the soil and a negative effect on crop production in Mediterranean areas. Under drought stress, many plants react by inducing a different series of responses at both physiological and molecular levels, allowing them to survive for a variable period of time. Therefore, in order to understand the response of roots to drought conditions, the genotypes peach × almond ‘Garnem’ [P. amygdalus Batsch × P. persica (L.) Batsch] and their progeny, the hybrid ‘P.2175’ × ‘Garnem’-3 and OP-‘P.2175’ (P. cerasifera Ehrh.) were subjected to a period of water deficit. Drought conditions with a subsequent re-watering period were tested for potted plants for one month. Stomatal conductance and leaf water potential were measured to monitor the plant physiological responses. Significant differences among the drought stress and drought stress recovery treatments and among the genotypes were observed. In addition, four genes related to the ABA biosynthesis pathway were studied for their expression by RT-qPCR: an AN20/AN1 zinc finger protein (ppa012373m); a bZIP transcription factor (ppa013046m); a dehydrin (ppa005514m) and a LEA protein (ppa008651m). Their expression profiles correlated with our physiological results of drought response, being higher in roots than in phloem tissue. In general, the expression of the four studied genes was higher after 15 days under drought conditions. Under drought and recovery conditions, the zinc finger and bZIP transcription factors showed significant differences in their relative expression levels from LEA and dehydrin. These results suggest the role of LEA and dehydrin in the regulatory response to drought stress in Prunus genotypes. Therefore, the dehydrin and the protein LEA might be potential biomarkers to select rootstocks for tolerance to drought conditions.
    Evolution of subcutaneous adipose tissue fibrosis after bariatric surgery
    M.S Chabot, Diabetes & Metabolism - 2016
    Obesity is associated with the development of metabolic complications such as insulin resistance (IR). The mechanisms leading to IR remain unclear. This study aimed to investigate the relationship between adipose tissue fibrosis and IR in obese patients before and after bariatric surgery.
    Ribosome biogenesis is dynamically regulated during osteoblast differentiation
    Cynthia L. Nebena,, Science Direct - 2016
    Changes in ribosome biogenesis are tightly linked to cell growth, proliferation, and differentiation. The rate of ribosome biogenesis is established by RNA Pol I-mediated transcription of ribosomal RNA (rRNA). Thus, rRNA gene transcription is a key determinant of cell behavior. Here, we show that ribosome biogenesis is dynamically regulated during osteoblast differentiation. Upon osteoinduction, osteoprogenitor cells transiently silence a subset of rRNA genes through a reversible mechanism that is initiated through biphasic nucleolar depletion of UBF1 and then RNA Pol I. Nucleolar depletion of UBF1 is coincident with an increase in the number of silent but transcriptionally permissible rRNA genes. This increase in the number of silent rRNA genes reduces levels of ribosome biogenesis and subsequently, protein synthesis. Together these findings demonstrate that fluctuations in rRNA gene transcription are determined by nucleolar occupancy of UBF1 and closely coordinated with the early events necessary for acquisition of the osteoblast cell fate.
    Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes
    Anis Ahmad, The FASEB Journal - 2016
    The molecular mechanisms responsible for the development of proteinuria and glomerulosclerosis in radiation nephropathy remain largely unknown. Podocytes are increasingly recognized as key players in the pathogenesis of proteinuria in primary and secondary glomerular disorders. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) is a key determinant of podocyte injury and a known off target of the anti-CD20 antibody rituximab (RTX). The current study investigates the role of sphingolipids in radiation-induced podocytopathy. After a single dose of radiation (8 Gy), several ceramide species were significantly elevated. In particular, C16:00, C24:00, and C24:1 ceramides were the most abundant ceramide species detected. These changes were paralleled by a time-dependent drop in SMPDL3b protein, sphingosine, and sphingosine-1-phosphate levels. Interestingly, SMPDL3b overexpressing podocytes had higher basal levels of sphingosine-1-phosphate and maintained basal ceramide levels after irradiation. Morphologically, irradiated podocytes demonstrated loss of filopodia and remodeling of cortical actin. Furthermore, the actin binding protein ezrin relocated from the plasma membrane to the cytosol as early as 2 h after radiation. In contrast, SMPDL3b overexpressing podocytes were protected from radiation-induced cytoskeletal remodeling. Treatment with RTX before radiation exposure partially protected podocytes from SMPDL3b loss, cytoskeletal remodeling, and caspase 3 cleavage. Our results demonstrate that radiation injury induces early cytoskeletal remodeling, down-regulation of SMPDL3b, and elevation of cellular ceramide levels. Overexpression of SMPDL3b and pretreatment with RTX confer a radioprotective effect in cultured podocytes. These findings indicate a potential role for SMPDL3b and RTX in radiation-induced podocytopathy.—Ahmad, A., Mitrofanova, A., Bielawski, J., Yang, Y., Marples, B., Fornoni, A., Zeidan, Y. H. Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.
    Comparison of miRNA signature versus conventional biomarkers before and after off-pump coronary artery bypass graft
    Fatemeh Pourrajab, Journal of Pharmaceutical and Biomedical Analysis - 2016
    Circulating levels of microRNAs (miRNAs) and their expression patterns are supposed to serve as signatures for diagnosis or prognosis of cardiovascular events. The present study aimed at determining if there is any correlation between the release pattern of 2 miRNAs and the plasma levels of conventional biomarkers cardiac troponin I (cTnI), creatine kinase (CK) and uric acid (UA) in patients undergoing their first off-pump coronary artery bypass graft (OCABG). Seventy OCABG patients (69% men, aged 59.2 ± 8.2 years) were enrolled. Emergencies, re-operations, abnormal preoperative serum cTnI and combined procedures were excluded from this study. Pre-operative mean ejection fraction was 45.8 ± 8.6%, the average number of grafts was 3 ± 0.87/patient, and the internal mammary artery was used for all. Beside conventional clinical assays, we performed real-time quantitative PCR to analyze the circulating levels of miR-155, miR-126 and miR-499 at 1 day before surgery as well as 4 days after surgery. Importantly, there was no report of myocardial infarction in our patients, pre- or post-operatively. In contrast to conventional biomarkers cTnI and CK, circulating levels of miRNAs decreased significantly (P < 0.01) after revascularization surgery. A significant positive correlation was seen between the cTnI and miR-499 (r ∼ 0.53, P < 0.01) and between miR-126 and UA (r ∼ 0.5, P < 0.01). Time course study of circulating miR-499, miR-126 and miR-155 in cardiac surgery clarified their advantage and correlations to the traditional biomarkers cTnI, total CK, CK-MB and UA. Our results suggest that this signature is a novel, early biomarker which indicates myocardial ischemia in cardiac surgery. It could be postulated that the application of these miRNAs may be considered for monitoring of response to pharmacological interventions aimed at reducing cardiac ischemia, especially in OCABG candidates.
    Secretoglobin Superfamily Protein SCGB3A2 Alleviates House Dust Mite-Induced Allergic Airway Inflammation in Mice
    M. Yoneda, International Archives of Allergy and Immunology - 2016
    Background: Secretoglobin (SCGB) 3A2, a novel, lung-enriched, cytokine-like, secreted protein of small molecular weight, was demonstrated to exhibit various biological functions includin
    Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hotspots
    Irina V.Getun, Molecular and Cellular Biology - 2016
    Meiotic recombination initiates following the formation of DNA double strand breaks (DSBs) by the Spo11 endonuclease early in prophase I at discrete regions in the genome coined hotspots. In mammals, meiotic DSB site selection is directed in part by sequence specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hotspot specification are largely unknown. Here, we show that the recombinogenic cores of active hotspots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene meiotic stage cells, which would facilitate PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at leptotene. Importantly, manipulating histone acetylase and deacetylase activity established that histone acetylation marks are necessary for both hotspot activity and crossover resolution. We conclude there are functional roles for histone acetylation marks at mammalian meiotic recombination hotspots.
    The Kallikrein-Kinin System: A Novel Mediator of IL-17-Driven Anti-Candida Immunity in the Kidney
    Kritika Ramani, PLOS Pathogens - 2016
    Author Summary Candida albicans is the causative agent of oropharyngeal candidiasis (OPC, thrush), dermal and vaginal candidiasis. However, the most severe C. albicans-induced disease is disseminated candidiasis, a frequent nosocomial infection associated with a high mortality rate. During disseminated candidiasis, C. albicans form invasive hyphae that damage target organs, particularly kidney and liver. Previous studies have identified an essential role of interleukin-17 (IL-17) in controlling systemic infection through regulation of neutrophils. We show here for the first time that IL-17 also regulates the renal protective Kallikrein-kinin system (KKS). Our discovery of a connection between IL-17 and the KKS suggests a new, previously unanticipated avenue for the treatment of renal damage in disseminated candidiasis. These findings have potential translational significance, as agonists of the KKS are in routine clinical use. Therefore, these results not only identify downstream mediators that could serve as novel drug targets, but could possibly be used to guide decisions on whether targeting these mediators could be a useful therapeutic option in conjunction with current antifungal therapies.
    Pharmacological treatment and BBB-targeted genetic therapy for MCT8-dependent hypomyelination in zebrafish
    David Zada, Disease Models & Mechanisms - 2016
    Skip to Next Section Hypomyelination is a key symptom of Allan-Herndon-Dudley syndrome (AHDS), a psychomotor retardation associated with mutations in the thyroid-hormone (TH) transporter MCT8 (monocarboxylate transporter 8). AHDS is characterized by severe intellectual deficiency, neuromuscular impairment and brain hypothyroidism. In order to understand the mechanism for TH-dependent hypomyelination, we developed an mct8 mutant (mct8−/−) zebrafish model. The quantification of genetic markers for oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes revealed reduced differentiation of OPCs into oligodendrocytes in mct8−/− larvae and adults. Live imaging of single glial cells showed that the number of oligodendrocytes and the length of their extensions are reduced, and the number of peripheral Schwann cells is increased, in mct8−/− larvae compared with wild type. Pharmacological analysis showed that TH analogs and clemastine partially rescued the hypomyelination in the CNS of mct8−/− larvae. Intriguingly, triiodothyronine (T3) treatment rescued hypomyelination in mct8−/− embryos before the maturation of the blood–brain barrier (BBB), but did not affect hypomyelination in older larvae. Thus, we expressed Mct8-tagRFP in the endothelial cells of the vascular system and showed that even relatively weak mosaic expression completely rescued hypomyelination in mct8−/− larvae. These results suggest potential pharmacological treatments and BBB-targeted gene therapy that can enhance myelination in AHDS and possibly in other TH-dependent brain disorders.
    The neuropeptide galanin modulates natural killer cell function
    Andreas Koller, Neuropeptides - 2016
    Natural killer (NK) cells are part of the innate immune system and combat pathogens and tumors by secreting pro-inflammatory cytokines like interferon gamma (IFN-γ) and by their cytotoxic action. Galanin is a neuropeptide also expressed in peripheral tissue where it impacts several physiological functions, including inflammation. The effects of galanin are mediated via three receptors, GAL1–3. Since other neuropeptides have been shown to regulate NK cell activity, we investigated the potential of galanin to modulate human NK cell function. NK cells were isolated from human peripheral blood mononuclear cells. mRNA expression was analyzed by qRT-PCR. The dynamic mass redistribution of NK cells upon regulatory peptide stimulation was determined by label-free biochip technology. IFN-γ producing NK cells were identified by flow cytometry analysis and IFN-γ secretion was measured by ELISA. NK cell cytotoxicity was analyzed by flow cytometry via CD107a mobilization. NK cells were found to express the receptor GAL2 but not GAL1, GAL3 or galanin. Galanin per se did not affect the dynamic mass redistribution of NK cells, but significantly enhanced the response of NK cells to IL-18. Galanin significantly modulated the IFN-γ production of the CD56bright NK cell population upon IL-12 and IL-18 stimulation. Furthermore, galanin significantly modulated the IL-12 and IL-18 stimulated IFN-γ secretion. NK cell cytotoxicity was not modulated by galanin treatment. Galanin can be classified as an immunomodulatory peptide as it is able to sensitize NK cells toward specific cytokines.
    Effects of soybean meal on digestive enzymes activity, expression of inflammation-related genes, and chromatin modifications in marine fish (Sparus aurata L.) larvae
    Erick Perera, Fish Physiology and Biochemistry - 2016
    The effects of soybean meal (SBM) in early diet of Sparus aurata larvae at two developmental windows were assessed. Prolonged (beyond 14 days post-hatch, dph) feeding with SBM decreased the activity of pancreatic enzymes of larvae. In the absence of SBM these larvae later resumed enzyme activities, but exhibited a significant delay in development. Larvae response to SBM involved up-regulation of extracellular matrix remodeling enzymes and pro-inflammatory cytokines, coupled with a drop in putative intestinal enzymes. Larvae receiving SBM at first feeding appear later to have lower expression of inflammation-related genes, especially those fed SBM until 14 dph. Multivariate analysis confirmed that the duration of the SBM early feeding period drives the physiology of larvae in different directions. Feeding larvae with SBM increased global histone H3 acetylation, whereas upon removal of SBM the process was reverted. A more in deep analysis revealed a dynamic interplay among several reversible histone modifications such as H3K14ac and H3K27m3. Finally, we showed that SBM feeding of larvae results in global hypomethylation that persist after SBM removal. This study is the first demonstrating an effect of diet on marine fish epigenetics. It is concluded that there are limitations for extending SBM feeding of S. aurata larvae beyond 14 dph even under co-feeding with live feed, affecting key physiological processes and normal growth. However, up to 14 dph, SBM does not affect normal development, and produces apparently lasting effects on some key enzymes, genes, and chromatin modifications.
    ADAM17 in tumor associated leukocytes regulates inflammatory mediators and promotes mammary tumor formation
    Laura R. Bohrer1,*, Genes and Cancer - 2016
    The presence of inflammatory cells within the tumor microenvironment has been tightly linked to mammary tumor formation and progression. Specifically, interactions between tumor cells and infiltrating macrophages can contribute to the generation of a pro-tumorigenic microenvironment. Understanding the complex mechanisms that drive tumor cell-macrophage cross-talk will ultimately lead to the development of approaches to prevent or treat early stage breast cancers. As described here, we demonstrate that the cell surface protease a disintegrin and metalloproteinase 17 (ADAM17) is expressed by macrophages in mammary tumors and contributes to regulating the expression of pro-inflammatory mediators, including inflammatory cytokines and the inflammatory mediator cyclooxygenase-2 (Cox-2). Furthermore, we demonstrate that ADAM17 is expressed on leukocytes, including macrophages, within polyoma middle T (PyMT)-derived mammary tumors. Genetic deletion of ADAM17 in leukocytes resulted in decreased onset of mammary tumor growth, which was associated with reduced expression of the Cox-2 within the tumor. These findings demonstrate that ADAM17 regulates key inflammatory mediators in macrophages and that leukocyte-specific ADAM17 is an important promoter of mammary tumor initiation. Understanding the mechanisms associated with early stage tumorigenesis has implications for the development of preventive and/or treatment strategies for early stage breast cancers.
    PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance
    Soto Romuald, PLOS Genet - 2016
    Author Summary Fibromuscular Dysplasia (FMD) is a vascular disease characterized by a succession of occlusions and dilatation of medium-sized arteries (e.g renal, carotid or coronary arteries) with important health consequences, mainly resistant hypertension and stroke. FMD is an atypical vascular disease because it is not associated with overweight or dyslipidemia and 80% of patients are early middle aged women. Our genetic study conducted in >1100 patients and >3800 controls demonstrate that a common variant rs9349379 located on chromosome 6 in the phosphatase and actin regulator 1 gene ( PHACTR1 ) increases by ~40% the risk of FMD. This is the first time a genetic risk factor is reported for FMD because it has been longtime considered rare and potentially under a Mendelian mode of inheritance. We also show that rs9349379 correlates with the expression of PHACTR1 in fibroblasts from FMD patients and controls. Interestingly, the same allele that increases the risk of FMD is at risk for cervical artery dissection and migraine, often reported in FMD patients but protective from myocardial infarction and coronary disease, where atherosclerosis is more common. The clear role of PHACTR1 in maintaining vascular well integrity is not fully elucidated. Using a specific antibody we detected PHACTR1 both on endothelial and smooth muscle cells of human FMD and control carotids, which suggests that PHACTR1 may have multiple functions depending on the cell type and the degree of atherosclerosis of the arteries.
    Gli transcription factors mediate the oncogenic transformation of prostate basal cells induced by a Kras-androgen receptor axis
    Meng Wu, Journal of Biological Chemistry - 2016
    Although the differentiation of oncogenically transformed basal progenitor cells is one of the key steps in prostate tumorigenesis, the mechanisms mediating this cellular process are still largely unknown. Here we demonstrated that an expanded p63+ and CK5+ basal/progenitor cell population, induced by the concomitant activation of oncogenic Kras(G12D) and androgen receptor (AR) signaling, underwent cell differentiation in vivo. The differentiation process led to suppression of the p63 expressing cells with a decreased number of CK5+ basal cells, but an increase of CK8+ luminal tumorigenic cells, and revealed a hierarchal lineage pattern consisting of p63+/CK5+ progenitor, CK5+/CK8+ transitional progenitor, and CK8+ differentiated luminal cells. Further analysis of the phenotype showed that the Kras-AR axis induced tumorigenesis was mediated by Gli transcription factors. Combined blocking of the activators of this family of proteins (Gli1 and Gli2) inhibited the proliferation of p63+ and CK5+ basal/progenitor cells and development of tumors. Finally, we identified that Gli1 and Gli2 exhibited different functions in regulation of p63 expression or proliferation of p63+ cells in Kras-AR driven tumors. Gli2, but not Gli1, transcriptionally regulated the expression levels of p63 and prostate sphere formation. Our study provides evidence of a novel mechanism mediating pathological dysregulation of basal/progenitor cells through the differential activation of the Gli transcription factors. Also, these findings define Gli proteins as new downstream mediators of the Kras-AR axis in prostate carcinogenesis and open a potential therapeutic avenue of targeting prostate cancer progression by inhibiting Gli signaling.
    CRH peptide evolution occurred in three phases: Evidence from characterizing sea lamprey CRH system members
    Matthew J. Endsin, General and Comparative Endocrinology - 2016
    The corticotropin releasing hormone (CRH) system, which includes the CRH family of peptides, their receptors (CRHRs) and a binding protein (CRHBP), has been strongly conserved throughout vertebrate evolution. The identification of invertebrate homologues suggests this system evolved over 500 million years ago. However, the early vertebrate evolution of the CRH system is not understood. Current theory indicates that agnathans (hagfishes and lampreys) are monophyletic with a conservative evolution over the past 500 million years and occupy a position at the root of vertebrate phylogeny. We isolated the cDNAs for three CRH family members, two CRHRs and a CRHBP from the sea lamprey, Petromyzon marinus. Two of the CRH peptides are related to the CRH/urotensin-1 (UI) lineage, whereas the other is a urocortin (Ucn) 3 orthologue. The predicted amino acid identity of CRH and UI is 61% but they possess distinct motifs indicative of each peptide, suggesting an early divergence of the two genes. Based on our findings we propose the CRH peptides evolved in at least 3 distinct phases. The first occurring prior to the agnathans gave rise to the CRH/UI-like and Ucn2/3-like paralogous lineages. The second was a partial sub-genomic duplication of the ancestral CRH/UI-like gene, but not the Ucn2/3-like gene, giving rise to the CRH and UI (Ucn) lineages. The third event which resulted in the appearance of Ucn2 and Ucn3 must have occurred after the evolution of the cartilaginous fishes. Interestingly, unlike other vertebrate CRHRs, we were unable to classify our two P. marinus receptors (designated CRHRα and CRHRβ) as either type 1 or type 2, indicating that this split evolved later in vertebrate evolution. A single CRHBP gene was found suggesting that either this gene has not been affected by the vertebrate genome duplications or there have been a series of paralogous gene deletions. This study suggests that P. marinus possess a functional CRH system that differs from that of the gnathostomes and may represent a model for the earliest functioning CRH system in vertebrates.
    Frontiers | The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses | Plant Physiology
    Kashif Mahmood, Frontiers in Plant Science - 2016
    Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light (HL) stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, HL and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX) and positive regulatory (TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under HL stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions
    Acute and long‐term effects of blood flow restricted training on heat shock proteins and endogenous antioxidant systems
    K. T. Cumming, Scandinavian Journal of Medicine & Science in Sports - 2016
    Blood flow restricted exercise (BFRE) with low loads has been demonstrated to induce considerable stress to exercising muscles. Muscle cells have developed a series of defensive systems against exercise-induced stress. However, little is known about acute and long-term effects of BFRE training on these systems. Nine previously untrained females trained low-load BFRE and heavy load strength training (HLS) on separate legs and on separate days to investigate acute and long-term effects on heat shock proteins (HSP) and endogenous antioxidant systems in skeletal muscles. BFRE and HLS increased muscle strength similarly by 12 ± 7% and 12 ± 6%, respectively, after 12 weeks of training. Acutely after the first BFRE and HLS exercise session, αB-crystallin and HSP27 content increased in cytoskeletal structures, accompanied by increased expression of several HSP genes. After 12 weeks of training, this acute HSP response was absent. Basal levels of αB-crystallin, HSP27, HSP70, mnSOD, or GPx1 remained unchanged after 12 weeks of training, but HSP27 levels increased in the cytoskeleton. Marked translocation of HSP to cytoskeletal structures at the commencement of training indicates that these structures are highly stressed from BFRE and HLS. However, as the muscle gets used to this type of exercise, this response is abolished.
    Mutations in TSPEAR , Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis
    Alon Peled, PLOS Genet - 2016
    Author Summary Ectodermal dysplasias refer to a large group of inherited disorders characterized by developmental defects in tissues of ectodermal origin. The study of these conditions has been instrumental in the discovery of biological pathways involved in the regulation of epithelial tissue morphogenesis. In this report, through the delineation of the molecular basis of a novel form of autosomal recessive ectodermal dysplasia, we identified a new key player in ectodermal development. We detected a number of mutations in TSPEAR co-segregating with abnormal hair and tooth development in three families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR was found to be strongly expressed in murine hair and tooth. Using a reporter assay, we showed that it regulates Notch activity. Accordingly, NOTCH1 expression was altered in patient skin, and NOTCH1, as well as many of its known targets, was down-regulated in TSPEAR deficient keratinocytes. Moreover, Tspear silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch pathway. As such, these new data are likely to lead to further investigations aimed at characterizing the role of Notch signaling pathway in other forms of ectodermal dysplasias as well as acquired hair and tooth pathologies.
    Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models
    Qiumin Tan, Human Molecular Genetics - 2016
    Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival.
    TGF-β1 promotes linear invadosome formation in hepatocellular carcinoma cells, through DDR1 up-regulation and collagen I cross-linking
    Zakaria Ezzoukhry, European Journal of Cell Biology - 2016
    Transforming growth factor-β1 (TGF-β1) is an important player in chronic liver diseases inducing fibrogenesis and hepatocellular carcinoma (HCC) development. TGF-β1 promotes pleiotropic modifications at the cellular and matrix microenvironment levels. TGF-β1 was described to enhance production of type I collagen and its associated cross-linking enzyme, the lysyl oxidase-like2 (LOXL2). In addition, TGF-β1 and type I collagen are potent inducers of invadosomes. Indeed, type I collagen fibers induce the formation of active linear invadosomes through the discoidin domain receptor 1 (DDR1). The goal of our study was to address the role of TGF-β1 in collagen cross-linking and its impact on the formation of linear invadosomes in liver cancer cells. We first report a significant correlation between expressions of TGF-β1, and type I collagen, LOXL2, DDR1 and MT1-MMP in human HCCs. We demonstrate that TGF-β1 promotes a Smad4-dependent up-regulation of DDR1, together with LOXL2, in cultured HCC cells. Moreover, we show that LOXL2-induced collagen cross-linking enhances linear invadosome formation. Altogether, our data demonstrate that TGF-β1 favors linear invadosome formation through the expressions of both the inducers, such as collagen and LOXL2, and the components such as DDR1 and MT1-MMP of linear invadosomes in cancer cells. Meanwhile, our data uncover a new TGF-β1-dependent regulation of DDR1 expression.
    Expression of T helper cell–associated inflammatory mediator mRNAs in cells of bronchoalveolar lavage fluid samples and oxygen concentration in arterial blood samples from healthy horses exposed to hyperbaric oxygen
    Maty G. P. Looijen, American Journal of Veterinary Research - 2016
    OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell–associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy. ANIMALS 8 healthy horses. PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell–derived, Th2 cell–derived (including eotaxin-2), Th17 cell–derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session. RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.
    Influence of Donor Age and Stimulation Intensity on Osteogenic Differentiation of Rat Mesenchymal Stromal Cells in Response to Focused Low-Intensity Pulsed Ultrasound
    Regina Puts, Ultrasound in Medicine & Biology - 2016
    A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm2 (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.
    Targeted Gene Activation Using RNA-Guided Nucleases
    Ulf Andersson Ørom, Methods in Molecular Biology - 2016
    The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.
    Expression of the aryl hydrocarbon receptor contributes to the establishment of intestinal microbial community structure in mice
    Iain A. Murray, Scientific Reports - 2016
    Environmental and genetic factors represent key components in the establishment/maintenance of the intestinal microbiota. The aryl hydrocarbon receptor (AHR) is emerging as a pleiotropic factor, modulating pathways beyond its established role as a xenobiotic sensor.
    Effect of rearing temperature on the hypoxia response of embryonic zebrafish
    Kelly Levesque, The University of Guelph - 2016
    Environmental stressors, such as warm temperatures and hypoxia, can interact and pose a threat to aquatic species. Cross-talk between the hypoxia and heat stress cellular pathways can lead to enhanced cross-tolerance between these environmental stressors. In this study, I asked whether elevated temperatures(from 27°C to 32°C)during rearing would enhance the hypoxia-inducible transcription factor-1(HIF-1) mediated transcriptional response to hypoxia in early stages of zebrafish development and whether these differences would be associated with enhanced larval tolerance and survivalto hypoxia. I found that embryos reared at 32°C had an enhanced cellular HIF-1 response and that acute hypoxia activated the heat-shock response. Rearing at 32°C and/ or embryonichypoxia exposure had no effect on thehypoxia tolerance(Pcrit) of four day-old larvae and did not protect larvae against the lethal effects of a second acute hypoxia exposure.Overall, cross-talk at the gene expression level did not predict whole animal responses when larvae were reexposed to hypoxic conditions .
    Potential vectors of Xylella fastidiosa: a study of leafhoppers and treehoppers in citrus agroecosystems affected by Citrus Variegated Chlorosis
    G. Dellapé, Entomologia Experimentalis et Applicata - 2016
    This study investigated the predominant leafhopper and treehopper (Hemiptera, Auchenorrhyncha) species in Citrus Variegated Chlorosis (CVC)-affected citrus agroecosystems in Argentina, their seasonal fluctuation, and their potential role as vectors of Xylella fastidiosa Wells et al., using molecular methods for detection. More than 6 000 Auchenorrhyncha were collected from three citrus agroecosystems over a period of 3 years using yellow sticky traps and entomological nets. Cicadellidae and Membracidae were the most abundant families. Of the 43 species identified, five were predominant in citrus orchards, and three were predominant in weeds surrounding citrus plants. All predominant species and another four non-predominant species tested positive for X. fastidiosa in PCR and real-time PCR assays. In a transmission assay, Dechacona missionum (Berg), Tapajosa rubromarginata (Signoret), and Cyphonia clavigera (Fabricius) transmitted X. fastidiosa successfully. Scaphytopius bolivianus Oman and Frequenamia spiniventris (Linnavuori) populations increased once (during the summer), possibly due to favorable weather conditions, and Bucephalogonia xanthophis (Berg), Molomea lineiceps Young, and T. rubromarginata populations increased twice a year: once in summer and once in winter, coinciding with the increase in early citrus shoots (flush). Among the X. fastidiosa-positive species, those with the higher population densities during the sprouting period, where trees are highly susceptible to infection, must be considered as most relevant vectors of CVC in the citrus-growing areas in Argentina.
    Biased Signalling is an Essential Feature of TLR4 in Glioma Cells
    Marie-Theres Zeuner, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research - 2016
    A distinct feature of the Toll-like receptor 4 (TLR4) is its ability to trigger both MyD88-dependent and MyD88-independent signalling, culminating in activation of pro-inflammatory NF-κB and/or the antiviral IRF3. Although TLR4 agonists (lipopolysaccharides; LPSs) derived from different bacterial species have different endotoxic activity, the impact of LPS chemotype on the downstream signalling is not fully understood. Notably, different TLR4 agonists exhibit anti-tumoural activity in animal models of glioma, but the underlying molecular mechanisms are largely unknown. Thus, we investigated the impact of LPS chemotype on the signalling events in the human glioma cell line U251. We found that LPS of Escherichia coli origin (LPSEC) leads to NF-κB-biased downstream signalling compared to Salmonella minnesota-derived LPS (LPSSM). Exposure of U251 cells to LPSEC resulted in faster nuclear translocation of the NF-κB subunit p65, higher NF-κB-activity and expression of its targets genes, and higher amount of secreted IL-6 compared to LPSSM. Using super-resolution microscopy we showed that the biased agonism of TLR4 in glioma cells is neither a result of differential regulation of receptor density nor of formation of higher order oligomers. Consistent with previous reports, LPSEC-mediated NF-κB activation led to significantly increased U251 proliferation, whereas LPSSM-induced IRF3 activity negatively influenced their invasiveness. Finally, treatment with methyl-β-cyclodextrin (MCD) selectively increased LPSSM-induced nuclear translocation of p65 and NF-κB activity without affecting IRF3. Our data may explain how TLR4 agonists differently affect glioma cell proliferation and migration.
    Recovery of antigen-specific T cell responses from dogs infected with Leishmania (L.) infantum by use of vaccine associated TLR-agonist adjuvant
    Robert G. Schaut, Vaccine - 2016
    Visceral leishmaniasis (VL), caused by infection with the obligate intracellular protozoan parasite Leishmania infantum, is a fatal disease of dogs and humans. Protection against VL requires a T helper 1 (Th1) skewed CD4+ T response, but despite this knowledge, there are currently no approved-to-market vaccines for humans and only three veterinary-use vaccines globally. As VL progresses from asymptomatic to symptomatic, L. infantum–specific interferon gamma (IFNγ) driven-Th1 responses become dampened and a state of immune exhaustion established. T cell exhaustion and other immunoregulatory processes, starting during asymptomatic disease, are likely to hinder vaccine-induced responses if vaccine is administered to infected, but asymptomatic and seronegative, individuals. In this study we evaluated how immune exhaustion, shown previously by our group to worsen in concert with VL progression, effected the capacity of vaccine candidate antigen/toll-like receptor (TLR) agonist combinations to promote protective CD4+ T cell responses during progressive VL. In conjunction with Th1 responses, we also evaluated concomitant stimulation of immune-balanced IL-10 regulatory cytokine production by these vaccine products in progressive VL canine T cells. Vaccine antigen L111f in combination with TLR agonists significantly recovered CD4+ T cell IFNγ intracellular production in T cells from asymptomatic VL dogs. Vaccine antigen NS with TLR agonists significantly recovered CD4+ T cell production in both endemic control and VL dogs. Combinations of TLR agonists and vaccine antigens overcame L. infantum induced cellular exhaustion, allowing robust Th1 CD4+ T cell responses from symptomatic dogs that previously had dampened responses to antigen alone. Antigen-agonist adjuvants can be utilized to promote more robust vaccine responses from infected hosts in endemic areas where vaccination of asymptomatic, L. infantum-infected animals is likely.
    Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures
    Andrew Holowiecki, Toxicology and Applied Pharmacology - 2016
    While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs.
    Toll-like receptor 4 mutation suppresses hyperhomocysteinemia-mediated hypertension. - viewcontent.cgi
    Anastasia Familtseva, University of Louisville - 2016
    Background: Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR4 activation has been shown to promote inflammation that has been associated with pathogenesis of hypertension. In this study, we hypothesize that HHcy induces hypertension by TLR-4 activation that promotes inflammatory cytokine up-regulation (IL1β, IL 6, TNF-α) and initiation of mitochondrial dysfunction leading to cell death and chronic vascular inflammation.
    Downregulation of NEDD9 by apigenin suppresses migration, invasion, and metastasis of colorectal cancer cells
    Jin Dai, Toxicology and Applied Pharmacology - 2016
    Apigenin is a natural flavonoid which possesses multiple anti-cancer properties such as anti-proliferation, anti-inflammation, and anti-metastasis in many types of cancers including colorectal cancer. Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a multi-domain scaffolding protein of the Cas family which has been shown to correlate with cancer metastasis and progression. The present study investigates the role of NEDD9 in apigenin-inhibited cell migration, invasion, and metastasis of colorectal adenocarcinoma DLD1 and SW480 cells. The results show that knockdown of NEDD9 inhibited cell migration, invasion, and metastasis and that overexpression of NEDD9 promoted cell migration and invasion of DLD1 cells and SW4890 cells. Apigenin treatment attenuated NEDD9 expression at protein level, resulting in reduced phosphorylations of FAK, Src, and Akt, leading to inhibition on cell migration, invasion, and metastasis of both DLD1 and SW480 cells. The present study has demonstrated that apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt cascade in colorectal cancer cells. NEDD9 may function as a biomarker for evaluation of cancer aggressiveness and for selection of drug target for therapeutic potential against cancer progression.
    Agkihpin, a novel SVAE may inhibit the migration and invasion of liver cancer cells associated with the inversion of EMT induced by Wnt/β-catenin signaling inhibition
    Miao Huang, Biochemical and Biophysical Research Communications - 2016
    In our previous work, agkihpin, a snake venom arginine esterase (SVAE), was isolated from the Gloydius halys Pallas, which could attenuate the migration of liver cancer cells. However, the mechanism of the effect of agkihpin on attenuating migration of liver cancer cell is unknown yet. Here, to learn more about agkihpin and explore the possibility of agkihpin as an anti-metastatic drug in the future, a series of experiments about the migration and invasion of liver cancer cells with agkihpin, HepG 2 and SMMC-7721, was conducted. Epithelial-mesenchymal transition (EMT) is an initial step and a major phenotype of cancer metastasis and invasion, while a number of EMT opposite phenomenons were observed, for example, epithelial marker E-cadherin was up-regulated, mesenchymal markers N-cadherin and Vimentin, and transcription regulators Snail and twist were down-regulated after treating with agkihpin in liver cancer cells; canonical Wnt/β-catenin pathway, one of the signals initiated EMT, was inhibited by decreased expressions of FZD7 and β-catenin, phosphorylation of GSK3β (Ser9), and nuclear β-catenin accumulation in agkihpin treated cancer cells. By using bioinformatics analysis and protease activity analysis in vitro we also found that agkihpin might bind and degrade FZD7. As a result, we hypothesized that agkihpin could inhibit the Wnt/β-catenin signaling pathway by cleaving FZD7, leading to the inactivation of the TCF/LEF transcription factor, which contributed to the inversion of EMT, and finally attenuated the migration and invasion of liver cancer cells. Therefore, our findings provided novel mechanistic insights into the role of SVAEs in liver cancer controlling, and raised the possibility that agkihpin may be used therapeutically in liver cancer.
    SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics
    Dana Chen, Scientific Reports - 2016
    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities.
    The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response
    Preeti Bharaj, PLOS Pathogens - 2016
    For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.
    NLRC4 suppresses melanoma tumor progression independently of inflammasome activation
    Ann Janowski, The Journal of Clinical Investigation - 2016
    Members of the NLR family can assemble inflammasome complexes with the adaptor protein ASC and caspase-1 that result in the activation of caspase-1 and the release of IL-1β and IL-18. Although the NLRC4 inflammasome is known to have a protective role in tumorigenesis, there is an increased appreciation for the inflammasome-independent actions of NLRC4. Here, we utilized a syngeneic subcutaneous murine model of B16F10 melanoma to explore the role of NLRC4 in tumor suppression. We found that NLRC4-deficient mice exhibited enhanced tumor growth that was independent of the inflammasome components ASC and caspase-1. Nlrc4 expression was critical for cytokine and chemokine production in tumor-associated macrophages and was necessary for the generation of protective IFN-γ–producing CD4+ and CD8+ T cells. Tumor progression was diminished when WT or caspase-1–deficient, but not NLRC4-deficient, macrophages were coinjected with B16F10 tumor cells in NLRC4-deficient mice. Finally, examination of human primary melanomas revealed the extensive presence of NLRC4+ tumor-associated macrophages. In contrast, there was a paucity of NLRC4+ tumor-associated macrophages observed in human metastatic melanoma, supporting the concept that NLRC4 expression controls tumor growth. These results reveal a critical role for NLRC4 in suppressing tumor growth in an inflammasome-independent manner.
    Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects
    Franziska Pietsch, Journal of Antimicrobial Chemotherapy - 2016
    Objectives Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the β-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. Methods Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. Results Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT–PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. Conclusions These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.
    Topography of microglial activation in sensory- and affect-related brain regions in chronic pain
    Anna M.W. Taylor, Journal of Neuroscience Research - 2016
    Microglial activation in the spinal cord plays a central role in the development and maintenance of chronic pain after a peripheral nerve injury (PNI). There has not yet been a thorough assessment of microglial activation in brain regions associated with pain and reward. To this end, this study uses a mouse model of neuropathic pain in which the left sciatic nerve of male C57Bl/6J mice is loosely constricted (chronic constriction injury) to assess microglial activation in several brain regions 2 weeks after injury, a time point at which pain hypersensitivity is well established. We found significant microglial activation in brain regions associated with sensory pain transmission and affect, including the thalamus, sensory cortex, and amygdala. Activation was consistently most robust in brain regions contralateral to the side of injury. Brain regions not directly involved in either sensory or affective dimensions of pain, such as the motor cortex, did not display microglial activation. This study confirms that PNI induces microglial activation in regions involved with both sensory and affective components of pain. © 2016 Wiley Periodicals, Inc.
    Deficiency of Stearoyl-CoA Desaturase-1 Aggravates Colitogenic Potential of Adoptively Transferred Effector T cells
    Beng San Yeoh, American Journal of Physiology - Gastrointestinal and Liver Physiology - 2016
    Stearoyl CoA Desaturase (SCD-1) is a lipogenic enzyme involved in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayers of cell membranes. Accordingly, Scd1KO mice display substantially reduced oleate in cell membranes. Altered SCD-1 level was observed during intestinal inflammation; however, its role in modulating inflammatory bowel disease remains elusive. Herein, we investigated the colitogenic capacity of Scd1KO effector T cells by employing the adoptive T-cell transfer colitis model. Splenic effector T-cells (CD4+CD25-) from age- and sex-matched WT and Scd1KO mice were isolated by FACS and intraperitoneally administered to Rag1KO mice, which were monitored for the development of colitis. At day 60 post-cell transfer, Rag1KO mice which received Scd1KO CD4+CD25- T cells displayed accelerated and exacerbated colitis than mice receiving WT cells. Intriguingly, Scd1KO CD4+CD25- T cells display augmented inflammatory cytokine profile and cellular membrane fluidity with concomitant increase in pro-inflammatory saturated fatty acids, which we postulate to potentially underlie their augmented colitogenic potential.
    Human primary airway epithelial cells isolated from active smokers have epigenetically impaired antiviral responses
    Wenxin Wu, Respiratory Research - 2016
    Cigarette smoking (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and most COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of the increased risk and severity of infections in smokers is likely multifactorial, but certainly includes changes in immunologic host defenses.
    Mechanisms and intervention strategies for alcohol and HIV-antiretroviral therapy-induced liver injury.
    Hridgandh Donde, University of Louisville - 2015
    Section I: Chronic alcohol consumption is a leading cause of liver disease and liver-related death worldwide. Alcoholic liver disease includes, hepatic steatosis, steatohepatitis and ultimately fibrosis and cirrhosis. Emerging evidence has established the important role of the “gut-liver” axis in the development of alcoholic liver disease (ALD). Our recent work indicated that chronic alcohol induced perturbations in the gut microbiome and consequent changes in fatty acids have a major impact on the development of intestinal barrier dysfunction and ALD. The aim of this study was to investigate whether treatment with tributyrin - a butyrate prodrug results in protection against ALD in terms of hepatic steatosis, inflammation and injury. Tributyrin is a triglyceride that is rapidly absorbed and metabolized to butyrate. Moreover, it has more favorable pharmacokinetics compared with butyrate with low toxicity.
    Jan Hall, United States Patent - 2016
    The present invention relates to a method for diagnosing bone loss rate, particularly in the field of bone anchored implants. The present patent provides with a method that comprises the steps of: a) quantifying the expression level of one or more markers or ratio thereof related to the activity of osteoclasts and/or osteoblasts in an ex vivo sample; and b) determining the bone loss rate as a function of ongoing loss of marginal bone level by interpolating the value obtained in step a) in one or more calibration curves. The invention also relates to a kit for performing said method.
    Jingwei Cai, Journal of Proteome Research - 2016
    Recent studies have identified the important role of the gut microbiota in the pathogenesis and progression of obesity and related metabolic disorders. The antioxidant tempol was shown to prevent or reduce weight gain and modulate the gut microbiota community in mice; however, the mechanism by which tempol modulates weight gain/loss with respect to the host and gut microbiota has not been clearly established. Here we show that tempol (0, 1, 10, and 50 mg/kg p.o. for 5 days) decreased cecal bacterial fermentation and increased fecal energy excretion in a dose-dependent manner. Liver 1H NMR-based metabolomics identified a dose-dependent decrease in glycogen and glucose, enhanced glucogenic and ketogenic activity (tyrosine and phenylalanine), and increased activation of the glycolysis pathway. Serum 1H NMR-based metabolomics indicated that tempol promotes enhanced glucose catabolism. Hepatic gene expression was significantly altered as demonstrated by an increase in Pepck and G6pase and a decrease in Hnf4a, ChREBP, Fabp1, and Cd36 mRNAs. No significant change in the liver and serum metabolomic profiles was observed in germ-free mice, thus establishing a significant role for the gut microbiota in mediating the beneficial metabolic effects of tempol. These results demonstrate that tempol modulates the gut microbial community and its function, resulting in reduced host energy availability and a significant shift in liver metabolism toward a more catabolic state.
    Significant Contribution of Mouse Mast Cell Protease 4 in Early Phases of Experimental Autoimmune Encephalomyelitis
    Louisane Desbiens, Hindawi Publishing - 2016
    Mast cells are integrally involved in cellular based immune responses to pathogens as well as inflammatory reactions prompted by pathogens or toxins [1, 2] and have been suggested for several years to play a part in the acute phase of Multiple Sclerosis (MS) [3–8]. Albeit the mechanisms by which mast cells influence MS are yet to be fully understood, trypsin-like proteases released from degranulating mast cells have been shown to trigger demyelination in a mouse model for MS-experimental autoimmune encephalomyelitis (EAE) [9]. Data from Secor et al. (2000), furthermore, support that mast cells are involved in the pathogenesis of EAE [3]. The same group has more recently suggested a significant role for mast cells in activation of inflammasomes localized within meninges [10]. In contrast, other groups have challenged the contribution of mast cells in EAE [11–13]. In particular, Feyerabend and colleagues show that the complete ablation of MCs in a “kit independent” MC-deficient strain does not affect EAE development [12]. Albeit targeting mast cells as a viable approach to alleviate the disease remains debatable, the contribution of mast cell-derived proteases in EAE is still largely uninvestigated. One of the many mast cell-expressed proteases that potentially may account for the contribution of mast cells to MS/EAE is mouse mast cell protease 4 (mMCP-4), a 𝛽-chymase predicted to be the murine functional counterpart to the single human chymase (CMA1) based on deduced amino acid sequence, tissue localization, and serglycin storage dependence [14, 15]. In support for a role of this chymase in mast cell-dependent inflammatory conditions, mMCP-4 plays a protective role in a mouse model of mechanically induced cerebral trauma [16], yet it is detrimental in lung inflammation and immune complexinduced glomerulonephritis [17, 18]. In further support for a role of mMCP-4 in regulating inflammatory mediators, our group has reported that mMCP-4 generates endothelin-1 (ET-1) from its precursor big-ET-1 [19, 20] and thatmMCP-4 knockout (KO) mice display a 40% reduction in pulmonary ET-1 levels when compared to wild type (WT) congeners [19]. The role of ET-1 as a marker in the etiology of MS has only been explored in a limited fashion. Haufschild et al. (2001) reported a significant increase in ET-1 plasma levels in untreated MS patients [21], an observation confirmed by Pache and colleagues (2003) [22]. In treated MS patients though, such increases were not found [23]. However, in further support for a role of ET-1 in MS, ET-1 was shown to be overexpressed in a murine model of EAE [24]. Based on the above-suggested links between ET-1 and MS and between chymase and ET-1 generation, respectively, we here asked whether chymase might have a role in EAE and whether that enzyme in this experimental setting has a regulatory effect on ET-1 production. Indeed, the findings presented here suggest that mMCP-4 has a significant detrimental impact on the course of EAE and plays role in the generation of ET-1 in this mouse model for MS. The impact of mMCP-4 in EAE introduces a potential role for mast cell chymase in MS and thereby identifies the inhibitors of this particular enzyme as potential targets for therapy of MS.
    Broad RTK-targeted therapy overcomes molecular heterogeneity-driven resistance to cetuximab via vectored immunoprophylaxis in colorectal cancer
    Shi Hu, Cancer Letters - 2016
    The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.
    Transcriptome analysis of tetraploid cells identifies Cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53
    Tamara A. Potapova, Molecular Biology of the Cell - 2016
    Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates changes in gene expression evoked in acute and adapted tetraploid cells and their impact on cell-cycle progression. Acute polyploidy was generated by knockdown of essential regulator of cytokinesis Anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology. Transcriptome analysis of these acute tetraploid cells revealed common signatures of activation of the tumor-suppressor protein p53. Suppression of proliferation in these cells was dependent on p53 and its transcriptional target - CDK inhibitor p21. Rare proliferating tetraploid cells can emerge from acute polyploid populations. Gene expression analysis of single-cell derived, adapted tetraploid clones showed up-regulation of several p53 target genes and cyclin D2, the activator of CDK4/6/2. Overexpression of cyclin D2 in diploid cells strongly potentiated the ability to proliferate with increased DNA content despite the presence of functional p53. These results point out that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as Cyclin D2, elucidating a possible route for tetraploidy-mediated genomic instability in carcinogenesis.
    ZpdN, a plasmid-encoded sigma factor homolog, induces pBS32-dependent cell death in Bacillus subtilis
    B-E Myagmarjav, Journal of Bacteriology - 2016
    The ancestral Bacillus subtilis strain 3610 contains an 84 kb plasmid called pBS32 that was lost during domestication of commonly used laboratory derivatives. Here we demonstrate that pBS32, normally present at 1-2 copies per cell, increases copy number nearly 100-fold when cells are treated with the DNA damaging agent mitomycin C. Mitomycin C treatment also caused cell lysis dependent on pBS32 encoded prophage genes. ZpdN, a sigma factor homolog encoded on pBS32, was required for the plasmid response to DNA damage and artificial expression of ZpdN was sufficient to induce pBS32 hyper-replication and cell death. Plasmid DNA released by cell death was protected by the capsid protein ZpbH suggesting that the plasmid was packaged into a phage-like particle. The putative particles were further indicated by CsCl sedimentation but were not observed by electron microscopy and were incapable of killing B. subtilis cells extracellularly. We hypothesize that pBS32-mediated cell death releases a phage-like particle that is defective and unstable.
    Regulators of coastal wetland methane production and responses to simulated global change
    Carmella Vizza, Biogeosciences Discuss - 2016
    Abstract.Wetlands are the largest natural source of methane (CH4) to the atmosphere, but their emissions vary along salinity and productivity gradients. Global change has the potential to reshape these gradients and therefore alter future contributions of wetlands to the global CH4 budget. Our study examined CH4 production along a natural salinity gradient in coastal Alaska wetlands. In the laboratory, we incubated natural sediments to compare CH4 production rates between freshwater and intertidal wetlands, and quantified the abundances of methanogens and sulfate reducing bacteria in these ecosystems. We also simulated sea-level rise and enhanced organic matter availability, which we predicted would have contrasting effects on coastal wetland CH4 production. Intertidal wetlands produced less CH4 than freshwater wetlands due to high sulfate availability and generally higher abundances of sulfate reducing bacteria, whereas freshwater wetlands had significantly greater methanogen abundances. Simulated sea level rise in freshwater sediments, however, did not reduce CH4 production, perhaps because the d incubation period was too short to elicit a shift in microbial communities. In contrast, increased organic matter generally enhanced CH4 production rates, but this response varied by the macrophyte species added. Our study suggests that CH4 production in coastal wetlands, and therefore their overall contribution to the global CH4 cycle, will be sensitive o increased organic matter availability and potentially sea level rise. To better predict future wetland contributions to the global CH4 budget, future studies and modeling efforts should investigate how multiple global change mechanisms will interact to impact CH4 dynamics
    Fructose Synthesis and Transport at the Uterine-Placental Interface of Pigs: Cell-Specific Localization of SLC2A5, SLC2A8, and Components of the Polyol Pathway
    McKinsey Landers, Biology of Reproduction - 2016
    The fetal fluids and uterine flushings of pigs contain higher concentrations of fructose than glucose, but fructose is not detected in maternal blood. Fructose can be synthesized from glucose via enzymes of the polyol pathway, aldose reductase (AKR1B1) and sorbitol dehydrogenase (SORD), transported across cell membranes by solute carriers SLC2A5 and SLC2A8, and converted to fructose-1-phosphate by keto-hexokinase (KHK). SLC2A8, SLC2A5, AKR1B1, SORD and KHK mRNAs and proteins were analyzed using qPCR and immunohistochemistry or in situ hybridization in endometria and placentae of cyclic and pregnant gilts, cyclic gilts injected with estrogen, and ovariectomized gilts injected with progesterone (P4). Progesterone up-regulated SLC2A8 protein in uterine luminal (LE) and glandular epithelia (GE) during the peri-implantation period, and expression became exclusively placental, chorion and blood vessels, after Day 30. P4 up-regulated SLC2A5 mRNA in uterine LE and GE after implantation, and the chorion expressed SLC2A5 between Days 30 and 85. AKR1B1 and SORD proteins localized to uterine LE during the peri-implantation period, but expression switched to chorion by Day 20 and was maintained through Day 85. Uterine expression of AKR1B1 mRNA was down-regulated by estrogen. KHK protein localized to trophectoderm/chorion throughout gestation. These results provide the evidence that components for conversion of glucose to fructose, and for fructose transport, are present at the uterine-placental interface of pigs. The shift in expression from LE to chorion during pregnancy suggests free-floating conceptuses are supported by fructose synthesized by the uterus, but after implantation, the chorion becomes self-sufficient for fructose synthesis and transport.
    Essential role for the planarian intestinal GATA transcription factor in stem cells and regeneration
    Natasha M. Flores, Developmental Biology - 2016
    The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians.
    Biosynthetic mechanism of very long chain polyunsaturated fatty acids in Thraustochytrium sp. 26185
    Dauenpen Meesapyodsuk, Journal of Lipid Research - 2016
    Thraustochytrium, a unicellular marine protist, has been used as a commercial source of very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3). Our recent work indicates coexistence of a ∆4-desaturation-dependent pathway (aerobic) and a polyketide synthase-like polyunsaturated fatty acid (PUFA) synthase pathway (anaerobic) to synthesize the fatty acids in Thraustochytrium sp. 26185. Heterologous expression of the Thraustochytrium PUFA synthase along with a phosphopantetheinyl transferase (PPTase) in E. coli showed the anaerobic pathway was highly active in the biosynthesis of VLCPUFAs. The amount of ∆4 desaturated VLCPUFAs produced reached about 18% of the total fatty acids in the transformant cells at Day 6 in a time course of the induced expression. In the Thraustochytrium, the expression level of the PUFA synthase gene was much higher than that of the ∆4 desaturase gene, and also highly correlated with the production of VLCPUFAs. On the other hand, ∆9 and ∆12 desaturations in the aerobic pathway were either ineffective or absent in the species, as evidenced by the genomic survey, heterologous expression of candidate genes and in vivo feeding experiments. These results indicate that the anaerobic pathway is solely responsible for the biosynthesis for VLCPUFAs in the Thraustochytrium.
    Reducing the time interval between concussion and voluntary exercise restores motor impairment, short-term memory, and alterations to gene expression
    Richelle Mychasiuk, European Journal of Neuroscience - 2016
    Despite the most common form of brain injury, there has been little progress in the prognosis and treatment of concussion/mild traumatic brain injury (mTBI). Current ‘return-to-play’ guidelines are conservative, deterring the initiation of physical and social activity until patients are asymptomatic; but the effects of post-injury exercise have not been adequately investigated. Therefore, this study examined the effects of voluntary exercise on concussion recovery. Using a translational rodent model of concussion, we examined the influence of exercise on injury-associated behaviours that comprise post-concussive syndrome (PCS) and gene expression changes (bdnf, dnmt1, Igf-1, pgc1-a, Tert) in prefrontal cortex and hippocampus. In addition, as we have previously demonstrated telomere length (TL) to be a reliable predictor of mTBI prognosis, TL was also examined. The results suggest that exercise initiated within 1–3 days post-concussion significantly improved motor and cognitive functioning, but had limited efficacy treating emotional impairments. What is more, when deprived of social interaction and exercise, a combination similar to clinical recommendations for rest until symptom resolution, animals did not recover and exhibited impairments similar to typical mTBI animals. Exercise aided in restoration of mTBI-induced modifications to gene expression in both brain regions. An inverse relationship between the exercise return interval and TL was identified, indicating greater recovery with acute exercise reinstatement. Although additional strategies may need to be employed for emotional functioning, these findings support re-evaluation of ‘return-to-play’ guidelines, suggesting that exercise is valuable for the treatment of concussion.
    EGFR expression is associated with poor outcome in cutaneous squamous cell carcinoma
    J. Cañueto, British Journal of Dermatology - 2016
    Introduction Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans, after basal cell carcinoma, and its incidence is dramatically rising. CSCC is rarely problematic, but given its high frequency, the absolute number of complicated cases is also high. It is necessary to identify molecular markers to recognize those CSCC with poor prognosis. There is controversy concerning the role of EGFR (epidermal growth factor receptor) as a marker of prognosis in CSCC. In addition, EGFR-targeted therapies have emerged in recent years and a better understanding of the role of EGFR in CSCC may help some patients in predicting prognosis and guide curative management. Patients and Methods We evaluated clinical and histopathological features, including events of bad clinical evolution, in a series of 94 CSCC. We also analysed EGFR expression by immunohistochemistry, FISH and QPCR. Results We detected EGFR in 85 (90.4%) cases, with overexpression in 33 (35.1%) cases, and aberrant EGFR expression in the cytoplasm in 50 (53.1%) cases. EGFR overexpression in the primary tumours was associated with lymph node progression, TNM stage progression and proliferation (Ki-67 staining) in CSCC. EGFR overexpression and poor grade of differentiation were the strongest independent variables defining lymph node metastasis and progression in CSCC in a logistic regression model. Conclusion We demonstrate that EGFR overexpression has prognostic implications associated with lymph node metastasis and progression in CSCC. This article is protected by copyright. All rights reserved.
    Factors controlling the co-occurrence of microbial sulfate reduction and methanogenesis in coal bed reservoirs
    Andrew W. Glossner, International Journal of Coal Geology - 2016
    Sulfate-reducing microorganisms (SRM) and methanogenic archaea have been previously observed in coal bed methane reservoirs, suggesting that the model for separation of these organisms based on sulfate concentration may not apply to such reservoirs. Using a methanogenic consortium enriched from coal, microcosm experiments showed simultaneous activity of methanogens and sulfate reducers at sulfate concentrations ranging from 50 to 1000 μM when coal was the sole substrate. These experiments revealed no apparent correlation between methanogenic potential and sulfate concentration. In other microcosm experiments with varying acetate amendments, concentrations of the phospholipid fatty acids (PLFAs) 14:0, 16:1ω5, 16:1ω7cis, 16:1ω7trans, and cy17:0 correlated strongly with the initial acetate concentration in microcosms with 500 μM sulfate, while i17:0 correlated strongly in microcosms with 200 μM sulfate. A significant portion of the acetate in these experiments went to microbial metabolisms other than dissimilatory sulfate reduction or methanogenesis, suggesting that some of these PLFAs were likely produced by some other unknown acetate-consuming micro-organisms. Copies of the dsrA gene increased at least 10-fold over initial levels in samples without molybdate (MoO42 −) across all experiments, indicating that SRM were active when not inhibited by MoO42 −. In experiments with < 300 μM acetate, copies of the mcrA gene increased over 49 days regardless of sulfate concentration. These results suggest that both SRM and methanogens are active at low acetate concentrations and may compete for available acetate with other acetate-consuming bacteria in coal bed methane reservoirs
    Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I
    Randall A. Dass, PLOS Genet - 2016
    Author Summary Synthesis of the translation machinery, including the mega-Dalton, RNA-protein ribosome complex, serves as a key driver of cellular growth and proliferation. It is therefore unsurprising that ribosomal biogenesis is under intricate regulation. The process through which ribosomes are made entails the coordination of components from diverse signaling pathways in both normal and diseased cells. Both oncogenes and tumor suppressors can influence this orchestration by impinging upon the rate-determining steps of RNA Polymerase I-mediated transcription of ribosomal RNA (rRNA) and the coupled process of ribosome assembly. In this study we investigated whether the secreted protein Wnt5a, an antagonist of mammary tumor growth, regulates rRNA synthesis in breast cancer cells. We find that the induction of Wnt5a signaling disturbs assembly of the RNA polymerase I machinery, leading to a repressive rDNA chromatin state that is not amenable to active rRNA gene transcription. Wnt5a signaling represses rRNA synthesis by stimulating nucleolar accumulation of Dishevelled1 (DVL1), a downstream effector of Wnt5a signaling, while having no such effect on DVL2 or DVL3. Wnt5a-induced accumulation of DVL1 in the nucleolus directly interferes with the synthesis of rRNA, suggesting that a tumor suppressive effect of Wnt5a in breast cancer cells is mediated by DVL1-dependent repression of rRNA synthesis.
    Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments
    Tetsuro Komatsu, Traffic - 2016
    In adenoviral virions, the genome is organized into a chromatin-like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized, and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatio-temporal regulation of viral genomes during subsequent steps remains to be elucidated. Here we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication-related factors. We show de novo formation of a subnuclear domain, which we termed Virus-induced Post-Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inwards. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.
    The Development of a Quantitative Assay for the detection of Grapevine Red Blotch-accociated Virus in Vitis vinifera Identifies Significant Differences in Virus Distribution
    Felicia Jesslyn Setiono, Cornell University Thesis - 2016
    Grapevine red blotch-associated virus (GRBaV) is associated with red blotch disease which undermines optimal growth and development of grapevine (Vitis vinifera). Despite GRBaV’s significant economical and biological impacts, existing diagnostic methods lack sensitivity and consistency. This study has developed, optimized and employed a reliable quantitative Real-Time PCR (qPCR) assay for the detection of GRBaV in a variety of host tissue types. Primers specific to GRBaV and internal host control (NADP-dependent Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) were selected for use in qPCR based on their performance in initial validation tests. Controls consisted of 1) the internal GAPDH which served as relative reference of total input DNA and as a marker for template quality, 2) a dilution series of cloned target viral DNA, and 3) negative controls of water and total nucleic acid from uninfected vines. This method was then used to quantify the amount of GRBaV in multiple infected greenhouse-(GG) and field-(FG) grown vines. Absolute and relative quantification methods were shown to be strongly correlated (R2> 0.84) for both GG and FG. Viral DNA quantities varied in different tissue types and from one plant to another between and within a location, but most significantly between GG and FG, where only 56.0% of the total samples from the latter were determined as positive compared to 98.4% for the former. Petioles were consistently found to contain higher amounts of GRBaV compared to their corresponding leaves (P<0.05). Leaves proximal to the main stem were found to contain higher amounts of GRBaV compared to leaves located in the apical part of the cane(P<0.01).Based on these findings, it is recommended that total nucleic acid extracted from multiple petioles of fully developed leaves are used for robust and reliable GRBaV diagnosis using qPCR. The described qPCR assay and recommended sampling procedures will contribute to efforts in GRBaV containment and red blotch disease control
    PLOS ONE: Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice
    Chun Fu, PLOS ONE - 2016
    After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.
    Predominant expression of Alzheimer’s disease-associated BIN1 in mature oligodendrocytes and localization to white matter tracts
    Pierre De Rossi, Molecular Neurodegeneration - 2016
    Genome-wide association studies have identified BIN1 within the second most significant susceptibility locus in late-onset Alzheimer’s disease (AD). BIN1 undergoes complex alternative splicing to generate multiple isoforms with diverse functions in multiple cellular processes including endocytosis and membrane remodeling. An increase in BIN1 expression in AD and an interaction between BIN1 and Tau have been reported. However, disparate descriptions of BIN1 expression and localization in the brain previously reported in the literature and the lack of clarity on brain BIN1 isoforms present formidable challenges to our understanding of how genetic variants in BIN1 increase the risk for AD.
    5-HT2A Receptor Binding in the Frontal Cortex of Parkinson’s Disease Patients and Alpha-Synuclein Overexpressing Mice: A Postmortem Study
    Nadja Bredo Rasmussen, Hindawi Publishing Corporation - 2016
    Parkinson’s disease (PD) is clinically characterized by motor symptoms consisting of bradykinesia, resting tremor, rigidity, and postural instability.One of the leading hypotheses for PD pathogenesis focuses on alterations in alpha-synuclein (AS) expression, neuronal accumulation, and aggregation of AS— including formation of Lewy bodies—as a main causative factor in the pathological cascade [1].Though PD principally is classified as amovement disorder, it has nowbecome recognised that PD features a complex burden of different motor and nonmotor symptoms (NMS) [1, 2]. NMS covers a range of symptoms including hyposmia, visual hallucinations, sleep disturbances, a variety of dysautonomic symptoms, depression and other mood disorders, and impairment of cognition and consequently affected executive function [3]. The key brain area involved in cognition and executive function is the prefrontal cortex (PFC). The serotonin 5-HT2A receptor is highly expressed in PFC areas, playing an important role in executive function [4] and in modulating the cognitive control of our emotional responses during decision-making [5], making them essential for inhibitory response control, which recently has been shown to be impaired in PD patients [6]. Alterations in cortical 5-HT2A receptor levels have been reported in Alzheimer’s disease patients [7].We have shown how this is associated with betaamyloid accumulation in transgenic mice models of betaamyloid overexpression [8]. Here we wanted to investigate whether this could also be the case for PD. The aimof this study was, on one side, to look for 5-HT2A receptor alterations in the frontal cortex of postmortembrain tissue from PD patients and, secondly, to investigate by use of transgenic mice whether overexpression of AS would lead to alterations in frontal cortical 5-HT2A receptors. Alterations in serotonergic innervation have previously been described in relation to PD [9], but information is lacking about the extent to which serotonin receptors, and more specifically 5-HT2A receptors in PFC, are affected in this disease. For the first approach, we used membrane suspensions of human frontal cortex tissue of PD and control brains in order to perform receptor binding studies with the 5-HT2Aspecific radioligand, [3H]-MDL 100907, and furthermore measured AS protein levels by western blotting. Second, we used a transgenic mouse model overexpressing human AS to examine region-specific 5-HT2A receptor changes by autoradiography analysis of [3H]-MDL 100.907 binding, followed by gene expression analysis.
    Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism
    C. Alexander Valencia, PLOS ONE - 2016
    Background & Aims The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological factors in patients with idiopathic ALF and impaired energy metabolism. Methods Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis. Results A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9 , POLG , POLG2 , DGUOK , and RRM2B ; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9 , which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy. Conclusion Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF.
    Nucleolin-Mediated RNA Localization Regulates Neuron Growth and Cycling Cell Size
    Rotem Ben-Tov Perry, Cell Reports - 2016
    Summary How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin β1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin β1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin β1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.
    A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents : Scientific Reports
    Yuanchao Zhan,, Scientific Reports - 2016
    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205–279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.
    A comparative examination of cortisol effects on muscle myostatin and HSP90 gene expression in salmonids
    Nicholas J.Galt, General and Comparative Endocrinology - 2016
    Cortisol, the primary corticosteroid in teleost fishes, is released in response to stressors to elicit local functions, however little is understood regarding muscle-specific responses to cortisol in these fishes. In mammals, glucocorticoids strongly regulate the muscle growth inhibitor, myostatin, via glucocorticoid response elements (GREs) leading to muscle atrophy. Bioinformatics methods suggest that this regulatory mechanism is conserved among vertebrates, however recent evidence suggests some fishes exhibit divergent regulation. Therefore, the aim of this study was to evaluate the conserved actions of cortisol on myostatin and hsp90 expression to determine if variations in cortisol interactions have emerged in salmonid species. Representative salmonids; Chinook salmon (Oncorhynchus tshawytscha), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar); were injected intraperitoneally with a cortisol implant (50 μg/g body weight) and muscle gene expression was quantified after 48 h. Plasma glucose and cortisol levels were significantly elevated by cortisol in all species, demonstrating physiological effectiveness of the treatment. HSP90 mRNA levels were elevated by cortisol in brook trout, Chinook salmon, and Atlantic salmon, but were decreased in cutthroat trout. Myostatin mRNA levels were affected in a species, tissue (muscle type), and paralog specific manner. Cortisol treatment increased myostatin expression in brook trout (Salvelinus) and Atlantic salmon (Salmo), but not in Chinook salmon (Oncorhynchus) or cutthroat trout (Oncorhynchus). Interestingly, the VC alone increased myostatin mRNA expression in Chinook and Atlantic salmon, while the addition of cortisol blocked the response. Taken together, these results suggest that cortisol affects muscle-specific gene expression in species-specific manners, with unique Oncorhynchus-specific divergence observed, that are not predictive solely based upon mammalian stress responses.
    Expression of progesterone receptor in the porcine uterus and placenta throughout gestation: correlation with expression of uteroferrin and osteopontin
    C. B. Steinhauser, Domestic Animal Endocrinology - 2016
    Progesterone (P4) stimulates production and secretion of histotroph, a mixture of hormones, growth factors, nutrients, and other substances required for growth and development of the conceptus (embryo/fetus and placental membranes). Progesterone acts through the progesterone receptor (PGR); however, there is a gap in our understanding of P4 during pregnancy because PGR have not been localized in the uteri and placentae of pigs beyond day 18. Therefore, we determined endometrial expression of PGR messenger RNA (mRNA) and localized PGR protein in uterine/placental tissues throughout the estrous cycle and through day 85 of pregnancy in pigs. Further, 2 components of histotroph, tartrate-resistant acid phosphatase 5 (ACP5; uteroferrin) and secreted phosphoprotein 1 (SPP1; osteopontin) proteins, were localized in relation to PGR during pregnancy. Endometrial expression of PGR mRNA was highest at day 5 of the estrous cycle, decreased between days 5 and 11 of both the estrous cycle and pregnancy, and then increased between days 11 and 17 of the estrous cycle (P < 0.01), but decreased from days 13 to 40 of pregnancy (P < 0.01). Progesterone receptor protein localized to uterine stroma and myometrium throughout all days of the estrous cycle and pregnancy. PGR were expressed by uterine luminal epithelium (LE) between days 5 and 11 of the estrous cycle and pregnancy, then PGR became undetectable in LE through day 85 of pregnancy. During the estrous cycle, PGR were downregulated in LE between days 11 and 15, but expression returned to LE on day 17. All uterine glandular epithelial (GE) cells expressed PGR from days 5 to 11 of the estrous cycle and pregnancy, but expression decreased in the superficial GE by day 12. Expression of PGR in GE continued to decrease between days 25 and 85 of pregnancy; however, a few glands near the myometrium and in close proximity to areolae maintained expression of PGR protein. Acid phosphatase 5 protein was detected in the GE from days 12 to 85 of gestation and in areolae. Secreted phosphoprotein 1 protein was detected in uterine LE in apposition to interareolar, but not areolar areas of the chorioallantois on all days examined, and in uterine GE between days 35 and 85 of gestation. Interestingly, uterine GE cells adjacent to areolae expressed PGR, but not ACP5 or SPP1, suggesting these are excretory ducts involved in the passage, but not secretion, of histotroph into the areolar lumen and highlighting that P4 does not stimulate histotroph production in epithelial cells that express PGR.
    Peroxiredoxin 1 interacts with and blocks the redox factor APE1 from activating interleukin-8 expression
    Hassan Nassour, Scientific Reports - 2016
    APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, PRDX1 knockdown greatly facilitates APE1 detection within the nucleus by indirect immunofluorescence analysis, even though APE1 level was unchanged. The loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB onto the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. Our findings suggest that the interaction of PRDX1 with APE1 represents a novel anti-inflammatory function of PRDX1, whereby the association safeguards APE1 from reducing transcription factors and activating superfluous gene expression, which otherwise could trigger cancer invasion and metastasis.
    Tryptophan Codon-dependent Transcription in Chlamydia pneumoniae during Interferon-γ-mediated Tryptophan Limitation
    Scot P. Ouellette, American Society for Microbiology - 2016
    In evolving to an obligate intracellular niche, Chlamydia has streamlined its genome by eliminating superfluous genes as it relies on the host cell for a variety of nutritional needs like amino acids. However, Chlamydia can experience amino acid starvation when the human host cell in which they reside is exposed to IFNγ, which leads to a tryptophan (trp)-limiting environment via induction of the enzyme IDO. The stringent response is used to respond to amino acid starvation in most bacteria but is missing from Chlamydia. Thus, how Chlamydia, a trp auxotroph, responds to trp starvation in the absence of a stringent response is an intriguing question. We previously observed that C. pneumoniae responds to this stress by globally increasing transcription while globally decreasing translation, an unusual response. Here, we sought to understand this and hypothesized that the trp codon content of a given gene would determine its transcription level. We quantified transcripts from C. pneumoniae genes that were either rich or poor in trp codons and found that trp codon-rich transcripts were increased whereas those that lacked trp codons were unchanged or even decreased. There were exceptions, and these involved operons or large genes with multiple trp codons: downstream transcripts were less abundant after trp codon-rich sequences. These data suggest that ribosome stalling on trp codons causes a negative polar effect on downstream sequences. Finally, re-assessing previous C. pneumoniae microarray data based on codon content, we found that upregulated transcripts were enriched in trp codons thus supporting our hypothesis.
    Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus
    Leandro Rodríguez-Viera, PLOS ONE - 2017
    Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low K m, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.
    Aimee L. Benjamin, University of Vermont Dissertation - 2016
    Mastitis represents one of the major economical and animal welfare concerns within the dairy industry. Animals affected with this disease can experience a range of clinical symptoms from mild discomfort and swelling of the udder to a severe systemic inflam matory response that could result in the death of the animal. This range of responses is due to differences in pathogen, environment, and inter - animal differences in their innate immune response. A dermal fibroblast model was used to predict the magnitude of an animal’s innate immune response towards an intra - mammary S. aureus challenge. Animals whose fibroblasts exhibited a low response phenotype, characterized by lower levels of IL - 8 following in vitro immune stimulation, suffered less mammary tissue dama ge and a less severe reduction in milk quality following the in vivo S. aureus challenge as compared to animals classified as high responders. Furthermore, the heightened inflammatory response of the high responders offered no advantage in bacterial cleara nce. For a S. aureus infection, the lower response phenotype is preferred. To further explore inter - animal variation in the innate immune response, fibroblast cultures were established and challenged with LPS from two breeds of cattle, Holsteins, a dairy breed and Angus, a beef breed. Cultures from Holstein animals exhibited a higher responding phenotype than cultures from Angus animals. As these two breeds undergo selection for different traits and are reared differently as calves, whole transcriptome an alysis (RNA - Seq) and DNA methylation analysis (Methylated CpG Island Recovery Assay; MIRA - Seq) of their fibroblasts was completed to examine the genetic and epigenetic basis for the contrasting responses. RNA - Seq revealed several immune associated genes th at were expressed at higher levels in Holstein cultures compared to Angus cultures, including TLR4, IL - 8, CCL5, and TNF - α , both basally and following LPS exposure. Although MIRA - Seq analysis revealed 49 regions with differential methylation between the Hol stein and Angus cultures, overall, the methylation of the fibroblast genome was similar between these breeds. A combination of genetic and epigenetic factors seems to contribute to the breed - dependent differences observed between Holstein and Angus fibrobl asts. Early life exposure to bacterial compounds or inflammatory mediators can have long - term effects on the magnitude of an animal’s innate immune response, and may contribute to inter - animal variation in this response. To determine if an early life expo sure to LPS would modify the response to a subsequent LPS challenge in dairy animals, neonatal Holstein calves were treated with LPS or saline at 7 days of age and subsequently challenged with LPS 25 days later. Calves that received LPS at 7 days of age ha d greatly elevated levels of plasma IL - 6 and TNF - α compared to calves that received saline, indicating a substantial inflammatory response. However, following the subsequent LPS challenge completed on all calves, there were no differences in plasma IL - 6 an d TNF - α between the LPS - and saline - treated calves. Alternative exposure strategies in calves may generate the long - term effects observed in other model systems. There is a wide range in the responses observed in the innate immune response of the bovine. Animals with a lower innate immune response effectively clear the infection, but avoid the collateral tissue damage from excessive inflammation. Therefore, it seems that a reduced innate immune response would be more beneficial to the dairy cow.
    Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica
    Karin Hjort, Molecular Microbiology - 2016
    Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria.
    MLP and CARP are linked to chronic PKCα signalling in dilated cardiomyopathy
    Stephan Lang, Nature Communications - 2016
    MLP (muscle LIM protein)-deficient mice count among the first mouse models for dilated cardiomyopathy (DCM), yet the exact role of MLP in cardiac signalling processes is still enigmatic. Elevated PKCα signalling activity is known to be an important contributor to heart failure. Here we show that MLP directly inhibits the activity of PKCα. In end-stage DCM, PKCα is concentrated at the intercalated disc of cardiomyocytes, where it is sequestered by the adaptor protein CARP in a multiprotein complex together with PLCβ1. In mice deficient for both MLP and CARP the chronic PKCα signalling chain at the intercalated disc is broken and they remain healthy. Our results suggest that the main role of MLP in heart lies in the direct inhibition of PKCα and that chronic uninhibited PKCα activity at the intercalated disc in the absence of functional MLP leads to heart failure.
    Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells: Molecular Endocrinology: Vol 30, No 8
    Kang Ho Kim, Endocrine Society - 2016
    Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GRLysM) or HSC (GRhGFAP): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GRLysM mice, whereas the suppression of fibrotic gene expression was diminished in GRhGFAP mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis. - See more at: http://press.endocrine.org/doi/abs/10.1210/me.2016-1029?journalCode=mend#sthash.5aFq7wnQ.dpuf
    Fibroblast Growth Factor 23 Directly Targets Hepatocytes to Promote Inflammation in Chronic Kidney Disease - viewcontent.cgi
    Saurav Singh, University of Miami Thesis - 2016
    Chronic kidney disease (CKD) is a global health problem that affects 10 - 15% of the adult population worldwide and significantly increases the risk of death. Patients with CKD develop marked elevations in circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23), which correlate with the stage of disease, and are associated with higher risk of mortality. Chronic inflammation is a hallmark of CKD. Circulating levels of inflammatory cytokines, such as C Reactive protein (CRP), interleukin6 (IL6)and tumor necrosis factor α (TNFα) are significantly elevated in CKD patients and increase with disease progression. This inflammatory burden has been suggested to be a significant contributor to the high mortality rate in CKD. Furthermore, recent clinical studies have demonstrated that in CKD elevated serum levels of FGF23 are independently associated with higher circulating levels of inflammatory cytokines, which can stimulate FGF23 production in osteocytes. These correlative studies suggest a causative role of FGF23 in the development of systemic inflammation in CKD; however, the sources of inflammation and precise molecular mechanisms underlying the pathological interrelationship between deterioration of renal function, elevated FGF23 production and amplification of the inflammatory state are unknown. This thesis project explores the direct effects of FGF23 on the liver. Our data indicate that FGF23 can directly stimulate hepatocytes to produce inflammatory cytokines in the absence of αklotho, which is the FGF23 coreceptor in the kidney that is not expressed by hepatocytes. By activating FGF receptor isoform 4 (FGFR4), FGF23 stimulates phospholipase Cγ (PLCγ)/calcineurin/nuclear factor of activated T cells (NFAT) signaling in hepatocytes, which increases expression and secretion of inflammatory cytokines, including CRP. We show that the elevation of serum FGF23 levels increases hepatic and circulating levels of CRP in wild type mice, but not in FGFR4 knockout mice. Furthermore, administration of an isoform specific FGFR4 blocking antibody reduces hepatic and circulating levels of CRP in the 5/6 nephrectomy rat model of CKD. By demonstrating that FGF23 can directly stimulate hepatic secretion of inflammatory cytokines,our findings suggest a novel mechanism of chronic inflammation in patients with CKD. We postulate that FGFR4 blockade might have therapeutic anti-inflammatory effects in CKD.
    Differential Expression of CYC2 Genes and the Elaboration of Floral Morphologies in Hiptage, an Old World Genus of Malpighiaceae
    Wenheng Zhang, International Journal of Plant Sciences - 2016
    Premise of research. The primarily Neotropical Malpighiaceae exhibit an elegant suite of floral morphological characteristics associated with a specialized mutualism with oil bee pollinators, including bilaterally symmetrical flowers and paired oil glands on the calyx. One clade within the family, Hiptage Gaertn., has migrated to the paleotropics and lost its association with oil bees. Corresponding to this transition, some members of Hiptage have evolved a highly elaborate zygomorphic corolla with strongly reflexed petals and striking dorsoventral heteranthery. Previously, we demonstrated that expression of CYCLOIDEA2-like (CYC2-like) genes is correlated with the evolution of floral symmetry in Malpighiaceae. Here, we examine CYC2 expression in relation to the evolution of elaborate floral zygomorphy in Hiptage benghalensis.Methodology. CYC2-like genes were cloned from H. benghalensis. The spatial pattern of CYC2 expression was examined with quantitative reverse-transcription PCR on the dissected floral organs.Pivotal results. While most Neotropical Malpighiaceae express two CYC2-like genes, CYC2A and CYC2B, we demonstrate that H. benghalensis has experienced further duplications yielding four copies, which are expressed in all four whorls of the flower. As in Neotropical Malpighiaceae, CYC2A homologs HbCYC2A-1 and HbCYC2A-2 are expressed broadly in the dorsal region of the flower, but unlike that in other Neotropical species, expression also extends to the dorsal stamens. The CYC2B copies HbCYC2B-1 and HbCYC2B-2 are intensely expressed in the single dorsal petal (as in Neotropical Malpighiaceae), but their expression is further detected in the other floral whorls, especially in the stamens of the dorsal region.Conclusions. The relaxation of the conserved expression of CYC2-like genes in Neotropical Malpighiaceae and the expansion to broader floral regions, including the dorsal androecium, correlate with the development of dorsoventral heteranthery in H. benghalensis. We propose that changes in the pattern of CYC2 expression may have contributed to the elaborated androecium of H. benghalensis, which was crucial for its adaptation to a novel pollination strategy.
    Underexpression of CACNA1C Caused by Overexpression of microRNA-29a Underlies the Pathogenesis of Atrial Fibrillation
    Yujie Zhao, Medical Science Monitor - 2016
    ACKGROUND: The objective of this study was to investigate the molecular mechanism of atrial fibrillation (AF), as well as the negative regulatory relationship between miR-29a-3p and CACNA1C. MATERIAL AND METHODS: We searched the online miRNA database (www.mirdb.org) and identified the miR-29a-3p binding sequence within the 3’-UTR of the target gene, and then conducted luciferase assay to verify it. The cells were transfected with miR-29a-3p and ICa,L was determined in those cells. RESULTS: We validated CACNA1C to be the direct target gene of miR-29a-3p. We also established the negative regulatory relationship between miR-29a-3p and CACNA1C via studying the relative luciferase activity. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CACNA1C among different groups of cells treated with scramble control, 30nM miR-29a-3p mimics, and 60nM miR-29a-3p mimics, indicating a negative regulatory relationship between miR-29a-3p and CACNA1C. We next analyzed whether miR-29a-3p transfection in cardiomyocytes produced the effects on the ICa,L induced by electrical remodeling, and found a tonic inhibition of IBa by endogenous miR-29a-3p in atrial myocytes. CONCLUSIONS: We validated the negative regulation between miR-29a-3p and CACNA1C, and found that miR-29a-3p might a potential therapeutic target in the treatment of AF.
    Metagenomics reveals the high PAH&#x2010;degradation potential of abundant uncultured bacteria from chronically&#x2010;polluted subantarctic and temperate coastal marine environments - 574ae66f08ae2e0dd30197ac.pdf
    Claudia L Loviso, Journal of Applied Microbiology - 2015
    Aims: To investigate the potential to degrade polycyclic aromatic hydrocarbons (PAHs) of yet-to-be cultured bacterial populations from chronically-polluted intertidal sediments. Methods and Results: A gene variant encoding the alpha subunit of the catalytic component of an aromatic ring-hydroxylating oxygenase (RHO) was abundant in intertidal sediments from chronically-polluted subantarctic and temperate coastal environments, and its abundance increased after PAH amendment. Conversely, this marker gene was not detected in sediments from a non-impacted site, even after a short-term PAH exposure. A metagenomic fragment carrying this gene variant was identified in a fosmid library of subantarctic sediments. This fragment contained five pairs of alpha and beta subunit genes and a lone alpha subunit gene of oxygenases, classified as belonging to three different RHO functional classes. In silico structural analysis suggested that two of these oxygenases contain large substrate-binding pockets, capable of accepting high molecular weight PAHs. Conclusions: The identified uncultured microorganism presents the potential to degrade aromatic hydrocarbons with various chemical structures, and could represent an important member of the PAH-degrading community in these polluted coastal environments. Significance and Impact of Study: This work provides valuable information for the design of environmental molecular diagnostic tools and for the biotechnological application of RHO enzymes. Keywords: intertidal sediments; polycyclic aromatic hydrocarbons; ring-hydroxylating oxygenases; qPCR; metagenomic library; protein modelling.
    Distribution of the DNA transposon family, Pokey in the Daphnia pulex species complex
    Shannon H. C. Eagle, - 2016
    The Pokey family of DNA transposons consists of two putatively autonomous groups, PokeyA and PokeyB, and two groups of Miniature Inverted-repeat Transposable Elements (MITEs), mPok1 and mPok2. This TE family is unusual as it inserts into a specific site in ribosomal (r)DNA, as well as other locations in Daphnia genomes. The goals of this study were to determine the distribution of the Pokey family in lineages of the Daphnia pulex species complex, and to test the hypothesis that unusally high PokeyA number in some isolates of Daphnia pulicaria is the result of recent transposition. To do this, we estimated the haploid number of Pokey, mPok, and rRNA genes in 45 isolates from five Daphnia lineages using quantitative PCR. We also cloned and sequenced partial copies of PokeyA from four isolates of D. pulicaria.
    Evaluation of macroscopic changes and the efficiency of DNA profiling from burnt teeth
    Joe Adserias Garriga, Science & Justice - 2016
    Identification of human remains subjected to incineration is extremely challenging. Our study evaluates the macroscopic changes and efficiency of DNA profiling in burnt teeth under controlled temperature and time conditions. 28 teeth were exposed to temperatures between 100 and 700 °C for a duration of 1–15 min. Two non-burnt teeth were used as control. Macroscopic changes were evaluated and recorded. DNA was extracted using a silica-based methodology. Efficiency of DNA profiling was assessed through Quantitative PCR for STRs. Burnt teeth reached chalky white appearance at 400 °C 5 min and fractures were observed from 300 °C 10 min. Amplification of STRs was very low from 300 °C and 1 or 5 min. In contrast, the housekeeping gene, GAPDH, was amplified in all combinations of temperatures and times. Although it is possible to amplify the housekeeping gene at high temperature, DNA profiling is difficult to obtain, probably due to small size of these regions making them more prone to degradation.
    MicroRNA-186 and metastatic prostate cancer. - viewcontent.cgi
    Dominique Zilpha Jones, University of Louisville Dissertation - 2016
    MicroRNA (miR) dysregulation alters cancer associated gene expression, which contributes to cancer pathogenesis. For example, miR 186 over expression lead to enhanced proliferation and migration in pancreatic cancer cell models. However, the role of miR 186 in prostate cancer (PCa)remains controversial. Previously, miR 1865p was up regulated in PCa patient serum(stage III/IV) compared to controls. Furthermore, miR 186-5p was up regulated in metastatic PCa (PC3 , MDAPCa2b, LNCaP) Relative to normal prostate epithelial cells (RWPE1).We hypothesized miR186 inhibition will reduce aggressive PCa using metastatic cell models. To test this, we evaluated whether miR-1865p inhibition would reduce aggressive PCa Behavior and overexpression induce malignant transformation in normal cells.
    Role of phosphodiesterase-4 in alcohol-induced organ injury
    Diana Veronica Avila, University of Lousiville Dissertation - 2016
    Alcoholic liver disease (ALD) remains a leading cause of death from liver disease in the U.S., and there is still no FDA approved therapy. Alcohol metabolism leads to generation of free radicals and oxidative stress with a resultant formation of lipid peroxidation products, which, in turn, contribute to the development of ALD. Alcohol induced hepatic steatosis is the earliest and most frequent manifestation of ALD and a significant risk factor for progressive liver disease. Cyclic adenosine monophosphate (cAMP) signaling has been shown to significantly regulate lipid metabolism. Moreover, agents that increase cAMP have been shown to effectively mitigate oxidative stress both in vivo and in vitro. Hence, the role of hepatic PDE4 and a resultant dysregulation of cAMP signaling in alcohol induced hepatic steatosis and lipid peroxidation was examined. C57BL/6 wild type (WT) and Pde4b knockout (Pde4b/) mice were pairfed control and ethanol liquid diets. One group of wild type mice received Rolipram, a PDE4 specific inhibitor, during alcohol feeding. Alcohol feeding resulted in a significant fat accumulation and oxidative stress in WT mice as demonstrated by increased hepatic free fatty acid levels and lipid peroxidation. This alcohol effect was associated with a significant decrease in hepatic carnitine palmitoyltransferase 1A (CPT1A) expression, a rate limiting enzyme in fatty acid β oxidation. Additionally, hepatic F4/80 staining was markedly increased in alcohol fed WT mice, indicating Kupffer cell activation. Importantly, alcohol feeding significantly increased hepatic PDE4 enzyme expression as early as in one week with the concomitant decrease in cAMP/pCREB levels. PDE4 inhibition in alcohol fed mice prevented the decrease in hepatic CPT1A expression and lipid accumulation. This effect on CPT1A expression was mediated by preventing the decrease in a critical transcription factor for CPT1A expression, peroxisome proliferator activated receptor (PPARα)and increase in PPAR α coactivators, peroxisome proliferato ractivated receptor gamma coactivator 1α and sirtuin 1(PGC-1αand SIRT1). Moreover, compared to wild type mice, Pde4b knockout and Rolipram treated alcohol fed mice had higher levels of antioxidant enzymes SOD1/2, and GPx1/2 and decreased 4HNE and F4/80 staining. In summary, these results demonstrate that the alcohol induced increase in hepatic PDE4, specifically PDE4B expression, and compromised cAMP signaling predisposes the liver to impaired fatty acid oxidation and increased oxidative stress. These data also suggest that hepatic PDE4 is a clinically relevant therapeutic target for the treatment of alcoholic fatty liver disease.
    BMP-TAK1 (MAP3K7) Induces Adipocyte Differentiation Through PPARγ Signaling
    Yongchun Zhang, Journal of Cellular Biochemistry - 2016
    BMPs have been shown to promote adipocyte differentiation through SMAD-dependent signaling. However, the role of TGF-β-activated kinase 1 (TAK1) in non-canonical BMP signaling in adipocyte differentiation remains unclear. Here, we show that TAK1 inhibition decreases lipid accumulation in C3H10T1/2 mesenchymal stem cells (MSCs) induced to differentiate into adipocytes. TAK1 knockdown by siRNA further confirms that TAK1 is required for adipocyte commitment of MSCs. Additionally, TAK1 knockdown inhibits adipogenesis of 3T3-L1 preadipocytes, indicating that TAK1 is not only needed for adipocyte commitment, but also required for adipocyte terminal differentiation. Furthermore, TAK1 ablation specifically in adipocytes reduced high fat diet-induced weight gain and improved glucose tolerance. Mechanistically, we demonstrate that TAK1 is required for PPARγ transactivation and promotes PPARγ transcriptional activity synergistically with TAK1 binding protein 1 (TAB1). Collectively, our results demonstrate that TAK1 plays a critical role in BMP-mediated adipocyte differentiation. J. Cell. Biochem. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.
    Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing
    Raghavan Chinnadurai, STEM CELLS - 2016
    We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016
    Antimicrobial Resistance of Escherichia fergusonii Isolated from Broiler Chickens
    Karen Simmons, Journal of Food Protection - 2016
    The objective of this study was to investigate the antibiotic resistance of Escherichia fergusonii isolated from commercial broiler chicken farms. A total of 245 isolates from cloacal and cecal samples of 28- to 36-day-old chickens were collected from 32 farms. Isolates were identified using PCR, and their susceptibility to 16 antibiotics was determined by disk diffusion assay. All isolates were susceptible to meropenem, amikacin, and ciprofloxacin. The most common resistances were against ampicillin (75.1%), streptomycin (62.9%), and tetracycline (57.1%). Of the 184 ampicillin-resistant isolates, 127 were investigated using a DNA microarray carrying 75 probes for antibiotic resistance genetic determinants. Of these 127 isolates, the β-lactamase bla CMY2, bla TEM, bla ACT, bla SHV, and bla CTX-M-15 genes were detected in 120 (94.5%), 31 (24.4%), 8 (6.3%), 6 (4.7%), and 4 (3.2%) isolates, respectively. Other detected genes included those conferring resistance to aminoglycosides (aadA1, strA, strB), trimethoprims (dfrV, dfrA1), tetracyclines (tetA, tetB, tetC, tetE), and sulfonamides (sul1, sul2). Class 1 integron was found in 35 (27.6%) of the ampicillin-resistant isolates. However, our data showed that the tested E. fergusonii did not carry any carbapenemase bla OXA genes. Pulsed-field gel electrophoresis revealed that the selected ampicillin-resistant E. fergusonii isolates were genetically diverse. The present study indicates that the monitoring of antimicrobial-resistant bacteria should include enteric bacteria such as E. fergusonii, which could be a reservoir of antibiotic resistance genes. The detection of isolates harboring extended-spectrum β-lactamase genes, particularly bla CTX-M-15, in this work suggests that further investigations on the occurrence of such genes in broilers are warranted.
    A lipoprotein/B-barrel complex monitors lipopolysaccaride integrity transducing information across the outer membrane
    Anna Konovalova, eLife - 2016
    Lipoprotein RcsF is the OM component of the Rcs envelope stress response. RcsF exists in complexes with b barrel proteins (OMPs) allowing it to adopt a transmembrane orientation with a lipidated N-terminal domain on the cell surface and a periplasmic C-terminal domain. Here we report that mutations that remove BamE or alter a residue in the RcsF trans-lumen domain specifically prevent assembly of the interlocked complexes without inactivating either RcsF or the OMP. Using these mutations we demonstrate that these RcsF/OMP complexes are required for sensing OM outer leaflet stress. Using mutations that alter the positively charged surface-exposed domain, we show that RcsF monitors lateral interactions between lipopolysaccharide (LPS) molecules. When these interactions are disrupted by cationic antimicrobial peptides, or by the loss of negatively charged phosphate groups on the LPS molecule, this information is transduced to the RcsF C-terminal signaling domain located in the periplasm to activate the stress response
    Gong Chen, United States Patent - 2016
    Provided are methods and compositions from reprogramming human glial cells into human neurons. The reprogramming is achieved using combinations of compounds that can modify signaling via Transforming growth factor beta (TGF-β), Bone morphogenetic protein (BMP), glycogen synthase kinase 3 (GSK-3), and γ-secretase/Notch pathways. The reprogramming is demonstrated using groups of three or four compounds that are chosen from the group thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, purmorphamine. Reprogramming is demonstrated using the group of LDN193189/CHIR99021/DAPT, the group of B431542/CHIR99021/DAPT, the group of LDN193189/DAPT/SB431542, the group of LDN193189/CHIR99021/SB431542, a three drug combination of SB431542/CHIR99021/DAPT. Reprogramming using functional analogs of the compounds is also provided, as are pharmaceutical formulations that contain the drug combinations.
    Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress
    Emilien Nicolas, Nature Communications - 2016
    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology.
    LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation
    Shubhangi Prabhudesai, Journal of Neuroscience Research - 2016
    Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of β-synuclein, PARK13, and SOD1 and causes β-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na+/K+ ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.
    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. Keywords: TRPA1; Transient Receptor Potential A1; Ion Channel; Keratinocytes; Skin; Sensory Neurons; qPCR
    Gonads and the evolution of hormonal phenotypes
    Kimberly A. Rosvall, Integrative and Comparative Biology - 2016
    Hormones are dynamic signaling molecules that influence gene activity and phenotype, and they are thus thought to play a central role in phenotypic evolution. In vertebrates, many fitness-related traits are mediated by the hormone testosterone (T), but the mechanisms by which T levels evolve are unclear. Here, we summarize a series of studies that advance our understanding of these mechanisms by comparing males from two subspecies of dark-eyed junco (Junco hyemalis) that differ in aggression, body size, and ornamentation. We first review our research demonstrating population differences in the time-course of T production, as well as findings that point to the gonad as a major source of this variation. In a common garden, the subspecies do not differ in pituitary output of luteinizing hormone, but males from the more androgenized subspecies have greater gonadal gene expression for specific steroidogenic enzymes, and they may be less sensitive to feedback along the hypothalamo-pituitary-gonadal (HPG) axis. Furthermore, we present new data from a common garden study demonstrating that the populations do not differ in gonadal sensitivity to gonadotropin-inhibitory hormone (i.e., GnIH receptor mRNA abundance), but the more androgenized subspecies expresses less gonadal mRNA for glucocorticoid receptor and mineralocorticoid receptor, suggesting altered cross-talk between the hypothalamo-pituitary-gonadal and -adrenal axes as another mechanism by which these subspecies have diverged in T production. These findings highlight the diversity of mechanisms that may generate functional variation in T and influence hormone-mediated phenotypic evolution.
    Gene expression profiling of midbrain dopamine neurons upon gestational nicotine exposure
    Pınar Kanlikilicer, Medical & Biological Engineering & Computing - 2016
    Maternal smoking during pregnancy is associated with low birth weight, increased risk of stillbirth, conduct disorder, attention-deficit/hyperactivity disorder and neurocognitive deficits. Ventral tegmental area dopamine (DA) neurons in the mesocorticolimbic pathway were suggested to play a critical role in these pathological mechanisms induced by nicotine. Nicotine-mediated changes in genetic expression during pregnancy are of great interest for current researchers. We used patch clamp methods to identify and harvest DA and non-DA neurons separately and assayed them using oligonucleotide arrays to elucidate the alterations in gene expressions in these cells upon gestational nicotine exposure. Microarray analysis identified a set of 135 genes as significantly differentially expressed between DA and non-DA neurons. Some of the genes were found to be related to neurological disease pathways, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Significantly up-/down-regulated genes found in DA neurons were mostly related to G-protein-coupled protein receptor signaling and developmental processes. These alterations in gene expressions may explain, partially at least, the possible pathological mechanisms for the diseases induced by maternal smoking.
    Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation.
    Svetlana Kalujnaia, American Journal of Physiology - Regulatory, Integrative and Comparative Physiology - 2016
    A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single MIPS transcript identified in all eel tissues, although two MIPS spliced variants (termed MIPS(s) and MIPS(l)) are found in all tilapia tissues. The larger tilapia transcript (MIPS(l)) results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes where the MIPS(l) transcript predominates. SW-acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested whereas in the eel changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW-acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67 kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells as well as unknown entero-endocrine-like cells.
    Dinesh S Rao, United States patent - 2016
    The instant disclosure relates to the finding that lincRNA expression is associated with particular cytogenetic abnormalities and is related to disease pathogenesis of certain cancers. Long intergenic non-coding RNAs (lincRNAs) have been found to play a role in gene regulation, but their expression has not been described in B acute lymphoblastic leukemia (B-ALL). Methods and compositions are provided regarding B-ALL associated long intergenic RNAs (BALIRs).
    A Novel Protocol of Culturing Adult Porcine Islets for Transplantation in Type 1 Diabet Patients
    Wilma Suarez-Pinzon, The Minnesota Academy of Science Journal of - 2016
    . This study shows that lengthening the culture period for pre-transplantation adult porcine islets from 7 days to 30 days significantly lessens the chance of immunorejection after xenotransplantation for type 1 diabetes.Immunofluorescence staining was chosen to analyze antigen expression, islet viability, and insulin production after culture periods of 7, 15, and 30 days. Because an inexpensive humidity chamber to provided 95% humidity was not available, an inexpensive humidity chamber was engineered from materials purchased at Target and found in the laboratory. Additionally, silane gold-coated slides were optimized for immunofluorescence staining. Immunocytochemistry staining showed that after 30 days, there was a significant reduction in Gal+cells and in CD45, Class I, and Class II antigens (p < 0.05), all of which are associated with immune rejection of porcine islets. Additionally, there was a significant increase in insulin production over a 30-day culture period (p < 0.05), as well as a significant reduction in mononuclear cells that are responsible for immunorejection. Furthermore, viability staining showed that a culture period of 30 days provides a rehabilitative environment for islets that experience stress after extraction (p < 0.05). Finally, RT-PCR was done to validate results. RT-PCR showed no significant change in gene expression of insulin over a 30 day culture period (p > 0.05). As a result of our work, our research site is now running quality control trials to determine efficacy of incubating all pre-transplantation adult porcine islets for 30 days.Suarez-Pinzon W, Clark B, Godishala P. A novel protocol for culturing adult porcine islets for transplantation in Type I diabetic patients.
    Activation of EGFR/p38/HIF-1α is pivotal for angiogenesis and tumorigenesis of malignantly transformed cells induced by hexavalent chromium
    Donghern Kim, Journal of Biological Chemistry - 2016
    Cr(VI)-containing compounds are well-established environmental carcinogens. Most mechanistic investigations of Cr(VI)-induced carcinogenesis focus on oxidative stress and various cellular responses, leading to malignant cell transformation, or the first stage of metal-induced carcinogenesis. The development of malignantly transformed cells into tumors which require angiogenesis is the second stage. This study focuses on the second stage, in particular, the role of epidermal growth factor receptor (EGFR) signaling in angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our preliminary studies have shown that EGFR is constitutively activated in Cr(VI)-transformed cells, in lung tissue from Cr(VI)-exposed animals, and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. Using in vitro and in vivo models, the present study has investigated the role of EGFR in angiogenesis of Cr(VI)-transformed cells. The results show that Cr(VI)-transformed cells are angiogenic. Hypoxia inducible factor (HIF) 1α, proangiogenic protein matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) are all highly expressed in Cr(VI)-transformed cells, in lung tissue from animals exposed to Cr(VI), and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. p38 MAPK was also activated in Cr(VI)-transformed cells and in the human lung tumor tissue. Inhibition of EGFR reduces p38 MAPK, resulting in decreased expressions of HIF-1α, MMP-1, and VEGF, leading to suppressions of angiogenesis and tumorigenesis. Overall, the present study has demonstrated that EGFR plays an important role in angiogenesis and tumorigenesis of Cr(VI)-transformed cells.
    Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa
    Ruimin Gao, BMC Genomics - 2016
    Medicago sativa (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in alfalfa.
    53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis
    Chii Shyang Fong, eLIFE - 2016
    Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.
    Perturbation of epigenetic processes by doxorubicin in the mouse testis
    Oluwajoba O.Akinjo, Toxicology Research - 2016
    Epigenetic processes play a major role in normal mammalian development, particularly during gametogenesis and early embryogenesis. Thus, perturbation of epigenetic processes in the testis by xenobiotics could have a major impact on testicular function and fertility, and potentially affect the development and health of subsequent generations. There has been substantial research into the epigenetic toxicity of environmental exposures over the last decade. However, few studies have focussed on pharmaceutical drugs, which due to the nature of their use are typically found at much higher concentrations within exposed individuals than environmental chemicals. Here, we investigated genome-wide changes in testicular mRNA transcription, microRNA expression and DNA methylation to assess the contribution of epigenetic mechanisms to the testicular toxicity induced by doxorubicin (DOX) as a representative, widely used and well-characterised anti-cancer drug. We demonstrated that DOX is able to induce transcriptional, microRNA and DNA methylation changes, which perturb pathways involved in stress/cell death and survival and testicular function and lead to germ cell loss and reproductive organ damage. This identified potential novel mechanisms of DOX-induced testicular toxicity for further focussed investigations. Such work is required to fully assess the role of epigenetics in toxicity, determine whether single and/or multigenerational epigenetic toxicity is a real public health concern, and begin to develop and incorporate relevant epigenetic endpoints into regulatory toxicology.
    The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination
    Benjamin M. Laitman, PLOS Biol - 2016
    During the development of white matter in the central nervous system, the transcription factor Klf6 coordinates pro-myelinating effects of gp130-Stat3 signaling, and promotes oligodendrocyte differentiation in part via control of nuclear trafficking.
    QSOX1 AS AN ANTI-NEOPLASTIC DRUG TARGET - Arizona Board of Regents, a Body Corporate of the State of Arizona Acting for and on Behalf of Arizo
    Douglas Lake, United States Patent - 2016
    The present invention provides methods for tumor treatment by administering an inhibitor of quiescin sulfhydryl oxidase 1 (QSOX1), compositions comprising such inhibitors, and methods for identifying such inhibitors.
    Glycogen Repletion in Brown Adipose Tissue upon Refeeding Is Primarily Driven by Phosphorylation-Independent Mechanisms
    Christopher M. Carmean, PLOS ONE - 2016
    Glycogen storage in brown adipose tissue (BAT) is generally thought to take place through passive, substrate-driven activation of glycogenesis rather than programmatic shifts favoring or opposing the storage and/or retention of glycogen. This perception exists despite a growing body of evidence suggesting that BAT glycogen storage is actively regulated by covalent modification of key glycogen-metabolic enzymes, protein turnover, and endocrine hormone signaling. Members of one such class of covalent-modification regulators, glycogen-binding Phosphoprotein Phosphatase-1 (PP1)-regulatory subunits (PPP1Rs), targeting PP1 to glycogen-metabolic enzymes, were dynamically regulated in response to 24 hr of starvation and/or 24 hr of starvation followed by ad libitum refeeding. Over-expression of the PPP1R Protein Targeting to Glycogen (PTG), under the control of the aP2 promoter in mice, inactivated glycogen phosphorylase (GP) and enhanced basal- and starvation-state glycogen storage. Total interscapular BAT glycogen synthase and the constitutive activity of GS were conditionally affected. During starvation, glucose-6-phosphate (G-6-P) levels and the relative phosphorylation of Akt (p-Ser-473-Akt) were both increased in PTG-overexpressing (Tg) mice, suggesting that elevated glycogen storage during starvation modifies broader cellular metabolic pathways. During refeeding, Tg and WT mice reaccumulated glycogen similarly despite altered GS and GP activities. All observations during refeeding suggest that the phosphorylation states of GS and GP are not physiologically rate-controlling, despite there being a clear balance of endogenous kinase- and phosphatase activities. The studies presented here reveal IBAT glycogen storage to be a tightly-regulated process at all levels, with potential effects on nutrient sensing in vivo .
    Alternating Diet as a Preventive and Therapeutic Intervention for High Fat Diet-induced Metabolic Disorder
    Ma Yongjie, Scientific Reports - 2016
    This study presents the alternating diet as a new strategy in combating obesity and metabolic diseases. Lean or obese mice were fed a high-fat diet (HFD) for five days and switched to a regular diet for one (5 + 1), two (5 + 2), or five (5 + 5) days before switching back to HFD to start the second cycle, for a total of eight weeks (for prevention) or five weeks (for treatment) without limiting animals’ access to food. Our results showed that animals with 5 + 2 and 5 + 5 diet alternations significantly inhibited body weight and fat mass gain compared to animals fed an HFD continuously. The dietary switch changed the pattern of daily caloric intake and suppressed HFD-induced adipose macrophage infiltration and chronic inflammation, resulting in improved insulin sensitivity and alleviated fatty liver. Alternating diet inhibited HFD-induced hepatic Pparγ-mediated lipid accumulation and activated the expression of Pparα and its target genes. Alternating diet in the 5 + 5 schedule induced weight loss in obese mice and reversed the progression of metabolic disorders, including hepatic steatosis, glucose intolerance, and inflammation. The results provide direct evidence to support that alternating diet represents a new intervention in dealing with the prevalence of diet-induced obesity.
    The Role of Anthocyanins and The GATA Transcription Factors GNC and CGAI in the Plant Response to Stress
    Zhenhua Xu, A Thesis from The University of Guelph - 2016
    Worldwide crop production is significantly affected by various abiotic and biotic stresses. Research on plant stress signaling and the resulting physiological response can provide insights into the development of crop cultivars with improved stress tolerance. In the current work, the role of anthocyanins in plant response to reactive oxygen species (ROS), high light and cold Stress is investigated. In addition, the global putative targets of two GATA transcription factors, CGA1 and GNC, known to modulate the chlorophyll content and natural senescence process in response to different environmental conditions is investigated. Anthocyanins are a class of secondary metabolites which in food have been shown to have antioxidant activity and thus potentially be beneficial for human health when present in sufficient quantities. First, the antioxidant role of anthocyanins in plant stress response is analysed. Ten Arabidopsis anthocyanin mutants, with varying levels of anthocyanin content, which are in the main regulatory and biosynthetic genes of the anthocyanin pathway were utilized. Their physiological, molecular and cellular responses were analyzed under ROS, high light and cold stress. The data shows that ROS is an important signal to trigger the accumulation of anthocyanins,and in turn anthocyanins function as an antioxidant to buffer ROS homeostasis and provide protection against the cellular damage caused by ROS. GNC and CGA1are two GATA transcription factors that play important roles in chlorophyll accumulation, flowering, hormone signaling and natural senescence as well as in the plant response to nutrient stress. We utilized a genome wide ChIP-Seq technique in Arabidopsis to discover the putative target genes of CGA1 and GNC and revealed the possible downstream genes controlling chlorophyll content and the natural senescence process. In addition, the pleotropic functions of rice OsGNC were explored through analyzing the rice OsGNC transgenic lines, suggesting the functional conservation of GNC and CGA1 across plant species. This study expanded the current knowledge of abiotic stress response in plants and hopefully this information can be applied for future crop improvement
    Masanori Aikawa, United States Patent - 2016
    The present invention is directed to methods of treating or preventing obesity by administering agents that inhibit the NOTCH signaling pathway. Antibodies that inhibit the binding of Delta like 4 ligand (Dll4) to NOTCH receptors may be used for this purpose.
    Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y1 receptor-cAMP signal pathway
    Jong Bae Seo, Cell Biology and Toxicology - 2016
    Extracellular adenosine-5′-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.
    LPS Induces Hyper-Permeability of Intestinal Epithelial Cells
    Amir Bein, Journal of Cellular Physiology - 2016
    Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder leading to high morbidity and mortality rates. A growing body of evidence demonstrate the key role of the Toll like receptor 4 (TLR4) in NEC. This membranal receptor recognizes lipopolysaccharides (LPS) from the bacterial wall and triggers an inflammatory response. The aim of the present study was to elucidate the effect of LPS on paracellular permeability known to be severely affected in NEC. IEC-18 cells were treated with LPS and the effects on morphology, paracellular permeability and their associated gene and protein expressions were measured. Our results show that LPS down regulated the expression of occludin and ZO-1 mRNAs while up regulating Cdkn1a. In addition LPS caused a significant increase in paracellular permeability and epithelial barrier damage. Finally ZO-1 protein was found to be spatially disarrayed in the intercellular junctions in response to LPS. We conclude that LPS adversely affected the functionality of the intestinal epithelial barrier suggesting a new mechanism by which bacterial infection may contribute to the development of NEC. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.
    Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer
    Yongjie Ma, Scientific Reports - 2016
    Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders.
    Divergence along the gonadal steroidogenic pathway: Implications for hormone-mediated phenotypic evolution
    Kimberly A. Rosvall, Hormones and Behavior - 2016
    Across a range of taxa, hormones regulate suites of traits that influence survival and reproductive success; however, the mechanisms by which hormone-mediated traits evolve are still unclear. We hypothesized that phenotypic divergence might follow from differential regulation of genes encoding key steps in hormone biosynthesis and thus the rate of hormone production. We tested this hypothesis in relation to the steroid hormone testosterone by comparing two subspecies of junco (Junco hyemalis) in the wild and in captivity. These subspecies have diverged over the last 10–15 k years in multiple testosterone-mediated traits, including aggression, ornamentation, and body size. We show that variation in gonadal gene expression along the steroid biosynthetic pathway predicts phenotypic divergence within and among subspecies, and that the more androgenized subspecies exhibits a more prolonged time-course of elevated testosterone following exogenous stimulation. Our results point to specific genes that fulfill key conditions for phenotypic evolution because they vary functionally in their expression among individuals and between populations, and they map onto population variation in phenotype in a common garden. Our findings therefore build an important bridge between hormones, genes, and phenotypic evolution.
    Induction of the type I interferon response in neurological forms of Gaucher disease
    Einat B. Vitner, Journal of Neuroinflammation - 2016
    Neuroinflammation is a key phenomenon in the pathogenesis of many neurodegenerative diseases. Understanding the mechanisms by which brain inflammation is engaged and delineating the key players in the immune response and their contribution to brain pathology is of great importance for the identification of novel therapeutic targets for these devastating diseases. Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the GBA1 gene and is a significant risk factor for Parkinson’s disease; in some forms of Gaucher disease, neuroinflammation is observed.
    Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome - dmm.025791.full.pdf
    Damon T. Jacobs, University of Kansas Medical - 2016
    Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter proopiomelanocortin nor orexo genic agouti-related peptide expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1 mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease.
    Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members
    Tara Mahoney, Journal of Bacteriology - 2016
    The biogenesis of the outer membrane (OM) of Escherichia coli is a conserved and vital process. The assembly of integral β-barrel proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric β-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or non-essential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple, abundant substrates that are not as dependent on SurA, but are strongly dependent on BamB for assembly. Here, we describe novel mutations in bamD that lower levels of BamD ten-fold and over twenty-five fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wild-type BamA levels to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM. Statement of importance The outer membrane (OM) of Gram-negative bacteria such as Escherichia coli serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral β-barrel proteins (OMPs) are assembled by the β-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.
    N-acetylcysteine Protects Mice from High Fat Diet-induced Metabolic Disorders
    Yongjie Ma, Pharmaceutical Research - 2016
    ABSTRACTPurposeTo study the effects of N-acetylcysteine (NAC, C5H9NO3S) on diet-induced obesity and obesity-related metabolic disorders.MethodsSix-week-old male C57BL/6 mice fed a chow or high-fat diet (HFD) were treated with NAC (2 g/L) in drinking water for 11 weeks. Its influences on body weight and food intake were manually measured, and influence on body composition were analyzed by magnetic residence imaging. Glucose meter and ELISA were used to determine serum glucose and insulin levels, as well as lipid content in the liver. The effects of NAC treatment on mRNA levels of genes involved in inflammation, thermogenesis, and lipid metabolism in various tissues were determined by real time PCR.ResultsNAC supplementation inhibited the increase of fat mass and the development of obesity when mice were fed an HFD. NAC treatment significantly lowered HFD-induced macrophage infiltration, and enhanced adiponectin gene expression, resulting in reduced hyperglycemia and hyperinsulinemia, and improvement of insulin resistance. NAC oral administration suppressed hepatic lipid accumulation, as evidenced by lower levels of triglyceride and cholesterol in the liver. The beneficial effects are associated with a decrease of hepatic Pparγ and its target gene expression, and an increase in the expression of genes responsible for lipid oxidation and activation of farnesoid X receptor. Furthermore, NAC treatment also stimulates expression of thermogenic genes.ConclusionThese results provide direct proof of the protective potential of NAC against HFD-induced obesity and obesity-associated metabolic disorders.
    Campylobacter jejuni in Musca domestica: An examination of survival and transmission potential in light of the innate immune responses of the house flies
    Carson Gill, Insect Science - 2016
    The house fly, Musca domestica, has been implicated as a vector of Campylobacter spp., a major cause of human disease. Little is known whether house flies serve as biological amplifying hosts or mechanical vectors for Campylobacter jejuni. We investigated the period after C. jejuni had been ingested by house flies in which viable C. jejuni colonies could be isolated from whole bodies, the vomitus and the excreta of adult M. domestica and evaluated the activation of innate immune responses of house flies to ingested C. jejuni over time. C. jejuni could be cultured from infected houseflies soon after ingestion but no countable C. jejuni colonies were observed > 24 h postingestion. We detected viable C. jejuni in house fly vomitus and excreta up to 4 h after ingestion, but no viable bacteria were detected ≥ 8 h. Suppression subtractive hybridization identified pathogen-induced gene expression in the intestinal tracts of adult house flies 4–24 h after ingesting C. jejuni. We measured the expression of immune regulatory (thor, JNK, and spheroide) and effector (cecropin, diptericin, attacin, defensing, and lysozyme) genes in C. jejuni-infected and -uninfected house flies using quantitative real time PCR. Some house fly factor, or combination of factors, eliminates C. jejuni within 24 h postingestion. Because C. jejuni is not amplified within the body of the housefly, this insect likely serves as a mechanical vector rather than as a true biological, amplifying vector for C. jejuni, and adds to our understanding of insect–pathogen interactions.
    Influence of feeding stearidonic acid (18:4n-3)-enriched soybean oil, as compared to conventional soybean oil, on tissue deposition of very long-chain omega-3 fatty acids in meat-type chickens
    Robert Elkin, Animal Feed Science and Technology - 2016
    In chickens, the desaturation of α-linolenic acid (ALA; 18:3n-3) to stearidonic acid (SDA; 18:4n-3) is considered to be rate-limiting for the hepatic conversion of ALA to very long-chain (VLC; i.e. >20 C) n-3 polyunsaturated fatty acids (PUFAs). Thus, we hypothesized that feeding broilers SDA plus ALA, as compared to ALA alone, would bypass this inefficient metabolic step and enrich meat with greater amounts of VLC n-3 PUFAs. Female Ross × Heritage broilers were fed mash diets containing 50 g/kg of conventional soy oil (CON) from hatch until d 28. On d 29, they were divided into two groups and fed diets containing either 50 g/kg CON or 50 g/kg of SDA-enriched oil derived from the genetic modification of the soybean (SDASOY) until d 42. Final (42 d) body weights, as well as weight gains and feed conversion values from 29 to 35 d and 36 to 42 d, were not different (P > 0.05) between treatments. Compared to the CON treatment, dietary SDASOY increased (P < 0.01) total VLC n-3 PUFA contents of skinless and boneless breasts, tenders, and thighs by almost 3-fold. However, the SDASOY diet also contained more total n-3 fatty acids (ALA + SDA) than the CON diet (ALA only), and it was estimated that ALA and SDA were metabolized to VLC n-3 PUFAs and deposited into breast, tenders, and thigh meat with equal efficiency. Docosapentaenoic acid (DPA; 22:5n-3) was the predominant VLC n-3 PUFA in all three muscles, suggesting that another control point downstream of the initial hepatic Δ6-desaturase reaction was rate-limiting in the biosynthesis of DHA from ALA. Alternately, since broilers have the capability to convert ALA to DHA in the liver, it is likely that the capacity of the VLC n-3 PUFA biosynthetic pathway is simply not great enough to allow for the deposition of DHA into muscle at levels equal to those attained by direct dietary supplementation. It is also possible that, rather than undergoing elongation and desaturation, some of the ALA and SDA pool underwent β-oxidation in the liver, as suggested by others, while a large portion of each fatty acid was not metabolized and was transported out of the liver to other tissues, such as adipose. However, the relative hepatic expression of genes whose protein products are involved in fatty acid oxidation (as well as in desaturation and elongation or lipogenesis) were not significantly affected by dietary treatment or age.
    The Bromodomain testis-specific
    M. Úbeda-Manzanaro,, European Journal of Histochemistry - 2016
    Multiple genes and transcription factors are involved in regulation and control of the complex process of sex determination and differentiation of fish species. Also more, several hormonal factors and some environmental conditions can also be adequate spawning strategies and stimuli for inducing reproduction of fish species. Brdt gene belongs to the bromodomain-extraterminal domain (BET) family of transcriptional coregulators. In mammals, Brdt gene is almost exclusively expressed in testis. Furthermore, Brdt protein is involved in elongating spermatids, and is required for proper spermatogenesis and male fertility. However, from our understanding of fish species, the role of this gene as key, during gametogenesis, still remains unknown. In this study, two Brdt mRNA transcripts were isolated from two teleostean fish species, gilthead seabream and European seabass. In both species the shorter form lacked a functional Cterminal domain, which may involve a different function as transcriptional regulator. The pattern of Brdt expression showed that the highest levels occurred in the gonads. Significantly lower levels of expression were detected in brain, pituitary and different organ systems (heart, kidney, gills, among other somatic tissues) from both studied species. In situ hybridization approach evidenced that Brdt mRNA expression was restricted to specific cell-types of the germ line, during both oogenesis and spermatogenesis processes.
    Expression of OsMYB55 in maize activates stress-responsive genes and enhances heat and drought tolerance
    José A.Casaretto, BMC Genomics - 2016
    Plant response mechanisms to heat and drought stresses have been considered in strategies for generating stress tolerant genotypes, but with limited success. Here, we analyzed the transcriptome and improved tolerance to heat stress and drought of maize plants over-expressing the OsMYB55 gene.
    Biology, Gene Flow, and Management of Glyphosate-Resistant Common Waterhemp (Amaranthus rudis Sauer) in Nebraska - viewcontent.cgi
    Debalin Sarangi, University of Nebraska- Lincoln - 2016
    Common waterhemp is the most troublesome weed in the midwestern United States. Growers from Nebraska reported failure to control common waterhemp following sequential applications of glyphosate in glyphosate-tolerant corn and soybean, which led to moderate to severe yield loss; justifying the needto confirm resistance and study the biology and management of common waterhemp. The objectivesof this research were: 1) to confirm the presence of glyphosate-resistant (GR) common waterhemp biotypes in Nebraska and to evaluate their sensitivity to herbicides belongingto alternativesites-of-action; 2) to evaluate the response of common waterhemp to water stress; 3) to quantify pollen-mediated gene flow from GR common waterhemp under field conditions; and 4) to evaluate different herbicide programs for season-long control of glyphosate-resistant common waterhemp in glyphosate tolerant soybean. Greenhouse dose response studies conducted to evaluate the response of suspected GR common waterhemp biotypes collected from seven eastern Nebraska counties (Antelope, Dodge, Fillmore, Lancaster, Pawnee, Seward, and Washington)revealed that the biotypes were 3-to 39-fold resistant to glyphosate. The GR biotypes also showed a reduced sensitivity to acetolactate synthase (ALS)-inhibiting herbicides (chlorimuron-ethyl,imazamox, imazaquin, imazethapyr, and thifensulfuron-methyl). Experiments conducted to evaluate the response of common waterhemp to water stress suggested that degree and duration of water stress can adversely affect the growth and seed production of common waterhemp. Highest plant height (≥ 150 cm), growth index (≥ 3.8 × 105cm3), and seed production (>34,000 seeds plant-1) were recorded with 100% pot water content applied at 2-d intervals. Pollen mediated gene flow studies from GR to GS biotypes were conducted under field conditions using a Nelder wheel design.Frequency of gene flow was found to be highest (up to 0.77) at the closer distances (0 to 0.1 m); whereas gene flow frequency declined by 50% at < 2.5 m and 90% at distances < 90 m from the pollen source. Field experiments conducted for management of GR common waterhemp in soybean showed that preemergenece (PRE) followed by postemergence (POST) herbicide programs with multiple sites of action provided season long control of GR common waterhemp and resulted in the highest soybean yield compared to the POST-only herbicide programs.
    Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals : Nature Communications
    Thomas R. Gawriluk,, nature Communication - 2016
    Why mammals have poor regenerative ability has remained a long-standing question in biology. In regenerating vertebrates, injury can induce a process known as epimorphic regeneration to replace damaged structures. Using a 4-mm ear punch assay across multiple mammalian species, here we show that several Acomys spp. (spiny mice) and Oryctolagus cuniculus completely regenerate tissue, whereas other rodents including MRL/MpJ ‘healer’ mice heal similar injuries by scarring. We demonstrate ear-hole closure is independent of ear size, and closure rate can be modelled with a cubic function. Cellular and genetic analyses reveal that injury induces blastema formation in Acomys cahirinus. Despite cell cycle re-entry in Mus musculus and A. cahirinus, efficient cell cycle progression and proliferation only occurs in spiny mice. Together, our data unite blastema-mediated regeneration in spiny mice with regeneration in other vertebrates such as salamanders, newts and zebrafish, where all healthy adults regenerate in response to injury.
    QSOX1 as an Anti-Neoplastic Drug Target - Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizo
    Douglas Lake,, US Patent - 2016
    CROSS REFERENCE This application is a continuation of U.S. patent application Ser. No. 14/169,612 filed Jan. 31, 2014, which is a continuation-in-part of U.S. patent application Ser. No. 13/847,930 filed Mar. 20, 2013, which claims priority to U.S. Provisional Patent Application Ser. No. 61/722,396 filed Nov. 5, 2012 and to PCT Application Serial No. PCT/US11/52122 filed Sep. 19, 2011, which claims priority to U.S. Provisional Patent Application Ser. No. 61/384,502 filed Sep. 20, 2010. Each application is incorporated by reference herein in its entirety. BACKGROUND Pancreatic ductal adenocarcinoma (PDA) is a disease that carries a poor prognosis. It is often detected in stage III resulting in an unresectable tumor at the time of diagnosis. However, even if pancreatic cancer is surgically resected in stage I or II, it may recur at a metastatic site (1, 2). Currently, patients diagnosed with pancreatic ductal adenocarcinoma have less than a 5% chance of surviving past five years (3). Breast adenocarcinoma is the most common cancer diagnosed in women throughout the world. In 2012, an estimated 226,870 new cases of invasive breast cancer are expected to occur among US women, and an estimated 39,510 breast cancer deaths. SUMMARY OF THE INVENTION In a first aspect, the present invention provides methods for tumor treatment, comprising administering to a subject having a tumor an amount effective of an inhibitor of quiescin sulfhydryl oxidase 1 (QSOX1) expression and/or activity, or a pharmaceutically acceptable salt thereof, to treat the tumor. In one embodiment, the inhibitor of QSOX1 is selected from the group consisting of anti-QSOX1 antibodies, QSOX1-binding aptamers, QSOX1 antisense oligonucleotides, QSOX1 siRNA, and QSOX1 shRNA. In another embodiment, the tumor is a tumor that over-expresses QSOX1 compared to control. In a further embodiment, the subject is one from which tumor-derived QSOX1 peptides can be obtained. In a further embodiment, the tumor is a pancreatic tumor, and preferably a pancreatic adenocarcinoma. In a still further embodiment, the method is for limiting tumor metastasis. In a second aspect, the present invention provides isolated nucleic acids, comprising or consisting of antisense, siRNA, miRNA, and/or shRNA molecules having a nucleic acid sequence that is perfectly complementary at least 10 contiguous nucleotides of QSOX1 as shown in SEQ ID NO:1 and SEQ ID NO:2 or RNA equivalents thereof; and/or fragments of the nucleic acid molecule. In a preferred embodiment, the isolated nucleic acids comprising sequences from the group consisting of
    Hepatic transcriptional responses in Atlantic salmon (Salmo salar) exposed to gamma radiation and depleted uranium singly and in combination
    You Song, Science of The Total Environment - 2016
    Radionuclides are a special group of substances posing both radiological and chemical hazards to organisms. As a preliminary approach to understand the combined effects of radionuclides, exposure studies were designed using gamma radiation (Gamma) and depleted uranium (DU) as stressors, representing a combination of radiological (radiation) and chemical (metal) exposure. Juvenile Atlantic salmon (Salmo salar) were exposed to 70 mGy external Gamma dose delivered over the first 5 h of a 48 h period (14 mGy/h), 0.25 mg/L DU were exposed continuously for 48 h and the combination of the two stressors (Combi). Water and tissue concentrations of U were determined to assess the exposure quality and DU bioaccumulation. Hepatic gene expression changes were determined using microarrays in combination with quantitative real-time reverse transcription polymerase chain reaction (qPCR). Effects at the higher physiological levels were determined as plasma glucose (general stress) and hepatic histological changes. The results show that bioaccumulation of DU was observed after both single DU and the combined exposure. Global transcriptional analysis showed that 3122, 2303 and 3460 differentially expressed genes (DEGs) were significantly regulated by exposure to gamma, DU and Combi, respectively. Among these, 349 genes were commonly regulated by all treatments, while the majority was found to be treatment-specific. Functional analysis of DEGs revealed that the stressors displayed similar mode of action (MoA) across treatments such as induction of oxidative stress, DNA damage and disturbance of oxidative phosphorylation, but also stressor-specific mechanisms such as cellular stress and injury, metabolic disorder, programmed cell death, immune response. No changes in plasma glucose level as an indicator of general stress and hepatic histological changes were observed. Although no direct linkage was successfully established between molecular responses and adverse effects at the organism level, the study has enhanced the understanding of the MoA of single radionuclides and mixtures of these.
    Impact of gut microbiota on the fly’s germ line
    Michael Elgart, Nature Communications - 2016
    Unlike vertically transmitted endosymbionts, which have broad effects on their host’s germ line, the extracellular gut microbiota is transmitted horizontally and is not known to influence the germ line. Here we provide evidence supporting the influence of these gut bacteria on the germ line of Drosophila melanogaster. Removal of the gut bacteria represses oogenesis, expedites maternal-to-zygotic-transition in the offspring and unmasks hidden phenotypic variation in mutants. We further show that the main impact on oogenesis is linked to the lack of gut Acetobacter species, and we identify the Drosophila Aldehyde dehydrogenase (Aldh) gene as an apparent mediator of repressed oogenesis in Acetobacter-depleted flies. The finding of interactions between the gut microbiota and the germ line has implications for reproduction, developmental robustness and adaptation.
    Role of Specificity Protein-1 and Activating Protein-2 Transcription Factors in the Regulation of the Gap Junction Protein Beta-2 Gene in the Epididymis
    Cecile Adam, Biology of Reproduction - 2016
    In prepubertal rats, connexin 26 (GJB2) is expressed between adjacent columnar cells of the epididymis. At 28 days of age, when columnar cells differentiate into adult epithelial cell types, Gjb2 mRNA levels decrease to barely detectable levels. There is no information on the regulation of GJB2 in the epididymis. The present objective was to characterize the regulation of the Gjb2 gene promoter in the epididymis. A single transcription start site at position -3829bp relative to the ATG was identified. Computational analysis revealed several TFAP2A, SP1, and KLF4 putative binding sites. A 1.5 kb fragment of the Gjb2 promoter was cloned into a vector containing a luciferase reporter gene. Transfection of the construct into immortalized rat caput epididymal (RCE-1) cells indicated that the promoter contained sufficient information to drive the expression of the reporter gene. Deletion constructs showed that the basal activity of the promoter resides in the first -230bp of the transcriptional start site. Two response elements necessary for GJB2 expression were identified: a TFAP2A/SP1 site (-136 to -126bp) and a SP1 site (-50bp). Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays confirmed that SP1 and TFAP2A bound to the promoter. ChIP analysis on chromatin from young and pubertal rats indicated that TFAP2A and SP1 binding decreased with age. SP1 and TFAP2A knockdown indicated that SP1 is necessary for Gjb2 expression. DNA methylation did not appear to involved in the regulation of GjbJB2 expression. Results indicate that SP1 and TFAP2A regulate Gjb2 promoter activity during epididymal differentiation in the rat.
    Reduced Epithelial Na + /H + Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis
    Daniel Laubitz, PLOS ONE - 2016
    Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na + /H + exchange. In mice, a selective disruption of NHE3 ( Slc9a3 ), a major apical Na + /H + exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na + /H + exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2 -/- and NHE3 -/- /Rag2 -/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25 - FoxP3 + Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.
    Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach - Journal of Proteome Research (ACS Publications)
    Katelyn R. Ludwig, †Department of Chemistry and Biochemistry and ‡Department of Biological Sciences, University of Notre Dame - 2016
    MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are implicated in a number of disease states. MiRNAs can exist as individual entities or may be clustered and transcribed as a single polycistron. The mirn23a cluster consists of three miRNAs: miR-23a, miR-24-2, and miR-27a. Although these miRNAs are transcribed together, they often exist at varying levels in the cell. Despite the fact that the mirn23a cluster is known to play a role in a number of diseases and developmental processes, few direct targets have been identified. In this study, we examined the effects of miR-23a, miR-24-2, miR-27a, or the mirn23a cluster overexpression on the proteome of 70Z/3 pre-B lymphoblast cells. Quantitative mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ) allowed for the global profiling of cell lines after miRNA overexpression. We identified a number of targets of each miRNA that contained predicted miRNA seed sequences and are likely direct targets. In addition, we discovered a cohort of shared miRNA targets and cluster targets, demonstrating the importance of studying miRNA clusters in their entirety.
    Modulation of the TNFα-induced gene expression profile of intestinal epithelial cells by soy fermented with lactic acid bacteria
    Qiuye Lin, Journal of Functional Foods - 2016
    The effect of soy ferments on gene expression induced by the proinflammatory cytokine tumour necrosis factor α (TNFα) at the intestinal epithelial cell (IEC) level was evaluated. Microarray-based transcriptional analysis revealed that soy fermented with Lactobacillus helveticus R0052 and Streptococcus thermophilus R0083 (SF-Lh) down-regulated 33 of 40 proinflammatory genes up-regulated by TNFα in HT-29 IEC, attenuating expression of genes encoding several proinflammatory cytokines and cell adhesion molecules. TNFα-mediated activation of gene expression associated with the NF-κB pathway was also repressed, as was Interleukin-8 (IL-8) production. In contrast, soy fermented with Bifidobacterium longum R0175 and S. thermophilus R0083 (SF-Bl) up-regulated expression of three proinflammatory genes induced by TNFα. Increased intracellular levels of hydrogen peroxide (H2O2) were detected in HT-29 IEC following incubation with SF-Lh. These results indicate that SF-Lh has immunomodulatory activity through reduction of proinflammatory gene expression at the IEC level.
    Re-induction of the cell cycle in the Arabidopsis post-embryonic root meristem is ABA-insensitive, GA-dependent and repressed by KRP6
    Jeroen Nieuwland, Scientific Reports - 2016
    Seeding establishment following seed germination requires activation of the root meristem for primary root growth. We investigated the hormonal and genetic regulation of root meristem activation during Arabidopsis seed germination. In optimal conditions, radicle cell divisions occur only after the completion of germination and require de novo GA synthesis. When the completion of germination is blocked by ABA, radicle elongation and cell divisions occurred in these non-germinating seeds. Conversely under GA-limiting conditions, ABA-insensitive mutants complete germination in the absence of radicle meristem activation and growth. Radicle meristem activation and extension can therefore occur independently of completion of the developmental transition of germination. The cell cycle regulator KRP6 partially represses GA-dependent activation of the cell cycle. Germination of krp6 mutant seeds occurs more rapidly, is slightly insensitive to ABA in dose-response assays, but also hypersensitive to the GA synthesis inhibitor PAC. These conflicting phenotypes suggest the cell cycle uncouples GA and ABA responses in germinating Arabidopsis seeds, and that KRP6 acts downstream of GA to inhibit mitotic cell cycle activation during germination.
    Lipocalin 2 alleviates iron toxicity by facilitating hypoferremia of inflammation and limiting catalytic iron generation
    Xia Xiao, BioMetals - 2016
    Iron is an essential transition metal ion for virtually all aerobic organisms, yet its dysregulation (iron overload or anemia) is a harbinger of many pathologic conditions. Hence, iron homeostasis is tightly regulated to prevent the generation of catalytic iron (CI) which can damage cellular biomolecules. In this study, we investigated the role of iron-binding/trafficking innate immune protein, lipocalin 2 (Lcn2, aka siderocalin) on iron and CI homeostasis using Lcn2 knockout (KO) mice and their WT littermates. Administration of iron either systemically or via dietary intake strikingly upregulated Lcn2 in the serum, urine, feces, and liver of WT mice. However, similarly-treated Lcn2KO mice displayed elevated CI, augmented lipid peroxidation and other indices of organ damage markers, implicating that Lcn2 responses may be protective against iron-induced toxicity. Herein, we also show a negative association between serum Lcn2 and CI in the murine model of dextran sodium sulfate (DSS)-induced colitis. The inability of DSS-treated Lcn2KO mice to elicit hypoferremic response to acute colitis, implicates the involvement of Lcn2 in iron homeostasis during inflammation. Using bone marrow chimeras, we further show that Lcn2 derived from both immune and non-immune cells participates in CI regulation. Remarkably, exogenous rec-Lcn2 supplementation suppressed CI levels in Lcn2KO serum and urine. Collectively, our results suggest that Lcn2 may facilitate hypoferremia, suppress CI generation and prevent iron-mediated adverse effects.
    Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome
    Aimee L. Benjamin, BMC Genomics - 2016
    We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis.
    Effects of clove oil, essential oil of Lippia alba and 2-phe anaesthesia on juvenile meagre, Argyrosomus regius (Asso, 1801) - Cárdenas - 2016 - Journal of Applied Ichthyology - Wiley Online Library
    C Cardenas, Journal of Applied Ichthyology - 2016
    The objectives of this experiment were to (i) determine the efficacy of essential oils of clove (CO) and Lippia alba (EOLA) to induce deep anaesthesia in juvenile specimens (49.0 ± 6.2 g body mass, 16.6 ± 0.8 cm; n = 8 per treatment) of meagre (Argyrosomus regius); and (ii) study the feasibility of these substances, together with 2-phenoxyethanol (2-PHE), as potential sedatives [low concentration: (i) EOLA: 12 mg L−1; (ii) CO: 1 mg L−1; and (iii) 2-PHE: 33 mg·L −1; n = 8 per treatment] for live fish transport of this species. All test were performed at a constant temperature (18°C). Thus, the main primary stress indicator (plasma cortisol) and secondary factors (plasma metabolites) were evaluated. In addition, growth hormone (GH) mRNA expression was also evaluated in the pituitary gland. The results indicated that EOLA is considered to be effective for deep anaesthesia when the concentration is close to 160 mg L−1, while CO produces the same effect when lower concentrations are added (40–50 mg L−1). Regarding sedative concentrations, a significant ~3-fold increase in plasma cortisol levels was detected in the EOLA group when compared to control specimens. In addition, glucose levels were not reduced and significantly increased (~1.6-fold) for 2-PHE in relation to the control fish. None of the anaesthetics promoted a significant difference for GH expression with respect to the control group, but a significant ~2-fold increase for 2-PHE treatment with respect to the EOLA exposition was found in this gene expression. Results show that none of the anaesthetics analysed, at least in the ranges of concentrations used in this study (EOLA 12 mg L−1, CO 1 mg L−1, 2-PHE 33 mg L−1), are recommended for live fish transport, as shown by the absence of inhibition on the stress parameters assessed.
    Pea (Pisum sativum L.) Seed Coats and Seed Coat Fractions - THE GOVERNORS OF THE UNIVERSITY OF ALBERTA
    Ozga, Jocelyn, United States Patent - 2016
    What is claimed is: 1. A method for improving health and/or other beneficial effects in a subject, comprising administering pea seed coat fractions to said subject. 2. The method of claim 1, wherein said subject is a human or animal. 3. The method of claim 1, wherein said health and/or beneficial effect is selected from retained PAC bioavailability, retained PAC bioactivity, improved insulin sensitivity, reduced glycemia, increased satiety, improved glucose tolerance, improved glucose control, improved glucose homeostasis, beneficial effects on pancreatic islet composition and insulin secretion. 4. The method of claim 1, wherein said health and/or beneficial effect is selected from PAC-derived products that have increased bioavailability, improved insulin sensitivity, reduced glycemia, increased satiety, improved glucose tolerance, improved glucose control, improved glucose homeostasis, beneficial effects on pancreatic islet composition and insulin secretion. 5. A composition comprising pea seed coat fractions. 6. The composition of claim 4, wherein said composition is selected from a food, animal feed, flour, fibre, and ingredient. 7. A method for improving health and/or other beneficial effects in a subject, comprising administering cooked pea seed coat fractions to said subject. 8. A method for improving health and/or other beneficial effects in a subject, comprising administering pea seed coat fractions processed by cooking followed by freeze-drying to said subject. 9. A method for increasing the bioavailability of proanthocyanidins (PAC), comprising hydrolyzing pea seed coat-derived PACs.
    Comprehensive Definition of the SigH Regulon of Mycobacterium tuberculosis Reveals Transcriptional Control of Diverse Stress Responses
    Jared Sharp, PLOS ONE - 2016
    Expression of SigH, one of 12 Mycobacterium tuberculosis alternative sigma factors, is induced by heat, oxidative and nitric oxide stresses. SigH activation has been shown to increase expression of several genes, including genes involved in maintaining redox equilibrium and in protein degradation. However, few of these are known to be directly regulated by SigH. The goal of this project is to comprehensively define the Mycobacterium tuberculosis genes and operons that are directly controlled by SigH in order to gain insight into the role of SigH in regulating M . tuberculosis physiology. We used ChIP-Seq to identify in vivo SigH binding sites throughout the M . tuberculosis genome, followed by quantification of SigH-dependent expression of genes linked to these sites and identification of SigH-regulated promoters. We identified 69 SigH binding sites, which are located both in intergenic regions and within annotated coding sequences in the annotated M . tuberculosis genome. 41 binding sites were linked to genes that showed greater expression following heat stress in a SigH-dependent manner. We identified several genes not previously known to be regulated by SigH, including genes involved in DNA repair, cysteine biosynthesis, translation, and genes of unknown function. Experimental and computational analysis of SigH-regulated promoter sequences within these binding sites identified strong consensus -35 and -10 promoter sequences, but with tolerance for non-consensus bases at specific positions. This comprehensive identification and validation of SigH-regulated genes demonstrates an extended SigH regulon that controls an unexpectedly broad range of stress response functions.
    Ho, Dean, United States Patent - 2016
    1. A composition comprising: a) an aqueous solution; b) a plurality of therapeutic agent molecules, wherein said therapeutic agent molecules comprise an anthracycline-class compound or tetracycline-class compound; c) a plurality of nanodiamond particles suspended in said aqueous solution, wherein said plurality of therapeutic agent molecules are adsorbed to at least a portion of said plurality of nanodiamond particles; and d) sodium hydroxide (NaOH) and/or potassium hydroxide (KOH) present in said aqueous solution. 2. The composition of claim 1, wherein said anthracycline-class compound or tetracycline-class compound is selected from: daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, and rolitetracycline. 3. The composition of claim 1, wherein said anthracycline-class compound or tetracycline-class compound is released from said plurality of nanodiamond particles upon cellular introduction. 4. The composition of claim 1, wherein said composition comprises said sodium hydroxide (NaOH). 5. The composition of claim 1, wherein said composition comprises said potassium hydroxide (KOH). 6. The composition of claim 1, wherein said therapeutic agent comprises an anthracycline-class compound. 7. The composition of claim 1, wherein said therapeutic agent comprises a tetracycline-class compound. 8. The composition of claim 1, wherein said anthracycline-class compound or tetracycline-class compound is selected from: daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, and tetracycline. 9. The composition of claim 1, wherein said anthracycline-class compound or tetracycline-class compound is selected from: chlortetracycline, oxytetracycline, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, and rolitetracycline. 10. A composition comprising: a) an aqueous solution; b) a plurality of therapeutic agent molecules, wherein said therapeutic agent molecules comprises an anthracycline-class compound or tetracycline-class compound; c) a plurality of nanodiamond particles suspended in said aqueous solution, wherein said plurality of therapeutic agent molecules are adsorbed to at least a portion of said plurality of nanodiamond particles; and d) a hydroxide compound present in said aqueous solution. 11. The composition of claim 10, wherein said hydroxide compound comprises sodium hydroxide (NaOH). 12. The composition of claim 10, wherein said hydroxide compound comprises potassium hydroxide (KOH).
    Vascular endothelial growth factor and angiopoietins during hen ovarian follicle development
    Dongwon Kim, General and Comparative Endocrinology - 2016
    Growth and maturation of ovarian follicles in the hen (Gallus gallus) requires a network of blood vessels that increases in complexity during development. The present studies investigate expression of vascular endothelial growth factor A (VEGF), angiopoietin1 (ANGPT1) and ANGPT2 mRNAs together with their associated receptors (VEGFR and TIE2, respectively) during maturation. Elevated expression of VEGF and its receptors is associated with healthy, compared to atretic, follicles. Levels of VEGF significantly increase, while antagonistic ANGPT2 decrease, in granulosa cells (GC) at follicle selection. By comparison, levels of VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and TIE2 within the theca layer do not change (P > 0.05) relative to developmental stages surrounding follicle selection (6–8 mm versus 9–12 mm follicles). Prior to selection, treatment with transforming growth factor β1 (TGFβ1) significantly increases levels of VEGF in undifferentiated GC from prehierarchal (6–8 mm) follicles and actively differentiating GC from selected (9–12 and F4) follicles. Moreover, subsequent to selection follicle stimulating hormone (FSH) increases VEGF expression in GC from 9 to 12 mm follicles, and eventually luteinizing hormone (LH) promotes VEGF expression in GC from more mature preovulatory follicles. It is concluded that prior to follicle selection VEGF expression is regulated by autocrine and paracrine actions of TGFβ1 (but not FSH), and that a comparatively limited extent of vasculature is sufficient to maintain prehierarchal follicles in a viable and undifferentiated state. At follicle selection, FSH- and subsequently LH-induced VEGF production within the GC layer enhance angiogenesis within the theca layer, which facilitates the rapid growth of preovulatory follicles via enhanced incorporation of yellow yolk.
    The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotype-phenotype correlations
    Chun-An Chen, Genetics in Medicine - 2016
    Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) is an autosomal-dominant disorder characterized by optic atrophy and intellectual disability caused by loss-of-function mutations in NR2F1. We report 20 new individuals with BBSOAS, exploring the spectrum of clinical phenotypes and assessing potential genotype–phenotype correlations. Clinical features of individuals with pathogenic NR2F1 variants were evaluated by review of medical records. The functional relevance of coding nonsynonymous NR2F1 variants was assessed with a luciferase assay measuring the impact on transcriptional activity. The effects of two start codon variants on protein expression were evaluated by western blot analysis. We recruited 20 individuals with novel pathogenic NR2F1 variants (seven missense variants, five translation initiation variants, two frameshifting insertions/deletions, one nonframeshifting insertion/deletion, and five whole-gene deletions). All the missense variants were found to impair transcriptional activity. In addition to visual and cognitive deficits, individuals with BBSOAS manifested hypotonia (75%), seizures (40%), autism spectrum disorder (35%), oromotor dysfunction (60%), thinning of the corpus callosum (53%), and hearing defects (20%). BBSOAS encompasses a broad range of clinical phenotypes. Functional studies help determine the severity of novel NR2F1 variants. Some genotype–phenotype correlations seem to exist, with missense mutations in the DNA-binding domain causing the most severe phenotypes. Genet Med advance online publication 17 March 2016
    Rashtchian, Ayoub, United States Patent - 2016
    Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.
    Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates distinct biological pathways in prostate carcinogenesis | Clinical Epigenetics | Full Text
    Shivani N. Kamdar, Clinical EpigeneticsThe official journal of the Clinical Epigenetics Society - 2016
    Despite the significant global loss of DNA hydroxymethylation marks in prostate cancer tissues, the locus-specific role of hydroxymethylation in prostate tumorigenesis is unknown. We characterized hydroxymethylation and methylation marks by performing whole-genome next-generation sequencing in representative normal and prostate cancer-derived cell lines in order to determine functional pathways and key genes regulated by these epigenomic modifications in cancer.
    Impact of probiotic supplements on microbiome diversity following antibiotic treatment of mice
    Hannah Grazul, Gut Microbes - 2016
    Shifts in microbial populations of the intestinal tract have been associated with a multitude of nutritional, autoimmune, and infectious diseases. The limited diversity following antibiotic treatments creates a window for opportunistic pathogens, diarrhea, and inflammation as the microbiome repopulates. Depending on the antibiotics used, microbial diversity can take weeks to months to recover. To alleviate this loss of diversity in the intestinal microbiota, supplementation with probiotics has become increasingly popular. However, our understanding of the purported health benefits of these probiotic bacteria and their ability to shape the microbiome is significantly lacking. This study examined the impact of probiotics concurrent with antibiotic treatment or during the recovery phase following antibiotic treatment of mice. We found that probiotics did not appear to colonize the intestine themselves or shift the overall diversity of the intestinal microbiota. However, the probiotic supplementation did significantly change the types of bacteria which were present. In particular, during the recovery phase the probiotic caused a suppression of Enterobacteriaceae outgrowth (Shigella and Escherichia) while promoting a blooming of Firmicutes, particularly from the Anaerotruncus genus. These results indicate that probiotics have a significant capacity to remodel the microbiome of an individual recovering from antibiotic therapy.
    Effects of Metabolic Programming on Juvenile Play Behavior and Gene Expression in the Prefrontal Cortex of Rats
    Harleen Hehar, Developmental Neuroscience - 2016
    Early developmental processes, such as metabolic programming, can provide cues to an organism, which allow it to make modifications that are predicted to be beneficial for survival. Similarly, social play has a multifaceted role in promoting survival and fitness of animals. Play is a complex behavior that is greatly influenced by motivational and reward circuits, as well as the energy reserves and metabolism of an organism. This study examined the association between metabolic programming and juvenile play behavior in an effort to further elucidate insight into the consequences that early adaptions have on developmental trajectories. The study also examined changes in expression of four genes (Drd2, IGF1, Opa1, and OxyR) in the prefrontal cortex known to play significant roles in reward, bioenergetics, and social-emotional functioning. Using four distinct variations in developmental programming (high-fat diet, caloric restriction, exercise, or high-fat diet combined with exercise), we found that dietary programming (high-fat diet vs. caloric restriction) had the greatest impact on play behavior and gene expression. However, exercise also induced changes in both measures. This study demonstrates that metabolic programming can alter neural circuits and bioenergetics involved in play behavior, thus providing new insights into mechanisms that allow programming to influence the evolutionary success of an organism.
    Epigallocatechin-3-Gallate Inhibition of Myeloperoxidase and Its Counter-Regulation by Dietary Iron and Lipocalin 2 in Murine Model of Gut Inflammation - The American Journal of Pathology
    Beng San Yeoh, The American Journal of Pathology - 2016
    Green tea-derived polyphenol (−)-epigallocatechin-3-gallate (EGCG) has been extensively studied for its antioxidant and anti-inflammatory properties in models of inflammatory bowel disease, yet the underlying molecular mechanism is not completely understood. Herein, we demonstrate that EGCG can potently inhibit the proinflammatory enzyme myeloperoxidase in vitro in a dose-dependent manner over a range of physiologic temperatures and pH values. The ability of EGCG to mediate its inhibitory activity is counter-regulated by the presence of iron and lipocalin 2. Spectral analysis indicated that EGCG prevents the peroxidase-catalyzed reaction by reverting the reactive peroxidase heme (compound I:oxoiron) back to its native inactive ferric state, possibly via the exchange of electrons. Further, administration of EGCG to dextran sodium sulfate–induced colitic mice significantly reduced the colonic myeloperoxidase activity and alleviated proinflammatory mediators associated with gut inflammation. However, the efficacy of EGCG against gut inflammation is diminished when orally coadministered with iron. These findings indicate that the ability of EGCG to inhibit myeloperoxidase activity is one of the mechanisms by which it exerts mucoprotective effects and that counter-regulatory factors such as dietary iron and luminal lipocalin 2 should be taken into consideration for optimizing clinical management strategies for inflammatory bowel disease with the use of EGCG treatment.
    Characterization of Notch Signaling During Osteogenic Differentiation in Human Osteosarcoma Cell Line MG63
    Alessia Ongaro, Journal of Cellular Physiology - 2016
    Osteogenic differentiation is a multi-step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.
    Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae
    Sirjana Devi Shrestha, PLOS ONE - 2016
    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a . P . sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a , genetic crosses and self-fertilizations were performed. A cross between P . sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F 5 generation. However, test-crosses of F 1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a . Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F 1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P . sojae strains.
    Faul, Christian, United States Patent - 2015
    Methods of inhibiting fibroblast growth factor mediated activation of fibroblast growth factor receptors for the treatment of chronic kidney disease, diabetes, obesity, and cardiac diseases are enclosed. Pharmaceutical compositions for the treatment of such diseases using the methods are also disclosed as are methods of determining whether a subject would benefit from the methods of treatment and pharmaceutical compositions.
    Evaluation of Gelatin Microparticles as Adherent-Substrates for Mesenchymal Stem Cells in a Hydrogel Composite
    Steven Lu, Annals of Biomedical Engineering - 2016
    Due to the lack of cell-adhesive moieties in traditional synthetic hydrogels, the present work investigated the use of degradable gelatin microparticles (GMPs) as temporary adherent substrates for anchorage-dependent mesenchymal stem cells (MSCs). MSCs were seeded onto GMPs of varying crosslinking densities and sizes to investigate their role on influencing MSC differentiation and aggregation. The MSC-seeded GMPs were then encapsulated in poly(ethylene glycol)-based hydrogels and cultured in serum-free, growth factor-free osteochondral medium. Non-seeded MSCs co-encapsulated with GMPs in the hydrogels were used as a control for comparison. Over the course of 35 days, MSCs seeded on GMPs exhibited more cell–cell contacts, greater chondrogenic potential, and a down-regulation of osteogenic markers compared to the controls. Although the factors of GMP crosslinking and size had nominal influence on MSC differentiation and aggregation, GMPs demonstrate potential as an adherent-substrate for improving cell delivery from hydrogel scaffolds by facilitating cell–cell contacts and improving MSC differentiation.
    Deepa Gurung- Thesis NMBU.pdf
    r. Åshild Andreassen, Norwegian University of Life Science Master Thesis - 2015
    Tick-borne disease in general is among the most important vector-borne diseases that are emerging as a threat to humans and is currently identified as a major health problem in many countries. Tick-borne encephalitis and lyme borreliosis are the main diseases transmitted by Ixodes ricinus ticks in Europe. Besides TBEV (Tick borne encephalitis virus) and Borrelia burgdorferi sensu lato,I.ricinus is known to transmit other pathogenic microorganisms like- louping ill virus, Anaplasma phagocytophilum, Francisella tularensis,Coxiella burnetii and endosymbionts like-Wolbachia pipientis and Midichloria mitochondrii to humans and animals. Factors like climate change, human behavior and migrating animals are to blame for the spread of tick-borne diseases. The main route of disease transmission is through tick bites, but there is also evidence of infection through alimentary system for serious infectious agents like TBEV. The knowledge of natural foci and prevalence of these infectious microorganisms is important for risk assessment of human disease. In this study, a total of 3240 nymph and 234 adult ticks were collected from six location sites of three counties of Norway (Hordaland, Vest-Agder and Østfold). The tick samples were analyzed and detected with Real-time PCR, pyrosequencing and direct sequencing to detect the microorganisms. The minimum infection rate or prevalence was calculated from the confirmed observations. Because body fluids of the host animals are valuable epidemiological parameters for TBEV, we also analyzed cow milk and serum from sheep and cows from Hordaland, Vest-Agder and Skedsmokorset. Cow milk was analyzed with PCR and serum with enzyme-linked immunosorbent assay. Two commercial ELISA kits were compared for their sensitivity and specificity. The study confirms the existence of TBEV endemic foci compared to earlier study. The overall estimated TBEV prevalence in nymphs was 0.12% and in adult 2.13%. The B. burgdorferi s.l prevalence in adult ticks was 6.41%. A. phagocytophilum prevalence in nymph was 7.96% and in adult 19.23%. Prevalence of W. pipientisin adult ticks was 10.68% and M. mitochondrii 83.33%. We also found TBEV prevalence in a new location in Hordaland county. The detection of TBEV in milk and serum from cows has never been reported in Norway before.
    The Macrophage Polarization Regulates MSC Osteoblast Differentiation in vitro
    Lei Gong, Annals of Clinical & Laboratory Science - 2016
    Bone repair is a complex yet highly organized process involving interactions between various cell types and the extracellular environment. Macrophages are not only activated in inflammation during early phases of repair processes, but they are also present in bone throughout the whole bone repair process. Bone marrow derived mesenchymal stem cells (MSCs) represent an attractive therapeutic for bone fracture with their expansion potential, osteogenic capability, and potential for injury. However, less is known about the interaction between macrophage and MSC during bone repair and regeneration. This study was aimed to investigate whether macrophages in different statuses can regulate MSC osteoblast differentiation in vitro. Using in vitro cell coculture of macrophage and MSC, it was shown that macrophage polarization can regulate MSC osteoblast differentiation. This was evidenced by increased alkaline phosphatase (ALP), osteogenic markers, and bone mineralization in M2 macrophage cocultured MSC but decreased in M1 counterpart. These results might be mediated by pro-regenerative cytokines, such as TGF-β, VEGF, and IFG-1, produced by M2 macrophages and detrimental inflammation cytokines, such as IL-6, IL-12, and TNF-α, produced by M1 macrophages. Taken together, this shows that macrophage polarization could be crucial for maintaining bone homeostasis and promoting bone repair by regulating the MSC osteoblast differentiation.
    A Neurobiological Pathway That Mediates Stress-Induced Drug Use - viewcontent.cgi
    Natalia Gomez-Ospina, Nature Communications - 2016
    Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection.
    A Neurobiological Pathway That Mediates Stress-Induced Drug Use - viewcontent.cgi
    Oliver Vranjkovic, Marquette University - 2009
    Cocaine addiction represents a tremendous health and financial burden on our society and the high rate of relapse to cocaine use in abstinent addicts represents a major barrier to effective t herapy. Thus, understanding the factors that contribute to relapse and the underlying neurobiological processes is important for guiding the development of treatment for addiction. Stressful life events often trigger drug use in recovering addicts. The contribution of stress to drug use is problematic due to the unpredictable and often uncontrollable nature of stress. A growing literature indicates that norepinephrine and corticotropin releasing factor (CRF) in the brain play key roles in stress intera ctions with motivational neurocircuitry that mediate stress - induced drug seeking . Previous work from our lab has demonstrated that activation of the CRFR1 receptor within the ventral tegmental area (VTA) is both necessary and sufficient for drug seeking behavior during periods of stress. However, the afferent CRF projection into the VTA, and how CRF affects the neurocircuitry of VTA to evoke stress induced relapse are poorly understood. We report that stress induced cocaine use involves a beta2 adrenergic c receptor regulated CRF pathway from the ventral bed nucleus of the striaterminalis to the VTA and a CRFR1 receptor regulated dopaminergic pathway to the prelimbic cortex. It is hypothesized that dopamine released into the prelimbic cortex activates dopamine D1 receptors on pyramidal neurons that comprise a glutamatergic projection to the nucleus accumbens core that is critical for relapse to drug use in abstinent cocaine addicts. It is also reported that the ability of stressors to trigger drug use is determined by the amount and pattern of prior drug use. Findings suggesting that excessive cocaine use establishes susceptibility to stress induced relapse by recruiting CRF regulation of this key stressor responsive mesocortical dopaminergic pathway through increased CRFR1 expression are described. This dissertation defines a key pathway through which stress can promotes relapse and describes its recruitment as result of repeated excessive drug use. Understanding the processes through which stress contributes to cocaine seeking in these rodent models should facilitate translational work aimed targeting these mechanisms clinically and therefore the development of new medications or approaches managing for addiction.
    Myocardial Structural and Biological Anomalies Induced by High Fat Diet in Psammomys obesus Gerbils - ProQuest
    Abdelhamid Sahraoui, Laboratory of Physiology and Pharmacology, Faculty of Medicine - 2015
    Psammomys obesusgerbils are particularly prone to develop diabetes and obesity after brief period of abundant food intake. A hypercaloric high fat diet has been shown to affect cardiac function. Here, we sought to determine whether a short period of high fat feeding might alter myocardial structure and expression of calcium handling proteins in this particular strain of gerbils
    Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice | Skeletal Muscle | Full Text
    Adeel Safdar, BioMed Central - 2016
    Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear.
    Notch Inhibition in the Prevention of Vein Graft Failure - THE BRIGHAM AND WOMEN'S HOSPITAL, INC.
    Aikawa Masanori, United States Patent - 2016
    The present invention is directed to methods of treating a patient to prevent vein graft failure by administering a compound that inhibits the Notch signaling pathway.
    Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease
    Shiri Levy, Oxford Journals - 2016
    Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-β-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.
    An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance
    Philippe Rousseau, Biology of the Cell - 2016
    Background Information Naturally occurring telomerase reverse transcriptase (TERT) isoforms may regulate telomerase activity, and possibly function independently of telomeres to modulate embryonic stem (ES) cell self-renewal and differentiation. Results We report the characterisation of two novel mouse TERT (mTERT) splice variants, Ins-i1[1-102] (Insi1 for short) and Del-e12[1-40] (Dele12 for short) that have not been previously described. Insi1 represents an in-frame insertion of nucleotides 1–102 from intron 1, encoding a 34 amino acid insertion at amino acid 73. Based on known functions of this region in human and Tetrahymena TERTs, the insertion interrupts the RNA interaction domain 1 implicated in low-affinity RNA binding and the telomerase essential N-terminal domain implicated in DNA substrate interactions. Dele12 contains a 40 nucleotide deletion of exon 12 which generates a premature stop codon, and possible protein lacking the C-terminus. We found Insi1 expressed in adult mouse brain and kidney and Dele12 expressed in adult mouse ovary. Dele12 was inactive in vitro and in mTERT−/− ES cells and Insi1 retained 26–48% of telomerase activity reconstituted by wild-type mTERT in vitro and in mTERT−/− ES cells. The Insi1 variant exhibited reduced DNA substrate binding in vitro and both variants exhibited a reduction in binding the telomerase RNA, mTR, when expressed in mTERT−/− ES cells. Stable expression of Dele12 in the mouse fibroblast CB17 cell line inhibited telomerase activity and slowed cell growth, suggesting a potential dominant-negative effect. Levels of signal-free ends, representing short telomeres, and end-to-end fusions were higher in mTERT–/– ES cells expressing mTERT-Insi1 and mTERT-Dele12, compared with levels observed in mTERT–/– ES cells expressing wild-type mTERT. In addition, in mTERT−/− cells expressing mTERT-Insi1, we observed chromosomes that were products of repeated breakage-bridge-fusion cycles and other telomere dysfunction-related aberrations. Conclusion and Significance An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.
    Dynamics of circulating hypoxia-mediated miRNAs and tumor response in patients with high-grade glioma treated with bevacizumab
    Tali Siegal, Journal of Neurosurgery - 2016
    Bevacizumab is an antiangiogenic agent under investigation for use in patients with high-grade glioma. It produces a high rate of radiological response; however, this response should be interpreted with caution because it may reflect normalization of the tumor vasculature and not necessarily a true antitumor effect. The authors previously demonstrated that 4 hypoxia-mediated microRNAs (miRNA)—miR-210, miR-21, miR-10b, and miR-196b—are upregulated in glioma as compared with normal brain tissue. The authors hypothesized that the regulation and expression of these miRNAs would be altered in response to bevacizumab treatment. The object of this study was to perform longitudinal monitoring of circulating miRNA levels in patients undergoing bevacizumab treatment and to correlate it with tumor response.
    Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum
    Juber Herrera Uribe, Veterinary Research - 2016
    Infected pork meat is an important source of non-typhoidal human salmonellosis. Understanding of molecular mechanisms involved in disease pathogenesis is important for the development of therapeutic and preventive strategies. Thus, hereby we study the transcriptional profiles along the porcine intestine during infection with Salmonella Typhimurium, as well as post-transcriptional gene modulation by microRNAs (miRNA). Sixteen piglets were orally challenged with S. Typhimurium. Samples from jejunum, ileum and colon, collected 1, 2 and 6 days post infection (dpi) were hybridized to mRNA and miRNA expression microarrays and analyzed. Jejunum showed a reduced transcriptional response indicating mild inflammation only at 2 dpi. In ileum inflammatory genes were overexpressed (e.g., IL-1B, IL-6, IL-8, IL1RAP, TNFα), indicating a strong immune response at all times of infection. Infection also down-regulated genes of the FXR pathway (e.g., NR1H4, FABP6, APOA1, SLC10A2), indicating disruption of the bile acid absorption in ileum. This result was confirmed by decreased high-density lipoprotein cholesterol in serum of infected pigs. Ileal inflammatory gene expression changes peaked at 2 dpi and tended to resolve at 6 dpi. Furthermore, miRNA analysis of ileum at 2 dpi revealed 62 miRNAs potentially regulating target genes involved in this inflammatory process (e.g., miR-374 and miR-451). In colon, genes involved in epithelial adherence, proliferation and cellular reorganization were down-regulated at 2 and 6 dpi. In summary, here we show the transcriptional changes occurring at the intestine at different time points of the infection, which are mainly related to inflammation and disruption of the bile acid metabolism.
    Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis
    Natasha Whibley, Journal of Leukocyte Biology - 2016
    Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency.
    Nerve Growth Factor Is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs
    Emerson C Krock, Journal of Biological Chemistry - 2016
    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-B, 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. Nerve Growth Factor is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs. Available from: https://www.researchgate.net/publication/286926475_Nerve_Growth_Factor_is_Regulated_by_Toll-Like_Receptor_2_in_Human_Intervertebral_Discs [accessed Dec 16, 2015].
    Gersbach, Charles A., United States Patent Application - 2016
    Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based system related compositions and methods of using said CRISPR/Cas9-based system related compositions for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using said compositions for altering gene expression and genome engineering in muscle, such as skeletal muscle and cardiac muscle
    Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis
    Jody Groenendyk, PLOS ONE - 2016
    Background Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. Methodology/Principal Findings We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. Conclusions We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.
    The auxin response factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes
    Naden T. 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.
    Changes in hormone flux and signaling in white spruce (Picea glauca) seeds during the transition from dormancy to germination in response to temperature cues
    Yang Liu, BioMed Central - 2015
    Background Seeds use environmental cues such as temperature to coordinate the timing of their germination, allowing plants to synchronize their life history with the seasons. Winter chilling is of central importance to alleviate seed dormancy, but very little is known of how chilling responses are regulated in conifer seeds. White spruce (Picea glauca) is an important conifer species of boreal forests in the North American taiga. The recent sequencing and assembly of the white spruce genome allows for comparative gene expression studies toward elucidating the molecular mechanisms governing dormancy alleviation by moist chilling. Here we focused on hormone metabolite profiling and analyses of genes encoding components of hormone signal transduction pathways, to elucidate changes during dormancy alleviation and to help address how germination cues such as temperature and light trigger radicle emergence. Results ABA, GA, and auxin underwent considerable changes as seeds underwent moist chilling and during subsequent germination; likewise, transcripts encoding hormone-signaling components (e.g. ABI3, ARF4 and Aux/IAA) were differentially regulated during these critical stages. During moist chilling, active IAA was maintained at constant levels, but IAA conjugates (IAA-Asp and IAA-Glu) were substantially accumulated. ABA concentrations decreased during germination of previously moist-chilled seeds, while the precursor of bioactive GA1 (GA53) accumulated. We contend that seed dormancy and germination may be partly mediated through the changing hormone concentrations and a modulation of interactions between central auxin-signaling pathway components (TIR1/AFB, Aux/IAA and ARF4). In response to germination cues, namely exposure to light and to increased temperature: the transfer of seeds from moist-chilling to 30 °C, significant changes in gene transcripts and protein expression occurred during the first six hours, substantiating a very swift reaction to germination-promoting conditions after seeds had received sufficient exposure to the chilling stimulus. Conclusions The dormancy to germination transition in white spruce seeds was correlated with changes in auxin conjugation, auxin signaling components, and potential interactions between auxin-ABA signaling cascades (e.g. the transcription factor ARF4 and ABI3). Auxin flux adds a new dimension to the ABA:GA balance mechanism that underlies both dormancy alleviation by chilling, and subsequent radicle emergence to complete germination by warm temperature and light stimuli. Keywords Seed dormancy – Auxin – ABA – GAs – Moist-chilling – Seed germination – White spruce
    Reduced levels of protein recoding by A-to-I RNA editing
    Khen Khermesh, RNA Society - 2015
    Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer’s disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing. Keywords: Alzheimer disease; epigenetics; RNA editing; targeted resequencing
    In vitro toxicity of the galanin receptor 3 antagonist SNAP 37889
    Andreas Koller, Neuropeptides - 2015
    Galanin and its receptors (GAL1, GAL2, GAL3) modulate a range of neuronal, immune and vascular activities. In vivo administration of SNAP 37889 (1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-1H-indol-2-one), a potent small non-peptidergic antagonist of GAL3, was reported to reduce anxiety- and depression-related behavior, ethanol consumption, and antagonizes the effect of galanin on plasma extravasation in rodent models. Accordingly, SNAP 37889 has been proposed as a potential therapeutic agent to treat anxiety and depression disorders. Therefore, we evaluated the toxicity of SNAP 37889 to different cell types. Our experiments revealed that SNAP 37889 (≥10 μM) induced apoptosis in epithelial (HMCB) and microglial (BV-2) cell lines expressing endogenous GAL3, in peripheral blood mononuclear cells and promyelocytic leukemia cells (HL-60) expressing GAL2, and in a neuronal cell line (SH-SY5Y) lacking galanin receptor expression altogether. In conclusion, SNAP 37889 is toxic to a variety of cell types independent of GAL3 expression. We caution that the clinical use of SNAP 37889 at doses that might be used to treat anxiety- or depression- related diseases could have unexpected non-galanin receptor-mediated toxicity, especially on immune cells.
    Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides
    Yehezkel Sztainberg, Nature - 2015
    Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders. MECP2 duplication syndrome is one of the most common genomic rearrangements in males and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders, including loss of MeCP2 in Rett syndrome, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2-overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it, and have been successfully used to correct deficits in different mouse models. We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult symptomatic transgenic MECP2 duplication mice (MECP2-TG), and corrected MECP2 levels in lymphoblastoid cells from MECP2 duplication patients in a dose-dependent manner.
    Identification and Partial Characterization of Potential FtsL and FtsQ Homologs of Chlamydia
    Scot P. Ouellette, Frontiers in Microbiology - 2015
    Chlamydia is amongst the rare bacteria that lack the critical cell division protein FtsZ. By annotation, Chlamydia also lacks several other essential cell division proteins including the FtsLBQ complex that links the early (e.g., FtsZ) and late (e.g., FtsI/Pbp3) components of the division machinery. Here, we report chlamydial FtsL and FtsQ homologs. Ct271 aligned well with Escherichia coli FtsL and shared sequence homology with it, including a predicted leucine-zipper like motif. Based on in silico modeling, we show that Ct764 has structural homology to FtsQ in spite of little sequence similarity. Importantly, ct271/ftsL and ct764/ftsQ are present within all sequenced chlamydial genomes and are expressed during the replicative phase of the chlamydial developmental cycle, two key characteristics for a chlamydial cell division gene. GFP-Ct764 localized to the division septum of dividing transformed chlamydiae, and, importantly, over-expression inhibited chlamydial development. Using a bacterial two-hybrid approach, we show that Ct764 interacted with other components of the chlamydial division apparatus. However, Ct764 was not capable of complementing an E. coli FtsQ depletion strain in spite of its ability to interact with many of the same division proteins as E. coli FtsQ, suggesting that chlamydial FtsQ may function differently. We previously proposed that Chlamydia uses MreB and other rod-shape determining proteins as an alternative system for organizing the division site and its apparatus. Chlamydial FtsL and FtsQ homologs expand the number of identified chlamydial cell division proteins and suggest that Chlamydia has likely kept the late components of the division machinery while substituting the Mre system for the early components.
    Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells
    Ornella I. Selmin, The Journal of Nutrition - 2015
    Background: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. Objective: We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. Methods: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (ApcMin/+) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. Results: In ApcMin/+ mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60–90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. Conclusions: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.
    Demonstration of the dynamic mass redistribution label-free technology as a useful cell-based pharmacological assay for endogenously expressed GABAA receptors - MedChemComm (RSC Publishing)
    Anders B. Klein, MedChemComm - 2015
    Within the continuous quest for the discovery of pharmacologically interesting compounds, the development of new and superior drug screening assays is desired. In recent years, the use of label-free techniques has paved the way for an alternative high-throughput screening method. An example is the Epic® optical-based biosensor that relies on dynamic mass redistribution (DMR) for detection. So far, DMR assays have been mostly used to study G protein-coupled receptor (GPCR) pharmacology. Here, we demonstrate the utility of this assay for investigating ligand-gated ion channel receptors. Using the immortalized IMR-32 neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits, we show that GABA produces concentration-dependent cellular responses that can be measured and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol and the selective antagonists gabazine and bicuculline, we confirm that the data corresponds to that of a GABAA receptor. Based on quantitative real-time PCR measurements, the subunits α3, α5, β3 and θ are the most likely candidates for integration into functional receptors. Our demonstration that label-free methods such as the Epic technology can be used to characterize endogenous GABAA receptors in the IMR-32 cell line is exemplary for the superfamily of ligand-gated ion channel receptors, and holds interesting perspectives in relation to identifying novel mechanisms of action.
    N-Docosahexaenoylethanolamine ameliorates ethanol-induced impairment of neural stem cell neurogenic differentiation
    Mohammad Abdur Rashid, Neuropharmacology - 2016
    Previous studies demonstrated that prenatal exposure to ethanol interferes with embryonic and fetal development, and causes abnormal neurodevelopment. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid highly enriched in the brain, was shown to be essential for proper brain development and function. Recently, we found that N-docosahexenoyethanolamine (synaptamide), an endogenous metabolite of DHA, is a potent PKA-dependent neurogenic factor for neural stem cell (NSC) differentiation. In this study, we demonstrate that ethanol at pharmacologically relevant concentrations downregulates cAMP signaling in NSC and impairs neurogenic differentiation. In contrast, synaptamide reverses ethanol-impaired NSC neurogenic differentiation through counter-acting on the cAMP production system. NSC exposure to ethanol (25–50 mM) for 4 days dose-dependently decreased the number of Tuj-1 positive neurons and PKA/CREB phosphorylation with a concomitant reduction of cellular cAMP. Ethanol-induced cAMP reduction was accompanied by the inhibition of G-protein activation and expression of adenylyl cyclase (AC) 7 and AC8, as well as PDE4 upregulation. In contrast to ethanol, synaptamide increased cAMP production, GTPγS binding, and expression of AC7 and AC8 isoforms in a cAMP-dependent manner, offsetting the ethanol-induced impairment in neurogenic differentiation. These results indicate that synaptamide can reduce ethanol-induced impairment of neuronal differentiation by counter-affecting shared targets in G-protein coupled receptor (GPCR)/cAMP signaling. The synaptamide-mediated mechanism observed in this study may offer a possible avenue for ameliorating the adverse impact of fetal alcohol exposure on neurodevelopment.
    Metallothionein and Hsp70 trade-off against one another in Daphnia magna cross-tolerance to cadmium and heat stress
    Timo Haap, Aquatic Toxicology - 2015
    The association between the insensitivity of adapted ecotypes of invertebrates to environmental stress, such as heavy metal pollution, and overall low Hsp levels characterizing these organisms has been attracting attention in various studies. The present study seeks to induce and examine this phenomenon in Daphnia magna by multigenerational acclimation to cadmium in a controlled laboratory setting. In this experiment, interclonal variation was examined: two clones of D. magna that have previously been characterized to diverge regarding their cadmium resistance and levels of the stress protein Hsp70, were continuously exposed to a sublethal concentration of Cd over four generations to study the effects of acclimation on Hsp70, metallothionein (MT), reproduction and cross-tolerance to heat stress. The two clones differed in all the measured parameters in a characteristic way, clone T displaying Cd and heat resistance, lower Hsp70 levels and offspring numbers on the one hand and higher MT expression on the other hand, clone S the opposite for all these parameters. We observed only slight acclimation-induced changes in constitutive Hsp70 levels and reproductive output. The differences in MT expression between clones as well as between acclimated organisms and controls give evidence for MT accounting for the higher Cd tolerance of clone T. Overall high Hsp70 levels of clone S did not confer cross tolerance to heat stress, contrary to common expectations. Our results suggest a trade-off between the efforts to limit the proteotoxic symptoms of Cd toxicity by Hsp70 induction and those to sequester and detoxify Cd by means of MT.
    Automated amplicon design suitable for analysis of DNA variants by melting techniques
    Per Olaf Ekstrøm, BMC Research Notes - 2015
    Background The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged. Results We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/. Conclusion Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants.
    Molecular systems evaluation of oligomerogenic APPE693Q and fibrillogenic APPKM670/671NL/PSEN1Δexon9 mouse models identifies shared features with human Alzheimer’s brain molecular pathology
    B. Readhead, Molecular Psychiatry - 2015
    Identification and characterization of molecular mechanisms that connect genetic risk factors to initiation and evolution of disease pathophysiology represent major goals and opportunities for improving therapeutic and diagnostic outcomes in Alzheimer's disease (AD). Integrative genomic analysis of the human AD brain transcriptome holds potential for revealing novel mechanisms of dysfunction that underlie the onset and/or progression of the disease. We performed an integrative genomic analysis of brain tissue–derived transcriptomes measured from two lines of mice expressing distinct mutant AD-related proteins. The first line expresses oligomerogenic mutant APPE693Q inside neurons, leading to the accumulation of amyloid beta (Aβ) oligomers and behavioral impairment, but never develops parenchymal fibrillar amyloid deposits. The second line expresses APPKM670/671NL/PSEN1Δexon9 in neurons and accumulates fibrillar Aβ amyloid and amyloid plaques accompanied by neuritic dystrophy and behavioral impairment. We performed RNA sequencing analyses of the dentate gyrus and entorhinal cortex from each line and from wild-type mice. We then performed an integrative genomic analysis to identify dysregulated molecules and pathways, comparing transgenic mice with wild-type controls as well as to each other. We also compared these results with datasets derived from human AD brain. Differential gene and exon expression analysis revealed pervasive alterations in APP/Aβ metabolism, epigenetic control of neurogenesis, cytoskeletal organization and extracellular matrix (ECM) regulation. Comparative molecular analysis converged on FMR1 (Fragile X Mental Retardation 1), an important negative regulator of APP translation and oligomerogenesis in the post-synaptic space. Integration of these transcriptomic results with human postmortem AD gene networks, differential expression and differential splicing signatures identified significant similarities in pathway dysregulation, including ECM regulation and neurogenesis, as well as strong overlap with AD-associated co-expression network structures. The strong overlap in molecular systems features supports the relevance of these findings from the AD mouse models to human AD.
    Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail Theba pisana in relation to genetic structure - Online First - Springer
    Tal Mizrahi, Cell Stress and Chaperones - 2015
    Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.
    Methylation-dependent regulation of hypoxia inducible factor-1 alpha gene expression by the transcription factor kaiso
    Christina C. Pierrea, Elsevier - 2015
    Low oxygen tension (hypoxia) is a common characteristic of solid tumours and strongly correlates with poor prognosis and resistance to treatment. In response to hypoxia, cells initiate a cascade of transcriptional events regulated by the hypoxia inducible factor-1 (HIF-1) heterodimer. Since the oxygen-sensitive HIF-1α subunit is stabilized during hypoxia, it functions as the regulatory subunit of the protein. To date, while the mechanisms governing HIF-1α protein stabilization and function have been well studied, those governing HIF1A gene expression are not fully understood. However, recent studies have suggested that methylation of a HIF-1 binding site in the HIF1A promoter prevents its autoregulation. Here we report that the POZ-ZF transcription factor Kaiso modulates HIF1A gene expression by binding to the methylated HIF1A promoter in a region proximal to the autoregulatory HIF-1 binding site. Interestingly, Kaiso's regulation of HIF1A occurs primarily during hypoxia, which is consistent with the finding that Kaiso protein levels peak after 4 h of hypoxic incubation and return to normoxic levels after 24 h. Our data thus support a role for Kaiso in fine-tuning HIF1A gene expression after extended periods of hypoxia.
    The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition
    Maria J. Aristizabal, PLoS Genet - 2015
    Author Summary RNA Polymerase II (RNAPII) is the enzyme responsible for the transcription of all protein-coding genes, many non-coding genes and retrotransposons. RNAPII has a unique C-terminal domain (CTD) that is composed of repeats of a seven amino acid sequence and is conserved across species. The CTD functions as a recruiting platform for regulatory and RNA processing factors, making the CTD a master orchestrator of transcription. Mutants containing reduced CTD length show defects in steady state transcription during growth in normal conditions, as well as defects in induced gene expression. However, how CTD length affects retrotransposon gene expression remains to be investigated. Here, we uncovered a direct role for CTD length in limiting retrotransposon gene expression and mobility, revealing a new role for the RNAPII-CTD in maintaining genome integrity.
    Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells
    Alexander Puck, Results in Immunology - 2015
    Schlafen (SLFN/Slfn) family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs) and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence.
    Transcriptional changes in Atlantic salmon (Salmo salar) after embryonic exposure to road salt
    Knut Erik Tollefsen, Aquatic Toxicology - 2015
    Road salt is extensively used as a deicing chemical in road maintenance during winter and has in certain areas of the world led to density stratifications in lakes and ponds, and adversely impacted aquatic organisms in the recipients of the road run-off. Aquatic vertebrates such as fish have been particularly sensitive during fertilisation, as the fertilisation of eggs involves rapid uptake of the surrounding water, reduction in egg swelling and in ovo exposure to high road salt concentrations. The present study aimed to identify the persistent molecular changes occurring in Atlantic salmon (Salmo salar) eggs after 24 h exposure to high concentrations (5000 mg/L) of road salt at fertilisation. The global transcriptional changes were monitored by a 60 k salmonid microarray at the eyed egg stage (cleavage stage, 255 degree days after fertilisation) and identified a high number of transcripts being differentially changed. Functional enrichment, pathway and gene–gene interaction analysis identified that the differentially expressed genes (DEGs) were mainly associated with toxiciologically relevant processes involved in osmoregulation, ionregulation, oxidative stress, metabolism (energy turnover), renal function and developmental in the embryos. Quantitative rtPCR analysis of selected biomarkers, identified by global transcriptomics, were monitored in the eggs for an extended range of road salt concentrations (0, 50, 100, 500 and 5000 mg/L) and revealed a positive concentration-dependent increase in cyp4A14, a gene involved in lipid turnover and renal function, and nav1, a gene involved in neuraxonal development. Biomarkers for osmoregulatory responses (atp1a2), the gene encoding the main sodium/potassium ATP-fueled transporter for chloride ions, and oxidative stress (txndc9), a gene involved in regulation of cell redox homeostasis, displayed apparent concentration-dependency with exposure, although large variance in the control group precluded robust statistical discrimination between the groups. A No Transcriptional Effect Level (NOTEL) of 50 mg/L road salt was found to be several orders of magnitude lower than the adverse effects documented in developing fish embryos elsewhere, albeit at concentrations realistic in lotic systems receiving run-off from road salt. It remains to be determined whether these transcriptional changes may cause adverse effects in fish at ecologically relevant exposure concentrations of road salt.
    Coupling Between Nutrient Availability and Thyroid Hormone Activation
    Lattoya J. Lartey, Journal of Biological Chemistry - 2015
    The activity of the thyroid gland is stimulated by food availability via leptin-induced TRH/TSH expression. Here we show that food availability also stimulates thyroid hormone activation by accelerating conversion of T4-to-T3 via type 2 deiodinase (D2) in mouse skeletal muscle and in a cell model transitioning from 0.1 to 10% FBS. The underlying mechanism is transcriptional de-repression of DIO2 through the mTORC2 pathway as defined in rictor knock down cells. In cells kept in 0.1% FBS there is DIO2 inhibition via FOXO1 binding to DIO2 promoter. Repression of DIO2 by FOXO1 was confirmed using its specific inhibitor AS1842856 or adenoviral infection of constitutively active FOXO1. ChIP studies indicate that 4h after 10% FBS-containing media FOXO1 binding markedly decreases and DIO2 promoter is activated. Studies in the insulin-receptor FOXO1 KO mouse indicate that insulin is a key signaling molecule in this process. We conclude that FOXO1 represses DIO2 during fasting and de-repression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway.
    Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm
    Hua Jiang, Proceedings of the National Academy of Sciences - 2015
    An Arabidopsis pollen grain (male gametophyte) consists of three cells: the vegetative cell, which forms the pollen tube, and two sperm cells enclosed within the vegetative cell. It is still unclear if there is intercellular communication between the vegetative cell and the sperm cells. Here we show that ABA-hypersensitive germination3 (AHG3), encoding a protein phosphatase, is specifically transcribed in the vegetative cell but predominantly translated in sperm cells. We used a series of deletion constructs and promoter exchanges to document transport of AHG3 transcripts from the vegetative cell to sperm and showed that their transport requires sequences in both the 5′ UTR and the coding region. Thus, in addition its known role in transporting sperm during pollen tube growth, the vegetative cell also contributes transcripts to the sperm cells.
    BCRP protein levels do not differ regionally in adult human livers, but decline in the elderly
    Zoe Riches, Chemico-Biological Interactions - 2015
    The aim of this study was to characterize the ontogeny and variability of the BCRP (ABCG2) transporter in healthy human liver. Levels of BCRP mRNA and protein were determined with q-RT-PCR and western blot in a cohort of 87 human livers aged from 7 days to 87 years. A study of the regional expression of BCRP within adult livers was also performed in a nested cohort of 14 individuals with multiple samples per person collected from pre-selected sites. Levels of BCRP mRNA were not significantly different at any age, but protein levels for BCRP were lower in the elderly compared with adults (p < 0.001) and children (p < 0.05). The intra-liver levels of BCRP protein ranged approximately 6.5-fold and inter-liver BCRP protein varied 8.5-fold in the cohort. No differences in BCRP mRNA or protein were observed with sex or ethnicity, although higher levels of BCRP mRNA were observed in livers from overweight individuals (Body Mass Index ≥ 25–29.9) as compared to underweight or ideal weight individuals. There were no differences in the levels of BCRP mRNA or protein in different regions of the large lobe (n = 3 regions), small lobe (n = 3 regions), directly adjacent to the portal vein or directly adjacent to the common bile duct. This indicates that BCRP researchers can source tissue from all parts of the adult liver without artificial bias in their results. Lower BCRP protein expression in the elderly may be associated with compromised xeno- and endobiotic transport.
    Uncovering Hidden Layers of Cell Cycle Regulation through Integrative Multi-omic Analysis
    Ranen Aviner, PLoS Genet - 2015
    Author Summary How the genetic program of a cell unfolds to execute complex functions depends on a dynamic interplay between multiple steps that include transcription of DNA into mRNA, translation of mRNA into protein and post-translational degradation of mature proteins. Profiling of gene expression is traditionally based on measurements of steady-state mRNA levels, but recent studies have shown that mRNA and protein levels are highly discordant, suggesting that post-transcriptional mechanisms play a dominant role in modulating protein abundance. Here we combine measurements of mRNA, translation and protein across the mammalian cell cycle to uncover the hidden complexity of cell cycle regulation. Using this approach, we gain insights into the dynamics of protein synthesis and degradation and identify new genes and functions that cycle through cell division by periodic changes in translation or degradation rates. Integrative multi-omic analyses combining information on the transcriptome, translatome and proteome hold great promise for providing transformative biological insights in a variety of model systems.
    The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype
    António Pinto-Almeida, PLoS ONE - 2015
    Background Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug. Methodology/Principal Findings Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR. Conclusions/Significance This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.
    Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors - Springer
    Pratiksha I. Thakore, TALENs - 2015
    Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.
    Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex
    Justin L. Bollinger, Brain, Behavior, and Immunity - 2015
    Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1–CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females.
    Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine - Hodge - 2015 - Biotechnology and Bioengineering - Wiley Online Library
    Alexander J. Hodge, Biotechnology and Bioengineering - 2015
    The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8–14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased βMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors, including S-nitrosocysteine, have advantages over other bioactive molecules for use in scalable culture systems in driving cardiac differentiation, since they are inexpensive and the diffusivity of nitric oxide is relatively high. By enabling maintenance of spontaneous contraction in suspension culture and progressing electrophysiological function of resulting SC-CMs toward a more mature phenotype, long-term application of S-nitrosocysteine was shown to be beneficial during cardiac differentiation. Taken together, these results demonstrate the efficiency of nitric oxide as a signaling compound, with implications in the improvement of pluripotent stem cell-derived cardiomyocyte maturation in large-scale culture systems. Biotechnol. Bioeng. 2015;9999: 1–13. © 2015 Wiley Periodicals, Inc.
    Hepatitis C virus upregulates B-cell receptor signaling: a novel mechanism for HCV-associated B-cell lymphoproliferative disorders
    B. Dai, Oncogene - 2015
    B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.
    ATP-binding cassette proteins BCRP, MRP1 and P-gp expression and localization in the human umbilical cord
    Zoe Riches, Xenobiotica - 2015
    1. The umbilical cord is a direct conduit to the fetus hence transporters could have roles in partitioning substances between the maternal-placental-fetal units. Here we determined the expression and localization of the ATP-Binding Cassette (ABC) transporters BCRP (ABCG2), P-gp (ABCB1) and MRP1 (ABCC1) in human umbilical cords.2. The mRNA for BCRP and MRP1 was detected in 25/25 samples, but P-gp was detected in only 5/25. ABC transporter mRNA expression relative to 18S was 25.6 ± 0.3, 26.5 ± 0.6 and 22.2 ± 0.2 cycles for BCRP, MRP1 and P-gp respectively.3. Using a subset of 10 umbilical cords, BCRP protein was present in all samples (immunoblot) with positive correlation between mRNA and proteins (p = 0.07, r = 0.62) and between immunoblotting and immunohistochemistry (IHC) (p = 0.03, r = 0.67). P-gp protein was observed in 4/10 samples by both immunoblot and IHC, with no correlation between mRNA and protein (p = 0.45, r = 0.55) or immunoblotting and IHC (p = 0.2, r = 0.72), likely due to small sample size. MRP1 protein was not observed.4. Localization of BCRP and P-gp proteins was to Wharton’s jelly with no specific staining in arterial or venous endothelia.5. Understanding ABC transporter expression in the umbilical cord may be useful for determining fetal exposures to xenobiotics if functional properties can be defined.
    PLOS ONE: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines
    Motoharu Ono, PLOS ONE - 2015
    We have previously reported an antisense technology, ‘snoMEN vectors’, for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.
    Shedding of soluble platelet-derived growth factor receptor-β from human brain pericytes
    Abhay P. Sagare, Neuroscience Letters - 2015
    Platelet-derived growth factor receptor-β (PDGFRβ) is expressed in the brain by vascular mural cells–brain capillary pericytes and arterial vascular smooth muscle cells (VSMCs). Recent evidence shows that blood–brain barrier (BBB) disruption and increased permeability, especially in the hippocampus, positively correlates with elevated levels of soluble PDGFRβ (sPDGFRβ) in cerebrospinal fluid (CSF) in patients with mild dementia. To determine which vascular cell type(s) contributes to increased sPDGFRβ in CSF, we compared PDGFRβ expression and sPDGFRβ shedding in response to injury in early passage primary cultures of human brain pericytes, brain arterial VSMCs, and brain endothelial cells. PDGFRβ protein was undetectable in endothelial cells, but was found both in pericytes and VSMCs. PDGFRβ relative protein abundance was by 4.2-fold (p < 0.05) higher in pericytes compared to VSMCs. Hypoxia (1% O2) or amyloid-β peptide (25 μM) compared to normoxia (21% O2) both increased over 48 h shedding of sPDGFRβ and its levels in the culture medium from pericytes cultures, but not from VSMCs cultures, by 4.3-fold and 4.6-fold, respectively, compared to the basal sPDGFRβ levels in the medium (1.43 ± 0.15 ng/ml). This was associated with the corresponding loss of cell-associated PDGFRβ from pericytes and no change in cellular levels of PDGFRβ in VSMCs. Thus, sPDGFRβ is a biomarker of pericyte injury, and elevated sPDGFRβ levels in biofluids in patients with dementia and/or other neurodegenerative disorders likely reflects pericyte injury, which supports the potential for sPDGFRβ to be developed and validated as a biomarker of brain pericyte injury and BBB dysfunction.
    Lactobacillus fermentum CECT 5716 Reduces Staphylococcus Load in the Breastmilk of Lactating Mothers Suffering Breast Pain: A Randomized Controlled Trial
    Jose A. Maldonado-Lobón, Breastfeeding Medicine - 2015
    Introduction: Recent results indicate that human mastitis and painful breastfeeding may be characterized by a mammary bacterial dysbiosis, a process in which the population of potential pathogens increases at the expense of the normal mammary microbiota. The objective of the present study is the evaluation of three different doses of Lactobacillus fermentum CECT5716 to reduce the load of Staphylococcus in the breastmilk of women suffering from painful breastfeeding. Materials and Methods: A randomized double-blinded controlled study with four study groups was performed. Three groups received the probiotic strain for 3 weeks at doses of 3×109 colony-forming units (CFU)/day, 6×109 CFU/day, or 9×109 CFU/day. The fourth group received a placebo of maltodextrin. The main outcome of the study was Staphylococcus counts in breastmilk. The secondary outcomes were Streptococcus, Lactobacillus, and total bacteria counts in breastmilk, immunoglobulin A and interleukin 8 concentrations in breastmilk, and breast pain scores. Results: At the end of the study, a significant decrease in the Staphylococcus load was observed in the probiotic groups compared with the baseline loads (p=0.045), whereas the control group maintained similar levels over time. A significant difference in the pain score was observed among the groups receiving the three probiotic doses compared with the control group (p=0.035, p=0.000, and p=0.028, respectively). A dose–response effect could not be observed because the three doses tested induced similar effects, and no significant differences were detected. Conclusions: We conclude that L. fermentum CECT5716 is an efficient treatment for breast pain during lactation associated with a high level of Staphylococcus in breastmilk.
    Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells
    Jin-Man Kim, Nucleic Acids Research - 2015
    SET and MYND domain containing protein 3 (SMYD3) is a histone methyltransferase, which has been implicated in cell growth and cancer pathogenesis. Increasing evidence suggests that SMYD3 can influence distinct oncogenic processes by acting as a gene-specific transcriptional regulator. However, the mechanistic aspects of SMYD3 transactivation and whether SMYD3 acts in concert with other transcription modulators remain unclear. Here, we show that SMYD3 interacts with the human positive coactivator 4 (PC4) and that such interaction potentiates a group of genes whose expression is linked to cell proliferation and invasion. SMYD3 cooperates functionally with PC4, because PC4 depletion results in the loss of SMYD3-mediated H3K4me3 and target gene expression. Individual depletion of SMYD3 and PC4 diminishes the recruitment of both SMYD3 and PC4, indicating that SMYD3 and PC4 localize at target genes in a mutually dependent manner. Artificial tethering of a SMYD3 mutant incapable of binding to its cognate elements and interacting with PC4 to target genes is sufficient for achieving an active transcriptional state in SMYD3-deficient cells. These observations suggest that PC4 contributes to SMYD3-mediated transactivation primarily by stabilizing SMYD3 occupancy at target genes. Together, these studies define expanded roles for SMYD3 and PC4 in gene regulation and provide an unprecedented documentation of their cooperative functions in stimulating oncogenic transcription.
    STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-associated Herpesvirus
    Christine A. King, Journal of Virology - 2015
    Lytic activation of Kaposi's Sarcoma-associated Herpesvirus (KSHV) from latency is a critical contributor to pathogenesis and progression of KSHV-mediated disease. Development of targeted treatment strategies and improvement of lytic phase-directed oncolytic therapies therefore hinge on gaining a better understanding of latency-to-lytic transition. A key observation in that regard, also common to other herpesviruses, is the partial permissiveness of latently-infected cells to lytic cycle inducing agents. Here, we address the molecular basis for why only some KSHV-infected cells respond to lytic stimuli. Since cellular Signal Transducer and Activator of Transcription 3 (STAT3) is overactive in KSHV-associated cancers, KSHV activates STAT3, and STAT3 has been found to regulate lytic activation of Epstein-Barr virus (EBV)-infected cells, we asked if STAT3 contributes similarly to the life cycle of KSHV. We found that high levels of STAT3 correlate with the refractory state at the single cell level under conditions of both spontaneous and induced lytic activation; importantly, STAT3 also regulates lytic susceptibility. Further, knockdown of STAT3 suppresses the cellular transcriptional co-repressor Krüppel-associated box domain-associated protein 1 (KAP1), and suppression of KAP1 activates lytic genes including the viral lytic switch RTA, thereby linking STAT3 via KAP1 to regulation of the balance between lytic and latent cells. These findings, taken together with those from EBV-infected, and more recently HSV-1-infected cells, cement the contribution of host STAT3 to persistence of herpesviruses, and simultaneously reveal an important lead to devise strategies to improve lytic phase-directed therapies for herpesviruses. IMPORTANCE Lytic activation of the cancer-causing Kaposi's Sarcoma-associated Herpesvirus (KSHV) is vital to its life cycle and causation of disease. Like other herpesviruses, however, a substantial fraction of latently-infected cells are resistant to lytic-inducing stimuli. Investigating the molecular basis for this refractory state is essential for understanding how the virus persists, how it causes disease, and to guide efforts to improve treatment of KSHV-mediated diseases. We find that like two other herpesviruses, EBV and HSV-1, KSHV exploits the cellular transcription factor STAT3 to regulate susceptibility of latently-infected cells to lytic triggers. These findings highlight a common STAT3-centered strategy used by herpesviruses to maintain persistence in their host, while also revealing a key molecule to pursue while devising methods to improve herpesvirus lytic phase-directed therapies.
    Genome-Wide Profiling of PARP1 Reveals an Interplay with Gene Regulatory Regions and DNA Methylation
    Narasimharao Nalabothula, PLoS ONE - 2015
    Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme involved in DNA repair, chromatin remodeling and gene expression. PARP1 interactions with chromatin architectural multi-protein complexes (i.e. nucleosomes) alter chromatin structure resulting in changes in gene expression. Chromatin structure impacts gene regulatory processes including transcription, splicing, DNA repair, replication and recombination. It is important to delineate whether PARP1 randomly associates with nucleosomes or is present at specific nucleosome regions throughout the cell genome. We performed genome-wide association studies in breast cancer cell lines to address these questions. Our studies show that PARP1 associates with epigenetic regulatory elements genome-wide, such as active histone marks, CTCF and DNase hypersensitive sites. Additionally, the binding of PARP1 to chromatin genome-wide is mutually exclusive with DNA methylation pattern suggesting a functional interplay between PARP1 and DNA methylation. Indeed, inhibition of PARylation results in genome-wide changes in DNA methylation patterns. Our results suggest that PARP1 controls the fidelity of gene transcription and marks actively transcribed gene regions by selectively binding to transcriptionally active chromatin. These studies provide a platform for developing our understanding of PARP1’s role in gene regulation.
    NUDT21-spanning CNVs lead to neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation
    Huda Y. Zoghbi, eLife - 2015
    The brain is sensitive to the dose of MECP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is know about its regulation. Here, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3'UTR, resulting in an enrichment of inefficiently translated long-mRNA isoforms. Importantly, normalization of NUDT21 via siRNA-mediated knockdown in duplication-patient lymphoblasts restores MeCP2 to normal levels. In this study, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation.To Top The brain is sensitive to the dose of MECP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is know about its regulation. Here, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3'UTR, resulting in an enrichment of inefficiently translated long-mRNA isoforms. Importantly, normalization of NUDT21 via siRNA-mediated knockdown in duplication-patient lymphoblasts restores MeCP2 to normal levels. In this study, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation.
    Circulating microRNA Profiling Needs Further Refinement Before Clinical Use in Patients With Aortic Stenosis
    Sean Coffey, Journal of the American Heart Association - 2015
    Background Aortic stenosis (AS) is a progressive condition leading to heart failure and death without treatment. No medical therapy currently exists for AS, and a major management challenge is deciding on the correct timing of aortic valve replacement. MicroRNAs (miRNAs) are short noncoding RNAs that are stable in the circulation. We wished to use miRNAs as biomarkers of disease in AS. Methods and Results We performed microarray‐based whole miRNome profiling of 24 participants with AS and 27 control participants. After adjustment for age and multiple testing, we identified 4 miRNAs significantly different between groups. These findings were then examined using quantitative polymerase chain reaction in a larger validation cohort of 101 controls and 94 participants with AS, stratified in a prespecified analysis by presence of coexisting coronary artery disease (CAD). We obtained mixed results for miR‐22‐3p, miR‐24‐3p, miR‐382‐5p, and miR‐451a in the validation cohort, with differing associations according to CAD status. miR‐21‐5p was increased in AS patients without CAD, but there was no difference between groups with CAD. Conclusion Despite holding great promise, circulating miRNA profiling requires further refinement before translation into clinical use as a biomarker in aortic stenosis.
    Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles
    Troy D. Hubbard, Scientific Reports - 2015
    Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.
    Corp Regulates P53 in Drosophila melanogaster via a Negative Feedback Loop
    Riddhita Chakraborty, PLoS Genet - 2015
    Author Summary Organisms have exquisitely sensitive mechanisms to detect and respond to DNA damage. If DNA damage in a cell can be repaired, then that cell may resume its normal function. In multi-cellular organisms, if a cell cannot repair DNA damage it usually undergoes programmed cell death. This prevents the proliferation of cells with damaged genomes, which might otherwise differentiate incorrectly or proliferate without limit as cancer. In Drosophila melanogaster we identified corp as a gene that promotes the survival of such cells. Transcription of corp is activated by the P53 tumor suppressor protein, known primarily for its induction of cell death in response to DNA damage. Our experiments show that P53 regulates both pro-death and anti-death genes, and that a competition between these opposing factors determines cell fate. We find that corp functions by down-regulating P53, establishing a negative feedback loop. In vertebrates an identical mode of regulation is known: P53 up-regulates Mdm2, which physically interacts with P53 and is its primary negative regulator. We identified a protein motif on Corp that is shared with Mdm2, and is required for physical interaction between Corp and Drosophila P53. These results reinforce and strengthen the similarities of the P53 pathways and their regulation in vertebrates and in Drosophila.
    Mammaglobin 1 promotes breast cancer malignancy and confers sensitivity to anticancer drugs
    Nadia Picot, Molecular Carcinogenesis - 2015
    Mammaglobin 1 (MGB1), a member of the secretoglobin family, is expressed in mammary epithelial tissues and is overexpressed in most mammary carcinomas. Despite the extensive research correlating MGB1 expression profiles to breast cancer pathogenesis and disease outcome, the biological significance of MGB1 in cancer processes is still unclear. We have thus set out to conduct a functional evaluation of the molecular and cellular roles of MGB1 in breast cancer processes leading to disease progression. Using a series of breast cancer cell models with conditional MGB1 expression, we demonstrate that MGB1 promotes cancer cell malignant features. More specifically, loss of MGB1 expression resulted in a decrease of cell proliferation, soft agar spheroid formation, migration, and invasion capacities of breast cancer cells. Concomitantly, we also observed that MGB1 expression activates signaling pathways mediated by MAPK members (p38, JNK, and ERK), the focal adhesion kinase (FAK), matrix metalloproteinases (MMPs) and NFκB. Moreover, MGB1 regulates epithelial to mesenchymal (EMT) features and modulates Snail, Twist and ZEB1 expression levels. Interestingly, we also observed that expression of MGB1 confers breast cancer cell sensitivity to anticancer drug-induced apoptosis. Together, our results support a role for MGB1 in tumor malignancy in exchange for chemosensitivity. These findings provide one of the first descriptive overview of the molecular and cellular roles of MGB1 in breast cancer processes and may offer new insight to the development of therapeutic and prognostic strategies in breast cancer patients. © 2015 Wiley Periodicals, Inc.
    Scrutinizing the immune defence inventory of Camponotus floridanus applying total transcriptome sequencing
    Shishir K. Gupta, BMC Genomics - 2015
    Background Defence mechanisms of organisms are shaped by their lifestyle, environment and pathogen pressure. Carpenter ants are social insects which live in huge colonies comprising genetically closely related individuals in high densities within nests. This lifestyle potentially facilitates the rapid spread of pathogens between individuals. In concert with their innate immune system, social insects may apply external immune defences to manipulate the microbial community among individuals and within nests. Additionally, carpenter ants carry a mutualistic intracellular and obligate endosymbiotic bacterium, possibly maintained and regulated by the innate immune system. Thus, different selective forces could shape internal immune defences of Camponotus floridanus. Results The immune gene repertoire of C. floridanus was investigated by re-evaluating its genome sequence combined with a full transcriptome analysis of immune challenged and control animals using Illumina sequencing. The genome was re-annotated by mapping transcriptome reads and masking repeats. A total of 978 protein sequences were characterised further by annotating functional domains, leading to a change in their original annotation regarding function and domain composition in about 8 % of all proteins. Based on homology analysis with key components of major immune pathways of insects, the C. floridanus immune-related genes were compared to those of Drosophila melanogaster, Apis mellifera, and other hymenoptera. This analysis revealed that overall the immune system of carpenter ants comprises many components found in these insects. In addition, several C. floridanus specific genes of yet unknown functions but which are strongly induced after immune challenge were discovered. In contrast to solitary insects like Drosophila or the hymenopteran Nasonia vitripennis, the number of genes encoding pattern recognition receptors specific for bacterial peptidoglycan (PGN) and a variety of known antimicrobial peptide (AMP) genes is lower in C. floridanus. The comparative analysis of gene expression post immune-challenge in different developmental stages of C. floridanus suggests a stronger induction of immune gene expression in larvae in comparison to adults. Conclusions The comparison of the immune system of C. floridanus with that of other insects revealed the presence of a broad immune repertoire. However, the relatively low number of PGN recognition proteins and AMPs, the identification of Camponotus specific putative immune genes, and stage specific differences in immune gene regulation reflects Camponotus specific evolution including adaptations to its lifestyle. Keywords: Camponotus floridanus ; Carpenter ant; Transcriptome; Re-annotation; Immune system
    Infestation and Quantification of Ochratoxigenic Fungi in Barley and Wheat Naturally Contaminated with Ochratoxin A
    Julie Kuruc, Journal of Food Protection - 2015
    Cereal grains are a significant source of ochratoxin A (OTA) in the human diet. Multiple ochratoxigenic Aspergillus and Penicillium spp. have been reported as contaminants on various cereal grains around the world, although relatively few species dominate in any given location. Efforts to mitigate the risk of fungal contamination and OTA accumulation can be made pre- and postharvest. Still, a rapid and reliable screening method is sought that can be used to predict the OTA level of a sample and to inform risk assessments prior to processing. In this study, we assessed the efficacy of two OTA-related indices for OTA level prediction. Infestation rates were determined by direct plating for freshly harvested and stored barley, durum, and hard red spring wheat samples (n = 139) with known OTA levels. Presumptive ochratoxigenic isolates were tested for their ability to produce OTA. The nonribosomal peptide synthase (otanpsPN) involved in OTA biosynthesis was used to quantify ochratoxigenic fungi in barley and wheat. Viable Penicillium verrucosum was present in 45% of the samples. In total, 62.7% (n = 110) of the P. verrucosum isolates tested produced OTA on dichloran yeast extract sucrose 18% glycerol agar. Both OTA level and infestation rate (r = 0.30), as well as OTA level and otanpsPN concentration (r = 0.56), were weakly correlated. Neither infestation rate nor otanpsPN concentration is a reliable predictor of OTA level in a sample.
    A High Fat Diet Caused Widespread Epigenomic Differences on Hepatic Methylome in Rat
    Yukun Zhang, Physiological Genomics - 2015
    A high fat (HF) diet is associated with progression of liver diseases. To illustrate genome-wide landscape of DNA methylation in liver of rats fed either a control or HF diet, two enrichment-based methods, namely methyl-DNA immunoprecipitation assay with high-throughput sequencing (MeDIP-seq) and methylation-sensitive restriction enzyme sequencing (MRE-seq), were performed in our study. Rats fed with the HF diet exhibited an increased body weight and liver fat accumulation compared to that of the control group when they were 12 weeks of age. Genome-wide analysis of differentially methylated regions (DMR) showed that 12494 DMRs induced by HF diet were hypomethylated and 6404 were hypermethylated. DMRs with gene annotations (Differentially methylated genes, DMG) were further analyzed to show gene-specific methylation profile. There were 88, 2680, and 95 hypomethylated DMGs identified with changes in DNA methylation in the promoter; intragenic and downstream regions, respectively, compared to fewer hypermethylated DMGs (45, 1623, and 50 in the respective regions). Some of these genes also contained an ACGT cis-acting motif whose DNA methylation status may affect gene expression. Pathway analysis showed that these DMGs were involved in critical hepatic signaling networks related to hepatic development. Therefore, HF diet had global impacts on DNA methylation profile in the liver of rats, leading to differential expression of genes in hepatic pathways that may involve in functional changes in liver development.
    Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing
    Priscilla Lau, Proceedings of the National Academy of Sciences - 2011
    Histone H3 phosphorylation is a critical step that couples signal transduction pathways to gene regulation. To specifically assess the transcriptional regulatory functions of H3 phosphorylation- we developed an in vivo targeting approach and found that the H3 kinase MSK1 is a direct and potent transcriptional activator. Targeting of this H3 kinase to the endogenous c-fos promoter is sufficient to activate its expression without the need of upstream signaling. Moreover- targeting MSK1 to the alpha-globin promoter induces H3 S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Importantly- we discovered a mechanism whereby H3 S28 phosphorylation not only displaces binding of the polycomb-repressive complexes- but it also induces a methyl-acetylation switch of the adjacent K27 residue. Our findings show that signal transduction activation can directly regulate polycomb silencing through a specific histone code-mediated mechanism.
    Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice
    Adeel Safdar, Proceedings of the National Academy of Sciences - 2011
    A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse- harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma- exhibits accelerated aging phenotypes characteristic of human aging- systemic mitochondrial dysfunction- multisystem pathology- and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis- prevented mtDNA depletion and mutations- increased mitochondrial oxidative capacity and respiratory chain assembly- restored mitochondrial morphology- and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection- reduced multisystem pathology- and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.
    Stimulation of the hypothalamic ventromedial nuclei by pituitary adenylate cyclase-activating polypeptide induces hypophagia and thermogenesis
    Jon Resch, American Journal of Physiology - Regulatory- Integrative and Comparative Physiology - 2011
    Numerous studies have demonstrated that the hypothalamic ventromedial nuclei (VMN) regulate energy homeostasis by integrating and utilizing behavioral and metabolic mechanisms. The VMN heavily express pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptors (PAC1R). Despite the receptor distribution- most PACAP experiments investigating affects on feeding have focused on intracerebroventricular administration or global knockout mice. To identify the specific contribution of PACAP signaling in the VMN- we injected PACAP directly into the VMN and measured feeding behavior and indices of energy expenditure. Following an acute injection of PACAP- nocturnal food intake was significantly reduced for 6 h after injections without evidence of malaise. In addition- PACAP-induced suppression of feeding also occurred following an overnight fast and could be blocked by a specific PAC1R antagonist. Metabolically- VMN-specific injections of PACAP significantly increased both core body temperature and spontaneous locomotor activity with a concurrent increase in brown adipose uncoupling protein 1 mRNA expression. To determine which signaling pathways were responsive to PACAP administration into the VMN- we measured mRNA expression of well-characterized hypothalamic neuropeptide regulators of feeding. One hour after PACAP administration- expression of pro-opiomelanocortin mRNA was significantly increased in the arcuate nuclei (ARC)- with no changes in neuropeptide Y and agouti-related polypeptide mRNA levels. This suggests that PAC1R expressing VMN neurons projecting to pro-opiomelanocortin neurons contribute to hypophagia by involving melanocortin signaling. While the VMN also abundantly express PACAP protein- the present study demonstrates that PACAP input to the VMN can influence the control of energy homeostasis.
    Silencing GADD153/CHOP Gene Expression Protects against Alzheimer's Disease-Like Pathology Induced by 27-Hydroxycholesterol in Rabbit Hippocampus
    Jaya Prasanthi, PLoS ONE - 2011
    Endoplasmic reticulum (ER) stress is suggested to play a key role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Sustained ER stress leads to activation of the growth arrest and leucine zipper transcription factor- DNA damage inducible gene 153 (gadd153, also called CHOP). Activated gadd153 can generate oxidative damage and reactive oxygen species (ROS)- increase Beta-amyloid (ABeta) levels- disturb iron homeostasis and induce inflammation as well as cell death- which are all pathological hallmarks of AD. Epidemiological and laboratory studies suggest that cholesterol dyshomeostasis contributes to the pathogenesis of AD. We have previously shown that the cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) triggers AD-like pathology in organotypic slices. However- the extent to which gadd153 mediates 27-OHC effects has not been determined. We silenced gadd153 gene with siRNA and determined the effects of 27-OHC on AD hallmarks in organotypic slices from adult rabbit hippocampus. siRNA to gadd153 reduced 27-OHC-induced ABeta production by mechanisms involving reduction in levels of Beta-amyloid precursor protein (APP) and Beta-secretase (BACE1)- the enzyme that initiates cleavage of APP to yield ABeta peptides. Additionally- 27-OHC-induced tau phosphorylation- ROS generation- TNF-alpha activation- and iron and apoptosis-regulatory protein levels alteration were also markedly reduced by siRNA to gadd153. These data suggest that ER stress-mediated gadd153 activation plays a central role in the triggering of AD pathological hallmarks that result from incubation of hippocampal slices with 27-OHC. Our results add important insights into cellular mechanisms that underlie the potential contribution of cholesterol metabolism in AD pathology- and suggest that preventing gadd153 activation protects against AD related to cholesterol oxidized products.
    AMP-activated protein kinase (AMPK) Beta1Beta2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise
    Hayley O'Neill, Proceedings of the National Academy of Sciences - 2011
    AMP-activated protein kinase (AMPK) Beta1 or Beta2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle- alpha2Beta2gamma3-containing heterotrimers predominate. However- compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK alpha2- Beta2- and gamma3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism- because these models have normal mitochondrial content- contraction-stimulated glucose uptake- and insulin sensitivity. In the current study- we generated mice lacking both AMPK Beta1 and Beta2 isoforms in skeletal muscle (Beta1Beta2M-KO). Beta1Beta2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly- young Beta1Beta2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake- findings that were not apparent in mice with single mutations or deletions in muscle alpha- Beta- or gamma subunits.
    Multi-species Analyses of Direct Activators of the Constitutive Androstane Receptor
    Curtis Omiecinski, Toxicological Sciences - 2011
    The constitutive androstane receptor (CAR, NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation- CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays- obscuring the detection of ligand activators. However- a human splice variant of CAR- termed CAR3- exhibits negligible constitutive activity. In addition- CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study- we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses- thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse- rat- and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays- the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.
    A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor
    Takeshi Murakami, Proceedings of the National Academy of Sciences - 2011
    Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB- and ZouA- a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4-12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA)- it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites- evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic- degradative- and photosynthetic pathways in a wide variety of organisms.
    Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome
    Mark McCauley, Science Translational Medicine - 2011
    AbstractBack to Top Rett syndrome is a neurodevelopmental disorder typically caused by mutations in methyl-CpG-binding protein 2 (MECP2) in which 26\% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction- we characterized the electrocardiograms in 379 people with Rett syndrome and found that 18.5\% show prolongation of the corrected QT interval (QTc)- an indication of a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function- Mecp2Null/Y- also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice- Mecp2Null/+- show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia- implicating neuronally mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome- Beta-adrenergic receptor blockers- did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate- we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current- which was normalized when cells were treated with the sodium channel-blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control- which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current to prevent lethal cardiac arrhythmias.
    Short Day-Mediated Cessation of Growth Requires the Downregulation of AINTEGUMENTALIKE1 Transcription Factor in Hybrid Aspen
    Anna Karlberg, PLoS Genetics - 2011
    Day length is a key environmental cue regulating the timing of major developmental transitions in plants. For example- in perennial plants such as the long-lived trees of the boreal forest- exposure to short days (SD) leads to the termination of meristem activity and bud set (referred to as growth cessation). The mechanism underlying SD-mediated induction of growth cessation is poorly understood. Here we show that the AIL1-AIL4 (AINTEGUMENTALIKE) transcription factors of the AP2 family are the downstream targets of the SD signal in the regulation of growth cessation response in hybrid aspen trees. AIL1 is expressed in the shoot apical meristem and leaf primordia- and exposure to SD signal downregulates AIL1 expression. Downregulation of AIL gene expression by SDs is altered in transgenic hybrid aspen plants that are defective in SD perception and/or response- e.g. PHYA or FT overexpressors. Importantly- SD-mediated regulation of growth cessation response is also affected by overexpression or downregulation of AIL gene expression. AIL1 protein can interact with the promoter of the key cell cycle genes- e.g. CYCD3.2- and downregulation of the expression of D-type cyclins after SD treatment is prevented by AIL1 overexpression. These data reveal that execution of SD-mediated growth cessation response requires the downregulation of AIL gene expression. Thus- while early acting components like PHYA and the CO/FT regulon are conserved in day-length regulation of flowering time and growth cessation between annual and perennial plants- signaling pathways downstream of SD perception diverge- with AIL transcription factors being novel targets of the CO/FT regulon connecting the perception of SD signal to the regulation of meristem activity.- Day length is a critical and robust environmental cue utilised by plants to modulate their patterns of growth to adapt to changing seasonal conditions. In perennial plants such as long-lived trees of the boreal forest- reduction in day length (short day signal/SD) induces the cessation of growth prior to the advent of winter. This developmental switch is of major adaptive significance as inability to undergo growth cessation leads to mortality in these plants. Our knowledge of how SD signal induces growth cessation is rudimentary. Here we show that AIL1 (AINTEGUMENTALIKE 1)- a plant-specific transcription factor- is a downstream target of the SD signal and can regulate the expression of key cell proliferation related genes. Intriguingly- the early acting components in day length-regulated processes such as flowering and growth cessation are conserved between annual and perennial plants. However our results show that the pathways downstream of short day perception diverge between these day length-controlled developmental transitions. These results have important implications for the evolution of the perennial life cycle and demonstrate how the same signal- namely day length- can regulate diverse developmental switches in annual and perennial plants.
    PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids
    Jason Foulks, Blood - 2009
    Platelet activating factor (PAF) and PAF-like lipids induce inflammatory responses in target cells. These lipid mediators are inactivated by PAF-acetylhydrolase (PAF-AH). The PAF signaling system affects the growth of hematopoietic CD34+ cells- but roles for PAF-AH in this process are unknown. Here- we investigated PAF-AH function during megakaryopoiesis and found that human CD34+ cells accumulate this enzymatic activity as they differentiate toward megakaryocytes- consistent with the expression of mRNA and protein for the plasma PAF-AH isoform. Inhibition of endogenous PAF-AH activity in differentiated megakaryocytes increased formation of lipid mediators that signaled the PAF receptor (PAFR) in fully differentiated human cells such as neutrophils- as well as megakaryocytes themselves. PAF-AH also controlled megakaryocyte alphaIIbBeta3-dependent adhesion- cell spreading- and mobility that relied on signaling through the PAFR. Together these data suggest that megakaryocytes generate PAF-AH to modulate the accumulation of intracellular phospholipid mediators that may detrimentally affect megakaryocyte development and function.
    Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation
    Hsin-Hung Chou, PLoS Genetics - 2009
    Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments- ranging from pathogens in the human body to phytoplankton in the oceans- imposes strong selection for improved metal acquisition systems. In this study- we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source- but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12- a cobalt-containing cofactor- to sustain two vitamin B12-dependent enzymatic reactions essential to methanol- but not succinate- metabolism. Interestingly- this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth- indicating growth-rate-dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism- suggest that certain molecular features may promote the emergence of parallel mutations- and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences.- Effects of mutations can change under different genetic backgrounds or environmental factors- also known as epistasis and genotype-by-environment interactions (G×E)- respectively. Though epistasis and G×E are traditionally treated as distinct phenomena- our study of a beneficial mutation highlights their commonality. This mutation resulted from insertion of the same transposable element upstream of a novel cobalt transport system in 30 of 32 independent populations during evolution in metal-limited media. The resulting increased cobalt uptake provided a selective benefit that depended upon two environmental factors: cobalt limitation and growth substrates whose metabolism requires a particular vitamin B12 (which contains cobalt) -dependent biochemical pathway. Furthermore- this mutation exhibited epistatic and G×E interactions with other cellular processes in a generic way- such that its selective advantage increased as cells were able to grow faster. This growth-rate dependence accords with a simple model: the slowest of multiple physiological processes needed for growth exerts the greatest control over an organism's growth rate. It suggests that as growth results from the performance of the entire physiological system- genes or environmental factors that affect distinct physiological processes may thus interact through their convergent effects on growth phenotypes.
    Reprogramming Murine Telomerase Rapidly Inhibits the Growth of Mouse Cancer Cells In vitro and In vivo
    Tong Xu, Molecular Cancer Therapeutics - 2010
    Telomerase plays a critical role in cancer- prompting the pursuit of various telomerase-based therapeutic strategies. One such strategy- telomerase interference- exploits the high telomerase activity in cancer cells and reprograms telomerase to encode “toxic� telomeres. To date- telomerase interference has been tested in human cancer cells xenografted into mice- an approach that does not recapitulate spontaneous malignancy and offers few insights about host toxicities- because human telomerase is targeted in a mouse host. To address these limitations- we designed and validated two new gene constructs specifically targeting mouse telomerase: mutant template mouse telomerase RNA (MT-mTer) and small interfering RNA against wild-type mouse telomerase RNA (alpha-mTer-siRNA). Using lentiviral delivery in mouse prostate cancer cells- we achieved alpha-mTer-siRNA-mediated knockdown of wild-type mTer (80\% depletion) and concurrent overexpression of MT-mTer (50-fold). We showed that the two constructs effectively synergize to reprogram murine telomerase to add mutant instead of wild-type telomeric repeats- resulting in rapid telomeric uncapping (5-fold increase in DNA damage foci). This- in turn- led to rapid and significant apoptosis ({\textgreater}90\% of cells) and growth inhibition in vitro (90\% reduction in viable cell mass) and in vivo (75\% reduction in tumor allograft wet weight). In summary- we have shown that mouse cancer cells are vulnerable to direct telomerase interference using novel murine telomerase-targeting constructs, this approach can now be used to study the true therapeutic potential of telomerase interference in mouse spontaneous cancer models. Mol Cancer Ther, 9(2), 438-49
    Regulation and Characterization of the ATP-Binding Cassette Transporter-B1 in the Epididymis and Epididymal Spermatozoa of the Rat
    Steven Jones, Toxicological Sciences - 2011
    It has been reported that following administration- alkylphenols- such as octylphenol- reach the testis and epididymis but fail to accumulate in these tissues- suggesting the rapid expulsion of these chemicals by transporters. Specialized transporters that function to restrict compounds that enter target cells have been identified. ABCB1 is a member of the ATP-binding cassette family of proteins capable of transporting a broad range of drugs and xenobiotics out of tissues. The objective of this study was to characterize the expression profile and functional role of ABCB1a and ABCB1b along the different regions (initial segment- caput- corpus [CS]- and cauda [CA]) of the adult rat epididymis. ABCB1a and ABCB1b transcripts were detected in all four regions of the epididymis. Immunolocalization revealed minimal ABCB1 staining in epithelial cells or spermatozoa of proximal regions of the epididymis, however- this progressively increased in the CS and CA epididymis. This expression gradient was confirmed by Western blot- suggesting that spermatozoa acquire ABCB1 during epididymal maturation. Multidrug resistance (MDR) assays revealed that rat epididymal cells and epididymal spermatozoa display an MDR phenotype that can be inhibited under control conditions. To assess whether or not the system was inducible by alkylphenols- cells from an immortalized epididymal cell line (RCE) were exposed to different concentrations of nonylphenol. Results revealed a significant induction of both ABCB1a and ABCB1b messenger RNA and ABCB1 protein in RCE cells. Our findings demonstrate a role for ABCB1 in protecting both epididymal principal cells and spermatozoa from xenobiotics.
    The Rubella Virus Capsid Is an Anti-Apoptotic Protein that Attenuates the Pore-Forming Ability of Bax
    Carolina Ilkow, PLoS Pathogens - 2011
    Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly- many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication- assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways- it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly- interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli- however- release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly- we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly- data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so- this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally- it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.- Among the variety of defense systems employed by mammalian cells to combat virus infection- apoptosis or programmed cell death is the most drastic response. Some large DNA viruses encode proteins whose sole function is to block apoptosis. Conversely- very little is known about whether RNA viruses encode analogous proteins. In many cases- RNA viruses are able to replicate before cell death occurs- which may be one reason why so little thought has been given to this topic. However- a number of RNA viruses- some of which are important human pathogens- have slow replication cycles and it stands to reason that they must block apoptosis during this time period. Here we show that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. Data from reverse genetic experiments suggest that the anti-apoptotic function of a virus-encoded protein is important for replication of an RNA virus. We anticipate that other slowly replicating RNA viruses may employ similar mechanisms and- as such- these studies have implications for development of novel anti-virals and vaccines.
    Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility
    Amelia Tomlinson, Microbiology - 2010
    The ubiquitous plant pathogen Agrobacterium tumefaciens attaches efficiently to plant tissues and abiotic surfaces and can form complex biofilms. A genetic screen for mutants unable to form biofilms on PVC identified disruptions in a homologue of the exoR gene. ExoR is a predicted periplasmic protein- originally identified in Sinorhizobium meliloti- but widely conserved among alphaproteobacteria. Disruptions in the A. tumefaciens exoR gene result in severely compromised attachment to abiotic surfaces under static and flow conditions- and to plant tissues. These mutants are hypermucoid due to elevated production of the exopolysaccharide succinoglycan- via derepression of the exo genes that direct succinoglycan synthesis. In addition- exoR mutants have lost flagellar motility- do not synthesize detectable flagellin and are diminished in flagellar gene expression. The attachment deficiency is- however- complex and not solely attributable to succinoglycan overproduction or motility disruption. A. tumefaciens ExoR can function independently of the ChvG-ChvI two component system- implicated in ExoR-dependent regulation in S. meliloti. Mutations that suppress the exoR motility defect suggest a branched regulatory pathway controlling succinoglycan synthesis- motility and biofilm formation.
    Polycomb Repressive Complex 2 and Trithorax modulate Drosophila longevity and stress resistance
    Alex Siebold, Proceedings of the National Academy of Sciences - 2010
    Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are key epigenetic regulators of global transcription programs. Their antagonistic chromatin-modifying activities modulate the expression of many genes and affect many biological processes. Here we report that heterozygous mutations in two core subunits of Polycomb Repressive Complex 2 (PRC2)- the histone H3 lysine 27 (H3K27)-specific methyltransferase E(Z) and its partner- the H3 binding protein ESC- increase longevity and reduce adult levels of trimethylated H3K27 (H3K27me3). Mutations in trithorax (trx)- a well known antagonist of Polycomb silencing- elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. Like many long-lived mutants- E(z) and esc mutants exhibit increased resistance to oxidative stress and starvation- and these phenotypes are also suppressed by trx mutations. This suppression strongly suggests that both the longevity and stress resistance phenotypes of PRC2 mutants are specifically due to their reduced levels of H3K27me3 and the consequent perturbation of Polycomb silencing. Consistent with this- long-lived E(z) mutants exhibit derepression of Abd-B- a well-characterized direct target of Polycomb silencing- and Odc1- a putative direct target implicated in stress resistance. These findings establish a role for PRC2 and TRX in the modulation of organismal longevity and stress resistance and indicate that moderate perturbation of Polycomb silencing can increase longevity.
    Meningeal Mast Cells Affect Early T Cell Central Nervous System Infiltration and Blood-Brain Barrier Integrity through TNF: A Role for Neutrophil Recruitment?
    Blayne Sayed, The Journal of Immunology - 2010
    Mast cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis- a rodent model of the human demyelinating disease multiple sclerosis. Yet their site and mode of action is unknown. In both diseases- myelin-specific T cells are initially activated in peripheral lymphoid organs. However- for disease to occur- these cells must enter the immunologically privileged CNS through a breach in the relatively impermeable blood-brain barrier. In this study- we demonstrate that a dense population of resident mast cells in the meninges- structures surrounding the brain and spinal cord- regulate basal CNS barrier function- facilitating initial T cell CNS entry. Through the expression of TNF- mast cells recruit an early wave of neutrophils to the CNS. We propose that neutrophils in turn promote the blood-brain barrier breach and together with T cells lead to further inflammatory cell influx and myelin damage. These findings provide specific targets for intervention in multiple sclerosis as well as other immune-mediated CNS diseases.
    A survey of the anti-apoptotic Bcl-2 subfamily expression in cancer types provides a platform to predict the efficacy of Bcl-2 antagonists in cancer therapy
    W Placzek, Cell Death & Disease - 2010
    We investigated the mRNA expression levels of all six antiapoptotic Bcl-2 subfamily members in 68 human cancer cell lines using qPCR techniques and measured the ability of known Bcl-2 inhibitors to induce cell death in 36 of the studied tumor cell lines. Our study reveals that Mcl-1 represents the anti-apoptotic Bcl-2 subfamily member with the highest mRNA levels in the lung- prostate- breast- ovarian- renal- and glioma cancer cell lines. In leukemia/lymphoma and melanoma cancer cell lines- Bcl-2 and Bfl-1 had the highest levels of mRNA- respectively. The observed correlation between the cell killing properties of known Bcl-2 inhibitors and the relative mRNA expression levels of anti-apoptotic Bcl-2 proteins provide critical insights into apoptosis-based anticancer strategies that target Bcl-2 proteins. Our data may explain current challenges of selective Bcl-2 inhibitors in the clinic- given that severe expression of Bcl-2 seems to be limited to leukemia cell lines. Furthermore- our data suggest that in most cancer types a strategy targeted to Mcl-1 inhibition- or combination of Bfl-1 and Mcl-1 inhibition for melanoma- may prove to be more successful than therapies targeting only Bcl-2.
    The adaptor molecule Act1 regulates BAFF responsiveness and self-reactive B cell selection during transitional B cell maturation
    Natalia Giltiay, Journal of immunology (Baltimore- Md. : 1950) - 2010
    The transitional stage is a key check-point for elimination of autoreactive B cells in the periphery. This selection process requires fine regulation of signals received through B-cell receptor (BCR) and B cell activating factor receptor (BAFFR). We previously identified the adaptor molecule Act1 as a negative regulator of BAFF-mediated signaling. Deficiency of Act1 in mice results in peripheral B cell hyperplasia and development of autoimmunity. In this study we demonstrate that Act1 plays a critical role in the regulation of transitional B cell survival and maturation. We found that the ratio of late-transitional (T2) to early-transitional (T1) cells was increased in spleens from Act1-deficient mice. Moreover- BAFF stimulation induced better T1 cell survival and promoted more efficient maturation of T1 cells into T2 cells ex vivo in the absence of Act1. BAFF stimulation induced higher levels of the anti-apoptotic Bcl2-member Mc1-l in Act1-deficient T1 than that in wild-type control cells- suggesting that Mcl1 might be one of the key effector molecules for BAFF-mediated survival in the Act1-deficient transitional B cells. Importantly- co-stimulation with BAFF was able to rescue Act1-deficient T1 cells from BCR-induced apoptosis more effectively than Act1-suffienct T1 B cells. Finally- by using double transgenic HEL mice- we demonstrated that Act1 deficiency can promote the maturation of HEL-specific autoreactive B cells. Taken together- our results suggest that the transitional stage is a critical point of action for Act1 in the elimination of autoreactive B cells and in the regulation of peripheral B cell homeostasis.
    The Fowler Syndrome-Associated Protein FLVCR2 Is an Importer of Heme
    Simon Duffy, Molecular and Cellular Biology - 2010
    Mutations in FLVCR2- a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1- have recently been associated with Fowler syndrome- a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However- the cellular function of FLVCR2 remains unresolved. Here- we report the cellular function of FLVCR2 as an importer of heme- based on the following observations. First- FLVCR2 binds to hemin-conjugated agarose- and binding is competed by free hemin. Second- mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third- heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally- cells overexpressing FLVCR2 are more sensitive to heme toxicity- a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues- including liver- placenta- brain- and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.
    Alterations in the Human Blood-Epididymis Barrier in Obstructive Azoospermia and the Development of Novel Epididymal Cell Lines from Infertile Men
    Evemie Dube, Biology of Reproduction - 2010
    Post-testicular sperm maturation requires a specific luminal environment in the epididymis that is created- in part- by the blood-epididymis barrier. There is limited information on gene expression in the epididymis of infertile obstructive azoospermia (OA) patients due to the difficulty in obtaining tissues. The objectives of this study were to determine if epididymal tight junction proteins are altered in OA and to develop cell lines that could serve to elucidate alterations in the epididymis of infertile men. Epididymal claudin (CLDN) 1- CLDN4- and CLDN10 mRNA levels were altered in OA downstream from the obstruction site. Epithelial cell lines derived from the caput epididymidis of one OA patient were developed (infertile human caput epididymal cell line [IHCE]). IHCEs were composed of homogenous populations of diploid cells that ultrastructurally resembled in vivo principal cells. The cells expressed cytokeratin- SPAG11B- CLDN2- CLDN3- desmoplakin- and vimentin. However- the cells did not express several other epididymal markers (CRISP1- SPINLW1- NPC2- CD52- or DCXR) or junctional proteins (CDH1- CDH2- CLDN1- CLDN4- CLDN7- or CLDN8). Further studies using IHCE1 and transepithelial resistance indicated that the cells were unable to form tight junctions. Microarray analyses comparing gene expression in IHCE1 and a recently developed fertile human caput epididymal cell line revealed differential expression of genes encoding junctional proteins- cell junction regulators- and epididymal proteins. Together- these data indicate that epididymal cellular junctions appear to be altered in OA.
    Ephrin-B1 forward signaling regulates craniofacial morphogenesis by controlling cell proliferation across Eph-ephrin boundaries
    Jeffrey Bush, Genes & Development - 2010
    Mutations in the X-linked human EPHRIN-B1 gene result in cleft palate and other craniofacial anomalies as part of craniofrontonasal syndrome (CFNS)- but the molecular and developmental mechanisms by which ephrin-B1 controls the underlying developmental processes are not clear. Here we demonstrate that ephrin-B1 plays an intrinsic role in palatal shelf outgrowth in the mouse by regulating cell proliferation in the anterior palatal shelf mesenchyme. In ephrin-B1 heterozygous mutants- X inactivation generates ephrin-B1-expressing and -nonexpressing cells that sort out- resulting in mosaic ephrin-B1 expression. We now show that this process leads to mosaic disruption of cell proliferation and post-transcriptional up-regulation of EphB receptor expression through relief of endocytosis and degradation. The alteration in proliferation rates resulting from ectopic Eph-ephrin expression boundaries correlates with the more severe dysmorphogenesis of ephrin-B1+/- heterozygotes that is a hallmark of CFNS. Finally- by integrating phosphoproteomic and transcriptomic approaches- we show that ephrin-B1 controls proliferation in the palate by regulating the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signal transduction pathway.
    Formate Acts as a Diffusible Signal To Induce Salmonella Invasion
    Yanyan Huang, Journal of Bacteriology - 2008
    To infect an animal host- Salmonella enterica serovar Typhimurium must penetrate the intestinal epithelial barrier. This process of invasion requires a type III secretion system encoded within Salmonella pathogenicity island I (SPI1). We found that a mutant with deletions of the acetate kinase and phosphotransacetylase genes (ackA-pta) was deficient in invasion and SPI1 expression but that invasion gene expression was completely restored by supplying medium conditioned by growth of the wild-type strain- suggesting that a signal produced by the wild type- but not by the ackA-pta mutant- was required for invasion. This mutant also excreted 68-fold-less formate into the culture medium- and the addition of sodium formate to cultures restored both the expression of SPI1 and the invasion of cultured epithelial cells by the mutant. The effect of formate was pH dependent- requiring a pH below neutrality- and studies in mice showed that the distal ileum- the preferred site of Salmonella invasion in this species- had the appropriate formate concentration and pH to elicit invasion- while the cecum contained no detectable formate. Furthermore- we found that formate affected the major regulators of SPI1- hilA and hilD- but that the primary routes of formate metabolism played no role in its activity as a signal.
    Three New O-Methyltransferases Are Sufficient for All O-Methylation Reactions of Ipecac Alkaloid Biosynthesis in Root Culture of Psychotria ipecacuanha
    Taiji Nomura, Journal of Biological Chemistry - 2010
    The medicinal plant Psychotria ipecacuanha produces ipecac alkaloids- a series of monoterpenoid-isoquinoline alkaloids such as emetine and cephaeline- whose biosynthesis derives from condensation of dopamine and secologanin. Here- we identified three cDNAs- IpeOMT1-IpeOMT3- encoding ipecac alkaloid O-methyltransferases (OMTs) from P. ipecacuanha. They were coordinately transcribed with the recently identified ipecac alkaloid Beta-glucosidase Ipeglu1. Their amino acid sequences were closely related to each other and rather to the flavonoid OMTs than to the OMTs involved in benzylisoquinoline alkaloid biosynthesis. Characterization of the recombinant IpeOMT enzymes with integration of the enzymatic properties of the IpeGlu1 revealed that emetine biosynthesis branches off from N-deacetylisoipecoside through its 6-O-methylation by IpeOMT1- with a minor contribution by IpeOMT2- followed by deglucosylation by IpeGlu1. The 7-hydroxy group of the isoquinoline skeleton of the aglycon is methylated by IpeOMT3 prior to the formation of protoemetine that is condensed with a second dopamine molecule- followed by sequential O-methylations by IpeOMT2 and IpeOMT1 to form cephaeline and emetine- respectively. In addition to this central pathway of ipecac alkaloid biosynthesis- formation of all methyl derivatives of ipecac alkaloids in P. ipecacuanha could be explained by the enzymatic activities of IpeOMT1-IpeOMT3- indicating that they are sufficient for all O-methylation reactions of ipecac alkaloid biosynthesis.
    Leishmania donovani Amastigotes Impair Gamma Interferon-Induced STAT1alpha Nuclear Translocation by Blocking the Interaction between STAT1alpha and Importin-alpha5
    Christine Matte, Infection and Immunity - 2010
    The protozoan parasite Leishmania donovani- the etiological agent of visceral leishmaniasis- is renowned for its capacity to sabotage macrophage functions and signaling pathways stimulated by activators such as gamma interferon (IFN-gamma). Our knowledge of the strategies utilized by L. donovani to impair macrophage responsiveness to IFN-gamma remains fragmentary. In the present study- we investigated the impact of an infection by the amastigote stage of L. donovani on IFN-gamma responses and signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in mouse bone marrow-derived macrophages. The levels of IFN-gamma-induced expression of major histocompatibility complex class II and inducible nitric oxide synthase (iNOS) were strongly reduced in L. donovani amastigote-infected macrophages. As the expression of those genes is mediated by the transcription factors STAT1alpha and IFN regulatory factor 1 (IRF-1)- we investigated their activation in amastigote-infected macrophages treated with IFN-gamma. We found that whereas STAT1alpha protein levels and the levels of phosphorylation on Tyr701 and Ser727 were normal- IRF-1 expression was inhibited in infected macrophages. This inhibition of IRF-1 expression correlated with a defective nuclear translocation of STAT1alpha- and further analyses revealed that the IFN-gamma-induced STAT1alpha association with the nuclear transport adaptor importin-alpha5 was compromised in L. donovani amastigote-infected macrophages. Taken together- our results provide evidence for a novel mechanism used by L. donovani amastigotes to interfere with IFN-gamma-activated macrophage functions and provide a better understanding of the strategies deployed by this parasite to ensure its intracellular survival.
    Genes Involved in Long-Chain Alkene Biosynthesis in Micrococcus luteus
    Harry Beller, Applied and Environmental Microbiology - 2010
    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels- as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698- a close relative of Sarcina lutea (now Kocuria rhizophila)- which 4 decades ago was reported to biosynthesize iso- and anteiso-branched- long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut\_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes- predominantly 27:3 and 29:3 (no. carbon atoms: no. Cï¼?C bonds). Heterologous expression of Mlut\_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones- predominantly 27:2- and in vitro studies with the purified Mlut\_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut\_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step- which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut\_13230 (OleA) to FabH (Beta-ketoacyl-ACP synthase III)- which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.
    A Novel Sphingosine Kinase Inhibitor Induces Autophagy in Tumor Cells
    Vladimir Beljanski, Journal of Pharmacology and Experimental Therapeutics - 2010
    The sphingolipids ceramide- sphingosine- and sphingosine 1-phosphate (S1P) regulate cell signaling- proliferation- apoptosis- and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P- shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage- morphological changes in lysosomes- formation of autophagosomes- and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine- an inhibitor of autophagy- switched the mechanism of toxicity to apoptosis- but decreased the potency of the SK2 inhibitor- indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts- daily administration of ABC294640 delayed tumor growth and elevated autophagy markers- but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy- which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently- ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.
    Innate immune processes are sufficient for driving silicosis in mice
    Celine Beamer, Journal of Leukocyte Biology - 2010
    The lung is constantly exposed to potentially pathogenic particles and microorganisms. It has become evident recently that not only innate but also adaptive immune responses to particulates- such as SiO2 entering the respiratory tract- are complex and dynamic events. Although the cellular mechanisms and anatomical consequences involved in the development of silicosis have been studied extensively- they still remain poorly understood. Based on their capacity for immune regulation- lymphocytes may play a key role in the respiratory response to environmental challenge by SiO2. The objective of this study was to characterize the impact of SiO2 exposure on respiratory immune processes- with particular emphasis on evaluating the importance of lymphocytes in the murine silicosis model. Therefore- lymphopenic mice- including NK-deficient- Rag1-/-- or a combination (Rag1-/- NK-depleted)- were used and demonstrated that SiO2-induced fibrosis and inflammation can occur independently of T- B- NK T- and NK cells. Studies in Rag1-/- mice suggest further that lymphocytes may participate in the regulation of SiO2-induced inflammation through modulation of the Nalp3 inflammasome. This observation may have clinical relevance in the treatment of inflammatory and fibrotic lung diseases that are refractory or respond suboptimally to current therapeutics.
    Men supplemented with 17Beta-estradiol have increased Beta-oxidation capacity in skeletal muscle
    Amy Maher, Physiological Genomics - 2010
    During endurance exercise women have lower carbohydrate and higher lipid oxidation compared with men. Supplementation of humans and rodents with 17Beta-estradiol (E2) lowers the respiratory exchange ratio- the glucose rate of appearance and disappearance- and the metabolic clearance rate. The mechanism(s) for the observed estrogen effects in substrate utilization remains to be determined. We hypothesized that estrogen would increase the mRNA and protein content for genes involved in the regulation of Beta-oxidation. Ten moderately active men were supplemented with placebo or E2 for 8 days in a randomized double-blind crossover design. After supplementation muscle biopsies were obtained from the vastus lateralis and examined for differences in mRNA- microRNA- and protein content of genes involved in lipid oxidation. E2 increased the protein abundance of medium-chain acyl-CoA dehydrogenase (MCAD) 42\% (P ≤ 0.05). Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) mRNA was significantly higher after E2 supplementation by 29\% (P ≤ 0.05)- and microRNA miR-29b (predicted to regulate PGC-1alpha) was significantly lower by 66\% (P ≤ 0.05). In conclusion- E2 might partially regulate lipid metabolism in skeletal muscle by altering the protein content of MCAD- which may be directly or indirectly regulated by an increase in PGC-1alpha and reduction in miR-29b.
    The Capsid-Binding Nucleolar Helicase DDX56 Is Important for Infectivity of West Nile Virus
    Zaikun Xu, Journal of Virology - 2011
    Recent findings suggest that in addition to its role in packaging genomic RNA- the West Nile virus (WNV) capsid protein is an important pathogenic determinant- a scenario that requires interaction of this viral protein with host cell proteins. We performed an extensive multitissue yeast two-hybrid screen to identify capsid-binding proteins in human cells. Here we describe the interaction between WNV capsid and the nucleolar RNA helicase DDX56/NOH61. Coimmunoprecipitation confirmed that capsid protein binds to DDX56 in infected cells and that this interaction is not dependent upon intact RNA. Interestingly- WNV infection induced the relocalization of DDX56 from the nucleolus to a compartment in the cytoplasm that also contained capsid protein. This phenomenon was apparently specific for WNV- as DDX56 remained in the nucleoli of cells infected with rubella and dengue 2 viruses. Further analyses showed that DDX56 is not required for replication of WNV, however- virions secreted from DDX56-depleted cells contained less viral RNA and were 100 times less infectious. Together- these data suggest that DDX56 is required for assembly of infectious WNV particles.
    IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-?1 resulting in epithelial to mesenchymal transition
    Logan Walsh, Cell Communication and Signaling : CCS - 2011
    Introduction TGF-Beta signaling has been extensively studied in many developmental contexts- amongst which is its ability to induce epithelial to mesenchymal transitions (EMT). EMTs play crucial roles during embryonic development and have also come under intense scrutiny as a mechanism through which breast cancers progress to become metastatic. Interestingly- while the molecular hallmarks of EMT progression (loss of cell adhesion- nuclear localization of Beta-catenin) are straightforward- the cellular signaling cascades that result in an EMT are numerous and diverse. Furthermore- most studies describing the biological effects of TGF-Beta have been performed using high concentrations of active- soluble TGF-Beta- despite the fact that TGF-Beta is produced and secreted as a latent complex. Methods MCF-7 breast cancer cells treated with recombinant IGF-1 were assayed for metalloproteinase activity and invasiveness through a matrigel coated transwell invasion chamber. IGF-1 treatments were then followed by the addition of latent-TGF-Beta1 to determine if elevated levels of IGF-1 together with latent-TGF-Beta1 could cause EMT. Results Results showed that IGF-1 - a molecule known to be elevated in breast cancer is a regulator of matrix metalloproteinase activity (MMP) and the invasive potential of MCF-7 breast cancer cells. The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways. In addition- increased IGF-1- together with latent TGF-Beta1 and active MMPs result in EMT. Conclusions Taken together our data suggest a novel a link between IGF-1 levels- MMP activity- TGF-Beta signaling- and EMT in breast cancer cells.
    Hsp90 Regulates the Function of Argonaute 2 and Its Recruitment to Stress Granules and P-Bodies
    Justin Pare, Molecular Biology of the Cell - 2009
    Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study- we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore- pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs- but interestingly- protein activator of the double-stranded RNA-activated protein kinase (PACT)- another Dicer cofactor- is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together- our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover- we propose that Ago2 and PACT form a complex that functions at the level of SGs.
    Inhibition of Hepatic Organic Anion-Transporting Polypeptide by RNA Interference in Sandwich-Cultured Human Hepatocytes: An In Vitro Model to Assess Transporter-Mediated Drug-Drug Interactions
    Mingxiang Liao, Drug Metabolism and Disposition - 2010
    Organic anion-transporting polypeptides (OATPs)- members of the SLCO/SLC21 family- mediate the transport of various endo- and xenobiotics. In human liver- OATP1B1- 1B3- and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However- there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study- a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction- microarray and immunoblotting analyses- along with uptake assays- illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30\% of the total uptake of cerivastatin into human hepatocytes- it caused a 50\% reduction in cerivastatin metabolism- which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.
    Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification
    Rahul Kohli, Journal of Biological Chemistry - 2010
    Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses- playing roles as target-directed- purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity- whereas its close relative- APOBEC3G (A3G)- lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential- as mistargeting poses significant risks- potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T- R = A/G) motifs- whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single- divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop- we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting- therefore- appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably- AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute- our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.
    Mast Cell-derived Prostaglandin D2 Controls Hyaluronan Synthesis in Human Orbital Fibroblasts via DP1 Activation IMPLICATIONS FOR THYROID EYE DISEASE
    Naxin Guo, Journal of Biological Chemistry - 2010
    Thyroid eye disease (TED) is a debilitating disorder characterized by the accumulation of adipocytes and hyaluronan (HA). Production of HA by fibroblasts leads to remarkable increases in tissue volume and to the anterior displacement of the eyes. Prostaglandin D2 (PGD2)- mainly produced by mast cells- promotes orbital fibroblast adipogenesis. The mechanism by which PGD2 influences orbital fibroblasts and their synthesis of HA is poorly understood. We report here that mast cell-derived PGD2 is a key factor that promotes HA biosynthesis by orbital fibroblasts. Primary orbital fibroblasts from TED patients were isolated and used to test the effects of PGD2- prostaglandin J2- as well as prostaglandin D receptor (DP) agonists and antagonists on HA synthesis. The expression of HA synthase (HAS)- hyaluronidase- DP1- and DP2 mRNA levels was assessed by PCR. Small interfering RNAs against HAS1 or HAS2 were used to assess the importance of HAS isoforms on HA production. Treatment of human orbital fibroblasts with PGD2 and PGJ2 increased HA synthesis and HAS mRNA. HAS2 was the dominant isoform responsible for HA production by PGD2. The effect of PGD2 on HA production was mimicked by the selective DP1 agonist BW245C. The DP1 antagonist MK-0524 completely blocked PGD2-induced HA synthesis. Human mast cells (HMC-1) produced PGD2. Co-culture of HMC-1 cells with orbital fibroblasts induced HA production and inhibition of mast cell-derived PGD2 prevented HA synthesis. Mast cell-derived PGD2 increased HA production via activation of DP1. Selectively targeting the production of PGD2 and/or activation of DP1 may prevent pathological changes associated with TED.
    Circulating MicroRNA-208b and MicroRNA-499 Reflect Myocardial Damage in Cardiovascular Disease
    Maarten Corsten, Circulation: Cardiovascular Genetics - 2010
    Background— Small RNA molecules- called microRNAs- freely circulate in human plasma and correlate with varying pathologies. In this study- we explored their diagnostic potential in a selection of prevalent cardiovascular disorders. Methods and Results— MicroRNAs were isolated from plasmas from well-characterized patients with varying degrees of cardiac damage: (1) acute myocardial infarction- (2) viral myocarditis- (3) diastolic dysfunction- and (4) acute heart failure. Plasma levels of selected microRNAs- including heart-associated (miR-1- -133a- -208b- and -499)- fibrosis-associated (miR-21 and miR-29b)- and leukocyte-associated (miR-146- -155- and -223) candidates- were subsequently assessed using real-time polymerase chain reaction. Strikingly- in plasma from acute myocardial infarction patients- cardiac myocyte-associated miR-208b and -499 were highly elevated- 1600-fold (P{\textless}0.005) and 100-fold (P{\textless}0.0005)- respectively- as compared with control subjects. Receiver operating characteristic curve analysis revealed an area under the curve of 0.94 (P{\textless}10-10) for miR-208b and 0.92 (P{\textless}10-9) for miR-499. Both microRNAs correlated with plasma troponin T- indicating release of microRNAs from injured cardiomyocytes. In viral myocarditis- we observed a milder but significant elevation of these microRNAs- 30-fold and 6-fold- respectively. Plasma levels of leukocyte-expressed microRNAs were not significantly increased in acute myocardial infarction or viral myocarditis patients- despite elevated white blood cell counts. In patients with acute heart failure- only miR-499 was significantly elevated (2-fold)- whereas no significant changes in microRNAs studied could be observed in diastolic dysfunction. Remarkably- plasma microRNA levels were not affected by a wide range of clinical confounders- including age- sex- body mass index- kidney function- systolic blood pressure- and white blood cell count. Conclusions— Cardiac damage initiates the detectable release of cardiomyocyte-specific microRNAs-208b and -499 into the circulation.
    Identification of a High-Affinity Ligand That Exhibits Complete Aryl Hydrocarbon Receptor Antagonism
    Kayla Smith, Journal of Pharmacology and Experimental Therapeutics - 2011
    The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity- e.g.- 2-3-7-8-tetrachlorodibenzo-p-dioxin and alpha-naphthoflavone- have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast- the recently identified selective AHR modulators (SAhRMs)- e.g.- 1-allyl-3-(3-4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360)- bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here- we present evidence for a third class of AHR ligand- “pure� or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions- which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays- in conjunction with quantitative polymerase chain reaction analysis of AHR targets- reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore- unlike many currently used AHR antagonists- e.g.- alpha-naphthoflavone- GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function- i.e.- suppression of cytokine-induced acute-phase gene expression- GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a pure antagonist with the capacity to inhibit both DRE-dependent and -independent activity.
    Flow-dependent Smad2 phosphorylation and TGIF nuclear localization in human aortic endothelial cells
    Robert Shepherd, American Journal of Physiology - Heart and Circulatory Physiology - 2011
    Endothelial cells respond to fluid flow stimulation through transient and sustained signal pathway activation. Smad2 is a signaling molecule and transcription factor in the Smad signaling pathway- traditionally associated with TGF-Beta. Although phosphorylation of Smad2 in the receptor-dependent COOH-terminal region is the most appreciated way Smad2 is activated to affect gene expression- phosphorylation may also occur in the MH1-MH2 linker region (L-psmad2). Here- we show that in human aortic endothelial cells (HAEC)- Smad2 was both preferentially phosphorylated in the linker region and localized to the nucleus in a flow-dependent manner. The Smad corepressor transforming growth interacting factor (TGIF) was also found to have flow-dependent nuclear localization. Tissue studies confirmed this L-psmad2 generation trend in rat aorta- indicating likely importance in arterial tissue. HAEC-based inhibitor studies demonstrated that L-psmad2 levels were not related to MAPK phosphorylation- but instead followed the pattern of pAkt473- both with and without the phosphatidylinositol 3-kinase inhibitor PI-103. Akt and Smad species were also shown to directly interact under flow relative to static controls. To further evaluate impacts of PI-103 treatment- expression profiles for two TGF-Beta and shear stress-dependent genes were determined and showed that mRNAs were lower from untreated 10 dyn/cm2 than 2 dyn/cm2 average shear stress cultures. However- upon exposure to PI-103- this trend was reversed- with a stronger response observed at 10 dyn/cm2. Taken together- the results of this work suggest that fluid flow exposure may influence endothelial gene expression by a novel mechanism involving Akt- L-psmad2- and TGIF.
    The ROOT DETERMINED NODULATION1 Gene Regulates Nodule Number in Roots of Medicago truncatula and Defines a Highly Conserved- Uncharacterized Plant Gene Family
    Elise Schnabel, Plant Physiology - 2011
    The formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. A screen for supernodulating Medicago truncatula mutants defective in this regulatory behavior yielded loss-of-function alleles of a gene designated ROOT DETERMINED NODULATION1 (RDN1). Grafting experiments demonstrated that RDN1 regulatory function occurs in the roots- not the shoots- and is essential for normal nodule number regulation. The RDN1 gene- Medtr5g089520- was identified by genetic mapping- transcript profiling- and phenotypic rescue by expression of the wild-type gene in rdn1 mutants. A mutation in a putative RDN1 ortholog was also identified in the supernodulating nod3 mutant of pea (Pisum sativum). RDN1 is predicted to encode a 357-amino acid protein of unknown function. The RDN1 promoter drives expression in the vascular cylinder- suggesting RDN1 may be involved in initiating- responding to- or transporting vascular signals. RDN1 is a member of a small- uncharacterized- highly conserved gene family unique to green plants- including algae- that we have named the RDN family.
    Exercise Increases Mitochondrial PGC-1alpha Content and Promotes Nuclear-Mitochondrial Cross-talk to Coordinate Mitochondrial Biogenesis
    Adeel Safdar, Journal of Biological Chemistry - 2011
    Endurance exercise is known to induce metabolic adaptations in skeletal muscle via activation of the transcriptional co-activator peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha). PGC-1alpha regulates mitochondrial biogenesis via regulating transcription of nuclear-encoded mitochondrial genes. Recently- PGC-1alpha has been shown to reside in mitochondria, however- the physiological consequences of mitochondrial PGC-1alpha remain unknown. We sought to delineate if an acute bout of endurance exercise can mediate an increase in mitochondrial PGC-1alpha content where it may co-activate mitochondrial transcription factor A to promote mtDNA transcription. C57Bl/6J mice (n = 12/group, ♀ = ♂) were randomly assigned to sedentary (SED)- forced-endurance (END) exercise (15 m/min for 90 min)- or forced endurance +3 h of recovery (END+3h) group. The END group was sacrificed immediately after exercise- whereas the SED and END+3h groups were euthanized 3 h after acute exercise. Acute exercise coordinately increased the mRNA expression of nuclear and mitochondrial DNA-encoded mitochondrial transcripts. Nuclear and mitochondrial abundance of PGC-1alpha in END and END+3h groups was significantly higher versus SED mice. In mitochondria- PGC-1alpha is in a complex with mitochondrial transcription factor A at mtDNA D-loop- and this interaction was positively modulated by exercise- similar to the increased binding of PGC-1alpha at the NRF-1 promoter. We conclude that in response to acute altered energy demands- PGC-1alpha re-localizes into nuclear and mitochondrial compartments where it functions as a transcriptional co-activator for both nuclear and mitochondrial DNA transcription factors. These results suggest that PGC-1alpha may dynamically facilitate nuclear-mitochondrial DNA cross-talk to promote net mitochondrial biogenesis.
    Pharmacologic Inhibition of ALK5 Causes Selective Induction of Terminal Differentiation in Mouse Keratinocytes Expressing Oncogenic HRAS
    Lauren Markell, Molecular Cancer Research - 2011
    TGFBeta has both tumor suppressive and oncogenic roles in cancer development. We previously showed that SB431542 (SB)- a small molecule inhibitor of the TGFBeta type I receptor (ALK5) kinase- suppressed benign epidermal tumor formation but enhanced malignant conversion. Here- we show that SB treatment of primary K5rTA/tetORASV12G bitransgenic keratinocytes did not alter HRASV12G-induced keratinocyte hyperproliferation. However- continuous SB treatment significantly enhanced HRASV12G-induced cornified envelope formation and cell death linked to increased expression of enzymes transglutaminase (TGM) 1 and TGM3 and constituents of the cornified envelope small proline-rich protein (SPR) 1A and SPR2H. In contrast- TGFBeta1 suppressed cornified envelope formation in HRASV12G keratinocytes. Similar results were obtained in HRASV12G transgenic mice treated topically with SB or by coexpressing TGFBeta1 and HRASV12G in the epidermis. Despite significant cell death- SB-resistant HRASV12G keratinocytes repopulated the primary culture that had overcome HRas-induced senescence. These cells expressed reduced levels of p16ink4a and were growth stimulated by SB but remained sensitive to a calcium-induced growth arrest. Together these results suggest that differential responsiveness to cornification may represent a mechanism by which pharmacologic blockade of TGFBeta signaling can inhibit the outgrowth of preneoplastic lesions but may cause a more progressed phenotype in a separate keratinocyte population. Mol Cancer Res, 9(6), 746-56. ©2011 AACR.
    Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells
    Mohammad Mandegar, Human Molecular Genetics - 2011
    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC)- which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore- and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines- but never in stem cells- thus limiting their potential therapeutic application. In this work- we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency- which were stably maintained without selection for 3 months. Importantly- no integration of the HAC DNA was observed in the hESc lines- compared with the fibrosarcoma-derived control cells- where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency- differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc- and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications.
    Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration
    Yann Malato, The Journal of Clinical Investigation - 2011
    Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury, rather- they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition- other evidence suggests that hepatocytes are capable of lineage conversion- acting as precursors of biliary epithelial cells during biliary injury. To test these concepts- we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further- we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition- our new vector system will be a valuable tool for timed- efficient- and specific loop out of floxed sequences in hepatocytes.
    Transstadial Transmission of Francisella tularensis holarctica in Mosquitoes- Sweden
    Jan Lundstrom, Emerging Infectious Diseases - 2011
    In Sweden- human cases of tularemia caused by Francisella tularensis holarctica are assumed to be transmitted by mosquitoes- but how mosquito vectors acquire and transmit the bacterium is not clear. To determine how transmission of this bacterium occurs- mosquito larvae were collected in an area where tularemia is endemic- brought to the laboratory- and reared to adults in their original pond water. Screening of adult mosquitoes by real-time PCR demonstrated F. tularensis lpnA sequences in 14 of the 48 mosquito pools tested, lpnA sequences were demonstrated in 6 of 9 identified mosquito species. Further analysis confirmed the presence of F. tularensis holarctica-specific 30-bp deletion region sequences (FtM19inDel) in water from breeding containers and in 3 mosquito species (Aedes sticticus- Ae. vexans- and Ae. punctor) known to take blood from humans. Our results suggest that the mosquitoes that transmit F. tularensis holarctica during tularemia outbreaks acquire the bacterium already as larvae.
    An acute bout of high-intensity interval training increases the nuclear abundance of PGC-1alpha and activates mitochondrial biogenesis in human skeletal muscle
    Jonathan Little, American Journal of Physiology - Regulatory- Integrative and Comparative Physiology - 2011
    Low-volume- high-intensity interval training (HIT) increases skeletal muscle mitochondrial capacity- yet little is known regarding potential mechanisms promoting this adaptive response. Our purpose was to examine molecular processes involved in mitochondrial biogenesis in human skeletal muscle in response to an acute bout of HIT. Eight healthy men performed 4 × 30-s bursts of all-out maximal intensity cycling interspersed with 4 min of rest. Muscle biopsy samples (vastus lateralis) were obtained immediately before and after exercise- and after 3 and 24 h of recovery. At rest- the majority of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha- a master regulator of mitochondrial biogenesis- was detected in cytosolic fractions. Exercise activated p38 MAPK and AMPK in the cytosol. Nuclear PGC-1alpha protein increased 3 h into recovery from exercise- a time point that coincided with increased mRNA expression of mitochondrial genes. This was followed by an increase in mitochondrial protein content and enzyme activity after 24 h of recovery. These findings support the hypothesis that an acute bout of low-volume HIT activates mitochondrial biogenesis through a mechanism involving increased nuclear abundance of PGC-1alpha.
    A Novel Aminosaccharide Compound Blocks Immune Responses by Toll-like Receptors and Nucleotide-binding Domain- Leucine-rich Repeat Proteins
    Kyoung-Hee Lee, Journal of Biological Chemistry - 2011
    Toll-like receptors (TLRs) and nucleotide-binding domain- leucine-rich repeat (NLR) proteins are two major forms of innate immune receptors that trigger inflammatory responses by various biological mechanisms such as cytokine production- recruitment of inflammatory cells- or activation of adaptive immunity. Although the innate immune system is designed to fight against infectious pathogens- excessive activation of TLR or NLR signaling pathways may lead to unwarranted inflammation with hazardous outcomes- including septic shock or inflammatory diseases. As part of the search for effective therapeutics to regulate these responses- here we show that a novel aminosaccharide compound- named DFK1012- inhibits immune responses caused by TLR and NLR activation. Treatment with DFK1012- but not its derivatives DFK845 or DFK846- strongly inhibited pro-inflammatory cytokine production upon stimulation via either TLR or NLR proteins in macrophages. Importantly- we have not observed cytotoxicity in any range of its working concentration. Treatment with DFK1012 did not interfere with TLR- or NLR-induced activation of p38 and JNK- phosphorylation/degradation of IkB- and subsequent nuclear translocation of NF-kB subunit p65- suggesting that the inhibitory activity of DFK1012 is not due to the suppression of downstream signaling. Indeed- DFK1012 did not impair transcription of pro-inflammatory cytokine genes but rather promoted post-translational degradation of pro-inflammatory cytokines. Therefore- DFK1012 is a novel anti-inflammatory compound that drives proteolysis of proinflammatory cytokines induced by TLR and NLR stimulation. DFK1012 may represent a novel class of potential therapeutic agents aimed at the treatment of inflammatory disorders.
    2-O-sulfotransferase regulates Wnt signaling- cell adhesion and cell cycle during zebrafish epiboly
    Erin Cadwalader, Development - 2012
    O-sulfotransferases modify heparan sulfate proteoglycans (HSPGs) by catalyzing the transfer of a sulfate to a specific position on heparan sulfate glycosaminoglycan (GAG) chains. Although the roles of specific HSPG modifications have been described in cell culture and invertebrates- little is known about their functions or abilities to modulate specific cell signaling pathways in vertebrate development. Here- we report that 2-O-sulfotransferase (2-OST) is an essential component of canonical Wnt signaling in zebrafish development. 2-OST-defecient embryos have reduced GAG chain sulfation and are refractory to exogenous Wnt8 overexpression. Embryos in which maternally encoded 2-OST is knocked down have normal activation of several zygotic mesoderm- endoderm and ectoderm patterning genes- but have decreased deep cell adhesion and fail to initiate epiboly- which can be rescued by re-expression of 2-OST protein. Reduced cell adhesion and altered cell cycle regulation in 2-OST-deficient embryos are associated with decreased Beta-catenin and E-cadherin protein levels at cell junctions- and these defects can be rescued by reactivation of the intracellular Wnt pathway- utilizing stabilized Beta-catenin or dominant-negative Gsk3- but not by overexpression of Wnt8 ligand. Together- these results indicate that 2-OST functions within the Wnt pathway- downstream of Wnt ligand signaling and upstream of Gsk3Beta and Beta-catenin intracellular localization and function.
    Genistein Affects Histone Modifications on Dickkopf-Related Protein 1 (DKK1) Gene in SW480 Human Colon Cancer Cell Line
    Huan Wang, PLoS ONE - 2012
    Genistein (GEN) is a plant-derived isoflavone and can block uncontrolled cell growth in colon cancer by inhibiting the WNT signaling pathway. This study aimed to test the hypothesis that the enhanced gene expression of the WNT signaling pathway antagonist- DKK1 by genistein treatment is associated with epigenetic modifications of the gene in colon cancer cells. Genistein treatment induced a concentration-dependent G2 phase arrest in the human colon cancer cell line SW480 and reduced cell proliferation. Results from several other human colon cancer cell lines confirmed the growth inhibitory effects of genistein. Overexpression of DKK1 confirmed its involvement in growth inhibition. Knockdown of DKK1 expression by siRNA slightly induced cell growth. DKK1 gene expression was increased by genistein in SW480 and HCT15 cells. DNA methylation at the DKK1 promoter was not affected by genistein treatment in all the cell lines tested. On the other hand- genistein induced histone H3 acetylation of the DKK1 promoter region in SW480 and HCT15 cells. This indicates that increased histone acetylation is associated with the genistein-induced DKK1 expression. The association between histone acetylation and DKK1 gene expression is confirmed by the histone deacetylase inhibitor trichostatin A (TSA) treatment. In conclusion- genistein treatment decreases cell growth and proliferation in colon cancer cell lines. The effect is associated with the increased DKK1 expression through the induction of histone acetylation at the DKK1 promoter region.
    Sedation or Inhalant Anesthesia before Euthanasia with CO2 Does Not Reduce Behavioral or Physiologic Signs of Pain and Stress in Mice
    Helen Valentine, Journal of the American Association for Laboratory Animal Science : JAALAS - 2012
    CO2 administration is a common euthanasia method for research mice- yet questions remain regarding whether CO2 euthanasia is associated with pain and stress. Here we assessed whether premedication with acepromazine- midazolam- or anesthetic induction with isoflurane altered behavioral and physiologic parameters that may reflect pain or stress during CO2 euthanasia. Mice were assigned to 1 of 6 euthanasia groups: CO2 only at a flow rate of 1.2 L/min which displaces 20\% of the cage volume per minute (V/min, control group), premedication with acepromazine (5 mg/kg)- midazolam (5 mg/kg)- or saline followed by 20\% V/min CO2, induction with 5\% isoflurane followed by greater than 100\% V/min CO2 ({\textgreater}6L/min), and 100\% V/min CO2 only (6 L/min). Measures included ultrasonic sound recordings- behavioral analysis of video recordings- plasma ACTH and corticosterone levels immediately after euthanasia- and quantification of c-fos from brain tissue. Compared with 20\% V/min CO2 alone- premedication with acepromazine or midazolam did not significantly alter behavior but did induce significantly higher c-fos expression in the brain. Furthermore- the use of isoflurane induction prior to CO2 euthanasia significantly increased both behavioral and neuromolecular signs of stress. The data indicate that compared with other modalities- 20\% V/min CO2 alone resulted in the least evidence of stress in mice and therefore was the most humane euthanasia method identified in the current study.
    Role of the Ah Receptor in Homeostatic Control of Fatty Acid Synthesis in the Liver
    Rachel Tanos, Toxicological Sciences - 2012
    We have previously demonstrated a role for the aryl hydrocarbon receptor (AHR) in the attenuation of the cholesterol biosynthesis pathway. This regulation did not require that the AHR binds to its cognate response element. Based on these observations and other reports depicting a role for AHR in lipid metabolism- we chose to investigate the involvement of the receptor in the regulation of the fatty acid synthesis pathway in mice and humans. For this purpose- C57BL/6J- liver-specific transgenic DRE-binding mutant AhR (A78D-Ahr Ttr Cre Alb Ahr fx/fx) and Cre Alb Ahr fx/fx mice were treated with an AHR ligand- and hepatic mRNA expression levels of key fatty acid genes (e.g.- Acaca- Fasn- Scd1) were measured. The basal levels of those genes were also compared between C57BL6/J and hepatic AHR-deficient mice- as well as between Ah b and Ah d congenic mice. To extend these results to humans- fatty acid gene expression in human cells were compared with AHR-silenced cells. In addition- primary human hepatocytes were treated with an AHR ligand to assess alterations in gene expression and fatty acid synthesis. These studies indicated that the AHR constitutively attenuates the expression of key fatty acid synthesis genes in the absence of binding to its cognate response element. In addition- activation of AHR led to further repression of the expression of these genes and a decrease in overall fatty acid synthesis and secretion in human hepatocytes. Based on our results- we can conclude that increased AHR activity represses fatty acid synthesis- suggesting it may be a future therapeutic target.
    Aryl Hydrocarbon Receptor (AhR) Regulates Silica-Induced Inflammation But Not Fibrosis
    Celine Beamer, Toxicological Sciences - 2012
    The aryl hydrocarbon receptor (AhR)- a ligand-activated transcription factor- is responsible for mediating a variety of pharmacological and toxicological effects caused by halogenated aromatic hydrocarbons such as 2-3-7-8-tetrachlorodibenzo-p-dioxin (TCDD). However- recent evidence has revealed that the AhR also has numerous physiological roles aside from xenobiotic metabolism- including regulation of immune and inflammatory signaling as well as normal development and homeostasis of several organs. To investigate the role of the AhR in crystalline silica (SiO2)-induced inflammation and fibrosis- C57Bl/6 and AhR-/- mice were exposed to SiO2 or vehicle. Similarly- C57Bl/6 mice were exposed to SiO2 and TCDD either simultaneously or sequentially to assess whether AhR activation alters inflammation and fibrosis. SiO2-induced acute lung inflammation was more severe in AhR-/- mice, however- the fibrotic response of AhR-/- mice was attenuated compared with C57Bl/6 mice. In a model of chronic SiO2 exposure- AhR activation by TCDD in C57Bl/6 mice resulted in reduced inflammation, however- the fibrotic response was not affected. Bone marrow-derived macrophages (BMM) from AhR-/- mice also produced higher levels of cytokines and chemokines in response to SiO2. Analysis of gene expression revealed that BMM derived from AhR-/- mice exhibit increased levels of pro-interleukin (IL)-1Beta- IL-6- and Bcl-2- yet decreased levels of signal transducers and activators of transcription (STAT)2- STAT5a- and serpin B2 (Pai-2) in response to SiO2.
    Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei
    Antonio Barquilla, Proceedings of the National Academy of Sciences - 2012
    African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms- the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases- TbTOR1 and TbTOR2- and two TOR complexes- TbTORC1 and TbTORC2. Surprisingly- two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor)- a major vault protein- and LST8 to form a unique TOR complex- TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.
    Deficiency of Angiotensin Type 1a Receptors in Adipocytes Reduces Differentiation and Promotes Hypertrophy of Adipocytes in Lean Mice
    Kelly Putnam, Endocrinology - 2012
    Adipocytes express angiotensin receptors- but the direct effects of angiotensin II (AngII) stimulating this cell type are undefined. Adipocytes express angiotensin type 1a receptor (AT1aR) and AT2R- both of which have been implicated in obesity. In this study- we determined the effects of adipocyte AT1aR deficiency on adipocyte differentiation and the development of obesity in mice fed low-fat (LF) or high-fat (HF) diets. Mice expressing Cre recombinase under the control of the aP2 promoter were bred with AT1aR-floxed mice to generate mice with adipocyte AT1aR deficiency (AT1aRaP2). AT1aR mRNA abundance was reduced significantly in both white and brown adipose tissue from AT1aRaP2 mice compared with nontransgenic littermates (AT1aRfl/fl). Adipocyte AT1aR deficiency did not influence body weight- glucose tolerance- or blood pressure in mice fed either LF or high-fat diets. However- LF-fed AT1aRaP2 mice exhibited striking adipocyte hypertrophy even though total fat mass was not different between genotypes. Stromal vascular cells from AT1aRaP2 mice differentiated to a lesser extent to adipocytes compared with controls. Conversely- incubation of 3T3-L1 adipocytes with AngII increased Oil Red O staining and increased mRNA abundance of peroxisome proliferator-activated receptor gamma (PPARgamma) via AT1R stimulation. These results suggest that reductions in adipocyte differentiation in LF-fed AT1aRaP2 mice resulted in increased lipid storage and hypertrophy of remaining adipocytes. These results demonstrate that AngII regulates adipocyte differentiation and morphology through the adipocyte AT1aR in lean mice.
    Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase
    Giulia Bandini, Molecular Microbiology - 2012
    The enzymes phosphomannomutase (PMM)- phospho-N-acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose- N-acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome- there is- surprisingly- no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Ã… resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite- while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose-phosphates- as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that- in vivo- PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why- uniquely- T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.
    Essential Role for IKK? in Production of Type 1 Interferons by Plasmacytoid Dendritic Cells
    Eduardo Pauls, The Journal of Biological Chemistry - 2012
    Background: The role of IKKBeta in the production of type 1 interferons by plasmacytoid dendritic cells (pDCs) is unknown.- Results: Inhibition of IKKBeta and its activator TAK1 prevents the production of IFNBeta in pDCs- and hence the production of IFNalpha.- Conclusion: Toll-like receptor 7/9-stimulated production of interferons in pDCs requires the canonical IKKs and TAK1.- Significance: IKKBeta inhibitors may have potential for the treatment of autoimmunity.- Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9- but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKBeta signaling pathway to induce the production of IFNBeta via a pathway that is independent of the degradation of IkBalpha. We also show that IKKBeta activity- as well as the subsequent IFNBeta-stimulated activation of the JAK-STAT1/2 signaling pathway- are essential for the production of IFNalpha by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNalpha because the activation of IKKBeta is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKBeta inhibitors than those needed to suppress the production of NFkB-dependent proinflammatory cytokines- such as IL-6- suggesting that drugs that inhibit IKKBeta may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9- respectively.
    The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses
    Russell Pachynski, The Journal of Experimental Medicine - 2012
    Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here- in a survey of public whole genome expression datasets we found that the gene for chemerin- a widely expressed endogenous chemoattractant protein- is down-regulated in melanoma as well as other human tumors. Moreover- high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma- tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1)- the chemoattractant receptor for chemerin- and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth- suggesting the potential for therapeutic application. These results show that chemerin- whether expressed by tumor cells or within the tumor environment- can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion.
    Developmental cis-regulatory analysis of the cyclin D gene in the sea urchin Strongylocentrotus purpuratus
    Christopher McCarty, Biochemical and Biophysical Research Communications - 2013
    Abstract Cyclin D genes regulate the cell cycle- growth and differentiation in response to intercellular signaling. While the promoters of vertebrate cyclin D genes have been analyzed- the cis-regulatory sequences across an entire cyclin D locus have not. Doing so would increase understanding of how cyclin D genes respond to the regulatory states established by developmental gene regulatory networks- linking cell cycle and growth control to the ontogenetic program. Therefore- we conducted a cis-regulatory analysis on the cyclin D gene- SpcycD- of the sea urchin- Strongylocentrotus purpuratus- during embryogenesis- identifying upstream and intronic sequences- located within six defined regions bearing one or more cis-regulatory modules each.
    One-step real-time PCR assay for detection and quantitation of hepatitis D virus RNA
    Maja Kodani, Journal of Virological Methods - 2013
    Abstract Hepatitis D virus (HDV) is a defective virus which requires hepatitis B virus (HBV) surface antigen (HBsAg) for its assembly. Hepatitis B infected individuals co-infected or superinfected with HDV often present with more severe hepatitis- progress faster to liver disease- and have a higher mortality rate than individuals infected with HBV alone. Currently- there are no commercially available clinical tests for the detection and quantitation of HDV RNA in the United States. A one-step TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for detection of HDV RNA- designing primers located in the region just downstream from the HDV antigen gene. The assay has the potential to detect all eight HDV genotypes. A quantifiable synthetic RNA control was also developed for use in the determination of HDV RNA titers in clinical samples. The limit of detection of this assay is 7.5 × 102 HDV RNA copies/ml with a dynamic range of six logs. Most clinical specimens tested (40/41) fell within the linear range of the assay. The median HDV RNA titer of the tested specimens was 6.24 × 106 copies/ml- with a range of 8.52 × 103-1.79 × 109 copies/ml. Out of 132 anti-HDV-positive specimens 41 (31.1\%) were positive for HDV RNA.
    Quantitative phenotyping of X-disease resistance in chokecherry using real-time PCR
    Danqiong Huang, Journal of Microbiological Methods - 2013
    A quantitative real-time SYBR Green PCR (qPCR) assay has been developed to detect and quantify X-disease phytoplasmas in chokecherry. An X-disease phytoplasma-specific and high sensitivity primer pair was designed based on the 16S rRNA gene sequence of X-disease phytoplasmas. This primer pair was specific to the 16SrIII group (X-disease) phytoplasmas. The qPCR method can quantify phytoplasmas from a DNA mix (a mix of both chokecherry and X-disease phytoplasma DNA) at as low as 0.001 ng- 10-fold lower than conventional PCR using the same primer pair. A significant correlation between the copy number of phytoplasmas and visual phenotypic rating scores of X-disease resistance in chokecherry plants was observed. Disease resistant chokecherries had a significantly lower titer of X-disease phytoplasmas than susceptible plants. This suggests that the qPCR assay provides a more objective tool to phenotype phytoplasma disease severity- particularly for early evaluation of host resistance, therefore- this method will facilitate quantitative phenotyping of disease resistance and has great potential in enhancing plant breeding.
    The TGFBeta1 pathway is required for NFkB dependent gene expression in mouse keratinocytes
    Kelly Hogan, Cytokine - 2013
    Abstract The transforming growth factor-beta 1 (TGFBeta1) and NFkB pathways are important regulators of epidermal homeostasis- inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent- but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes- TGFBeta1 induces NFkB-luciferase reporter activity that is dependent on both NFkB and Smad3. TGFBeta1-induced NFkB-luciferase activity was blocked by the IkB inhibitor parthenolide- the IkB super-repressor- a dominant negative TGFBeta1-activated kinase 1 (TAK1) and genetic deletion of NFkB1. Coexpression of NFkB p50 or p65 subunits enhanced NFkB-luciferase activity. Similarly- inhibition of the TGFBeta1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFBeta1 induction of NFkB-luciferase. TGFBeta1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IkB levels or nuclear translocation of NFkB subunits- although EMSA showed rapid NFkB nuclear binding activity that could be blocked by SB431542 treatment. TNFalpha- a well characterized NFkB target gene was also induced by TGFBeta1 and this was blocked in NFkB+/- and -/- keratinocytes and by the IkB super-repressor. To test the effects of the TGFBeta1 pathway on a biologically relevant activator of NFkB- we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5- but no significant increase in SBE-dependent gene expression. Inhibition of TGFBeta1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFalpha message. Similarly- UVB induction of TNFalpha mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGFBeta1 signaling is required for NFkB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFBeta1.
    Prolactin receptor attenuation induces zinc pool redistribution through ZnT2 and decreases invasion in MDA-MB-453 breast cancer cells
    Zeynep Bostanci, Experimental Cell Research - 2013
    Prolactin receptor (PRL-R) activation regulates cell differentiation- proliferation- cell survival and motility of breast cells. Prolactin (PRL) and PRL-R over-expression are strongly implicated in breast cancer- particularly contributing to tumor growth and invasion in the more aggressive estrogen-receptor negative (ER-) disease. PRL-R antagonists have been suggested as potential therapeutic agents, however- mechanisms through which PRL-R antagonists exert their actions are not well-understood. Zinc (Zn) is a regulatory factor for over 10\% of the proteome- regulating critical cell processes such as proliferation- cell signaling- transcription- apoptosis and autophagy. PRL-R signaling regulates Zn metabolism in breast cells. Herein we determined effects of PRL-R attenuation on cellular Zn metabolism and cell function in a model of ER-- PRL-R over-expressing breast cancer cells (MDA-MB-453). PRL-R attenuation post-transcriptionally increased ZnT2 abundance and redistributed intracellular Zn pools into lysosomes and mitochondria. ZnT2-mediated lysosomal Zn sequestration was associated with reduced matrix metalloproteinase 2 (MMP-2) activity and decreased invasion. ZnT2-mediated Zn accumulation in mitochondria was associated with increased mitochondrial oxidation. Our results suggest that PRL-R antagonism in PRL-R over-expressing breast cancer cells may reduce invasion through the redistribution of intracellular Zn pools critical for cellular function.
    Lipocalin 2 is a novel regulator of angiogenesis in human breast cancer
    Jiang Yang, The FASEB Journal - 2013
    Lipocalin 2 (Lcn2)- a member of the lipocalin family- is up-regulated in a variety of epithelial cancers. We have previously reported that Lcn2 induces the epithelial to mesenchymal transition in breast cancer through the estrogen receptor alpha/Slug axis and that it is a potential noninvasive biomarker of this disease. Here- we report the novel finding that Lcn2 regulates breast cancer angiogenesis. Vascular endothelial growth factor (VEGF)- a key angiogenic activator- was significantly increased with Lcn2 expression in MCF-7 human breast cancer cells as well as in an angiogenic line derived from MDA-MB-436 cells. Treatment with a VEGF-neutralizing antibody demonstrates that VEGF is essential for the angiogenic activity of Lcn2. We further demonstrate that Lcn2-induced VEGF is mediated through hypoxia-inducible factor 1alpha (HIF-1alpha) and that Lcn2 regulates HIF-1alpha through extracellular signal-regulated kinase (Erk). The regulation of HIF-1alpha and VEGF by Lcn2 was also demonstrated in the aggressive MDA-MB-231 cell line. Using the mouse corneal pocket assay- we found that Lcn2 significantly enhanced the angiogenesis induced by VEGF. Taken together- these results are the first to demonstrate that Lcn2 promotes angiogenesis in vitro and in vivo and suggest a novel mechanism through which Lcn2 may promote tumor progression.—Yang- J.- McNeish- B.- Butterfield- C.- Moses- M. A. Lipocalin 2 is a novel regulator of angiogenesis in human breast cancer.
    ICOS-Expressing Lymphocytes Promote Resolution of CD8-Mediated Lung Injury in a Mouse Model of Lung Rejection
    Qiang Wu, PLoS ONE - 2013
    Acute rejection- a common complication of lung transplantation- may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes- CD8+ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8+ T cell-mediated lung rejection- we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG-/- mice- we found that CD4+ T cells and ICOS+/+ T cells were required for protection against lethal lung injury- while neutrophil depletion was not protective. In addition- CD4+Foxp3 + ICOS+ T cells were enriched in the lungs of animals surviving lung injury and ICOS+/+ Tregs promoted survival in animals that received ICOS-/- T cells. Direct comparison of ICOS-/- Tregs to ICOS+/+ Tregs found defects in vitro but no differences in the ability of ICOS-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.
    miR-200b Inhibits Prostate Cancer EMT- Growth and Metastasis
    LaTanya Williams, PLoS ONE - 2013
    miRNA regulate gene expression at post-transcriptional level and fine-tune the key biological processes- including cancer progression. Here- we demonstrate the involvement of miR-200b in the metastatic spread of prostate cancer. We identified miR-200b as a downstream target of androgen receptor and linked its expression to decreased tumorigenicity and metastatic capacity of the prostate cancer cells. Overexpression of miR-200b in PC-3 cells significantly inhibited their proliferation and the formation of subcutaneous tumors. Moreover- in an orthotopic model- miR-200b blocked spontaneous metastasis and angiogenesis by PC-3 cells. This decreased metastatic potential was likely due to the reversal of the epithelial-to-mesenchymal transition- as was evidenced by increased pan-epithelial marker E-cadherin and specific markers of prostate epithelium- cytokeratins 8 and 18. In contrast- mesenchymal markers- fibronectin and vimentin- were significantly downregulated by miR-200b. Our results suggest an important role for miR-200b in prostate cancer progression and indicate its potential utility for prostate cancer therapy.
    Disruption of Glycerol Metabolism by RNAi Targeting of Genes Encoding Glycerol Kinase Results in a Range of Phenotype Severity in Drosophila
    Patrick Wightman, PLoS ONE - 2013
    In Drosophila- RNAi targeting of either dGyk or dGK can result in two alternative phenotypes: adult glycerol hypersensitivity or larval lethality. Here we compare these two phenotypes at the level of glycerol kinase (GK) phosphorylation activity- dGyk and dGK-RNA expression- and glycerol levels. We found both phenotypes exhibit reduced but similar levels of GK phosphorylation activity. Reduced RNA expression levels of dGyk and dGK corresponded with RNAi progeny that developed into glycerol hypersensitive adult flies. However- quantification of dGyk/dGK expression levels for the larval lethality phenotype revealed unexpected levels possibly due to a compensatory mechanism between dGyk and dGK or RNAi inhibition. The enzymatic role of glycerol kinase converts glycerol to glycerol 3-phosphate. As expected- elevated glycerol levels were observed in larvae that went on to develop into glycerol hypersensitive adults. Interestingly- larvae that died before eclosion revealed extremely low glycerol levels. Further characterization identified a wing phenotype that is enhanced by a dGpdh null mutation- indicating disrupted glycerol metabolism underlies the wing phenotype. In humans- glycerol kinase deficiency (GKD) exhibits a wide range of phenotypic variation with no obvious genotype-phenotype correlations. Additionally- disease severity often does not correlate with GK phosphorylation activity. It is intriguing that both human GKD patients and our GKD Drosophila model show a range of phenotype severity. Additionally- the lack of correlation between GK phosphorylation and dGyk/dGK-RNA expression with phenotypic severity suggests further study including understanding the alternative functions of the GK protein- could provide insights into the complex pathogenic mechanism observed in human GKD patients.
    A Meiosis-Specific Form of the APC/C Promotes the Oocyte-to-Embryo Transition by Decreasing Levels of the Polo Kinase Inhibitor Matrimony
    Zachary Whitfield, PLoS Biol - 2013
    {\textless}p{\textgreater}During the oocyte-to-embryo transition in Drosophila- degradation of the Polo kinase inhibitor- Matrimony- depends on Cortex- a meiosis-specific form of the Anaphase Promoting Complex/Cyclosome that is required for the oocyte's normal transition from meiosis to mitosis.{\textless}/p{\textgreater}{\textless}/sec{\textgreater}
    A Nontoxic Additive to Introduce X-Ray Contrast into Poly(Lactic Acid). Implications for Transient Medical Implants Such as Bioresorbable Coronary Vascular Scaffolds
    Yujing Wang, Advanced Healthcare Materials - 2013
    Bioresorbable coronary vascular scaffolds are about to revolutionize the landscape of interventional cardiology. These scaffolds- consisting of a poly(l-lactic acid) interior and a poly(d-l-lactic acid) surface coating- offer a genuine alternative for metallic coronary stents. Perhaps the only remaining drawback is that monitoring during implantation is limited to two X-ray contrast points. Here- a new approach to make the biodegradable scaffolds entirely radiopaque is explored. A new contrast agent is designed and synthesized. This compound is miscible with poly(d-l-lactic acid) matrix- and nontoxic to multiple cell types. Blends of poly(d-l-lactic acid) and the contrast agent are found to be hemocompatible- noncytotoxic- and radiopaque. The data show that it is possible to manufacture fully radiopaque bioresorbable coronary vascular scaffolds. Whole-stent X-ray visibility helps interventionalists ensure that the scaffold deploys completely. This important advantage may translate into improved safety- accuracy- and clinical performance of cardiac stents.
    Î’eta-naphthoflavone (DB06732) Mediates Estrogen Receptor-Positive Breast Cancer Cell Cycle Arrest through AhR-Dependent Regulation of PI3K/AKT and MAPK/ERK Signaling
    Chun Wang, Carcinogenesis - 2013
    Beta-naphthoflavone (BNF- DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However- the mechanism by which BNF exerts this antitumor effect remains unclear. Thus- we explored mechanisms of BNF’s antitumor effects in human breast cancer cells. BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21Cip1/Waf1- leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells- but not in ER-negative MDA-MB-231 cells. In addition- BNF inhibited PI3K/AKT signaling- and the PI3K inhibitor- LY294-002- exhibited the same inhibitory effects on cyclinD1/D3- CDK4 and the cell cycle as BNF. Interestingly- BNF activated mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK-ERK) signaling- and more notably- the MEK inhibitor PD98059 significantly blocked BNF-induced cell cycle arrest and upregulation of p21Cip1/Waf1. Furthermore- specific ERalpha and AhR siRNA studies indicate that ERalpha is required in BNF-induced p21Cip1/Waf1 expression- and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR dependent. Taken together- AhR-dependent inhibition of the PI3K/AKT pathway- activation of MAPK/ERK and modulation of ERalpha is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer- which may represent a promising strategy to be exploited in future clinical trials.
    Cotinine in Human Placenta Predicts Induction of Gene Expression in Fetal Tissues
    Carrie Vyhlidal, Drug Metabolism and Disposition - 2013
    Maternal cigarette smoking during pregnancy is associated with increased risk of perinatal morbidity and mortality. However- the mechanisms underlying adverse birth outcomes following prenatal exposure to cigarette smoke remain unknown due- in part- to the absence or unreliability of information regarding maternal cigarette smoke exposure during pregnancy. Our goal was to determine if placental cotinine could be a reliable biomarker of fetal cigarette smoke exposure during pregnancy. Cotinine levels were determined in placentas from 47 women who reported smoking during pregnancy and from 10 women who denied cigarette smoke exposure. Cotinine levels were significantly higher in placentas from women reporting cigarette smoking (median = 27.2 ng/g) versus women who reported no smoke exposure (2.3 ng/g- P {\textless} 0.001). Receiver operating characteristic curve analysis identified an optimal cut point of 7.5 ng/g (sensitivity = 78.7\%- specificity = 100\%) to classify placenta samples from mothers who smoked versus those from mothers who did not. Among 415 placentas for which maternal cigarette smoking status was unavailable- 167 had cotinine levels {\textgreater} 7.5 ng/g and would be considered positive for cigarette smoke exposure. Data from quantitative reverse-transcription polymerase chain reaction analyses demonstrated that in utero cigarette smoke exposure predicted by cotinine in placenta is associated with changes in the expression of xenobiotic-metabolizing enzymes in fetal tissues. CYP1A1 mRNA in fetal lung and liver tissue and CYP1B1 mRNA in fetal lung tissue were significantly induced when cotinine was detected in placenta. These findings indicate that cotinine in placenta is a reliable biomarker for fetal exposure and response to maternal cigarette smoking during pregnancy.
    ABCA7 expression is associated with Alzheimer's disease polymorphism and disease status
    Jared Vasquez, Neuroscience Letters - 2013
    Genome-wide association studies (GWAS) have implicated a series of single nucleotide polymorphisms (SNPs) in Alzheimer's disease (AD) risk. Elucidating the function of these SNPs is critical to identify the underlying pathways and- potentially- novel therapeutic agents. SNPs within the gene ATP binding cassette A7 (ABCA7) reached significance in these studies- warranting investigation into their actions. Here- we analyzed ABCA7 expression in a set of human brain samples as a function of AD-associated SNPs and AD status. We report that the rs3764650T allele that decreases AD risk is associated with increased ABCA7 expression. However- ABCA7 expression is increased in AD individuals. We interpret our findings as suggesting a model wherein increased ABCA7 expression reduces AD risk and that the increased ABCA7 observed in AD reflects an inadequate compensatory change.
    Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease
    Orkhontuya Tsedensodnom, Disease Models & Mechanisms - 2013
    Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However- the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here- we examined the relationship between alcohol metabolism- oxidative stress- secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae- such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol- with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1)- the major enzymes that metabolize ethanol- prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus- we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol- whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly- these treatments also blocked ethanol-induced steatosis and reduced UPR activation- whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively- these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD- and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes.
    Development of Translating Ribosome Affinity Purification for Zebrafish
    Robert Tryon, Genesis (New York- N.Y. : 2000) - 2013
    The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish- we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes- and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish.
    Transcription factor AP4 modulates reversible and epigenetic silencing of the Cd4 gene
    Takeshi Egawa, Proceedings of the National Academy of Sciences - 2011
    CD4 coreceptor expression is negatively regulated through activity of the Cd4 silencer in CD4-CD8- double-negative (DN) thymocytes and CD8+ cytotoxic lineage T cells. Whereas Cd4 silencing is reversed during transition from DN to CD4+CD8+ double-positive stages- it is maintained through heritable epigenetic processes following its establishment in mature CD8+ T cells. We previously demonstrated that the Runx family of transcription factors is required for Cd4 silencing both in DN thymocytes and CD8+ T cells. However- additional factors that cooperate with Runx proteins in the process of Cd4 silencing remain unknown. To identify collaborating factors- we used microarray and RNAi-based approaches and found the basic helix-loop-helix ZIP transcription factor AP4 to have an important role in Cd4 regulation. AP4 interacts with Runx1 in cells in which Cd4 is silenced- and is required for Cd4 silencing in immature DN thymocytes through binding to the proximal enhancer. Furthermore- although AP4-deficient CD8+ T cells appeared to normally down-regulate CD4 expression- AP4 deficiency significantly increased the frequency of CD4-expressing effector/memory CD8+ T cells in mice harboring point mutations in the Cd4 silencer. Our results suggest that AP4 contributes to Cd4 silencing both in DN and CD8+ T cells by enforcing checkpoints for appropriate timing of CD4 expression and its epigenetic silencing.
    Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion
    Rebecca Duncan, BMC Evolutionary Biology - 2011
    Background A major goal of molecular evolutionary biology is to understand the fate and consequences of duplicated genes. In this context- aphids are intriguing because the newly sequenced pea aphid genome harbors an extraordinary number of lineage-specific gene duplications relative to other insect genomes. Though many of their duplicated genes may be involved in their complex life cycle- duplications in nutrient amino acid transporters appear to be associated rather with their essential amino acid poor diet and the intracellular symbiosis aphids rely on to compensate for dietary deficits. Past work has shown that some duplicated amino acid transporters are highly expressed in the specialized cells housing the symbionts- including a paralog of an aphid-specific expansion homologous to the Drosophila gene slimfast. Previous data provide evidence that these bacteriocyte-expressed transporters mediate amino acid exchange between aphids and their symbionts. Results We report that some nutrient amino acid transporters show male-biased expression. Male-biased expression characterizes three paralogs in the aphid-specific slimfast expansion- and the male-biased expression is conserved across two aphid species for at least two paralogs. One of the male-biased paralogs has additionally experienced an accelerated rate of non-synonymous substitutions. Conclusions This is the first study to document male-biased slimfast expression. Our data suggest that the male-biased aphid slimfast paralogs diverged from their ancestral function to fill a functional role in males. Furthermore- our results provide evidence that members of the slimfast expansion are maintained in the aphid genome not only for the previously hypothesized role in mediating amino acid exchange between the symbiotic partners- but also for sex-specific roles.
    USP10 deubiquitylates the histone variant H2A.Z and both are required for androgen receptor-mediated gene activation
    Ryan Draker, Nucleic Acids Research - 2011
    H2A.Z- a variant of H2A- is found at the promoters of inducible genes in both yeast and higher eukaryotes. However- its role in transcriptional regulation is complex since it has been reported to function both as a repressor and activator. We have previously found that mono-ubiquitylation of H2A.Z is linked to transcriptional silencing. Here- we provide new evidence linking H2A.Z deubiquitylation to transcription activation. We found that H2A.Z and ubiquitin-specific protease 10 (USP10) are each required for transcriptional activation of the androgen receptor (AR)-regulated PSA and KLK3 genes. USP10 directly deubiquitylates H2A.Z in vitro and in vivo- and reducing USP10 expression in prostate cancer cells results in elevated steady-state levels of mono-ubiquitylated H2A.Z (H2A.Zub1). Moreover- knockdown of USP10 ablates hormone-induced deubiquitylation of chromatin proteins at the AR-regulated genes. Finally- by sequential ChIP assays- we found that H2A.Zub1 is enriched at the PSA and KLK3 regulatory regions- and loss of H2A.Zub1 is associated with transcriptional activation of these genes. Together- these data provide novel insights into how H2A.Z ubiquitylation/deubiquitylation and USP10 function in AR-regulated gene expression.
    Expression of Truncated Eukaryotic Initiation Factor 3e (eIF3e) Resulting from Integration of Mouse Mammary Tumor Virus (MMTV) Causes a Shift from Cap-dependent to Cap-independent Translation
    David Chiluiza, Journal of Biological Chemistry - 2011
    Integration of mouse mammary tumor virus (MMTV) at the common integration site Int6 occurs in the gene encoding eIF3e- the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation- but by an unknown mechanism. Because eIF3e makes up at least part of the binding site for eIF4G- we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G- signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43 S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP- c-Myc- CYR61- and Pim-1- which are translated in a cap-independent manner- were shifted to heavier polysomes whereas mRNAs encoding GAPDH- actin- L32- and L34- which are translated in a cap-dependent manner- were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between up-regulation of cap-independent translation and MMTV-induced tumorigenesis contrasts with the well established model for malignant transformation involving up-regulation of highly cap-dependent translation.
    Dietary Ligands of the Aryl Hydrocarbon Receptor Induce Anti-Inflammatory and Immunoregulatory Effects on Murine Dendritic Cells
    Jenna Benson, Toxicological Sciences - 2011
    Activation of the aryl hydrocarbon receptor (AhR) in immune cells- such as dendritic cells (DCs)- can lead to suppressed immune responses. Although AhR activation is most recognized for mediating the effects of its prototypical ligand 2-3-7-8-tetrachlorodibenzo-p-dioxin (TCDD)- many compounds existing in dietary sources can also bind the AhR. Because the immunomodulatory effects of indole-3-carbinol (I3C) and indirubin-3'-oxime (IO) have yet to be investigated in DCs- we evaluated the potential immunomodulatory effects of these compounds on murine DCs. We hypothesized that I3C and IO suppress immune and inflammatory responses in DCs. We found that both I3C and IO decreased the expression of CD11c- CD40- and CD54 while they increased expression of MHC2 and CD80. Following lipopolysaccharide (LPS)-activation- I3C and IO suppressed the production of pro-inflammatory mediators including tumor necrosis factor-- interleukin (IL)-1\{beta\}- IL-6- IL-12- and nitric oxide but increased IL-10 levels. These effects of I3C and IO were partially mediated by the AhR. Additionally- immunoregulatory genes- such as ALDH1A- IDO and TGFB- were upregulated following treatment with I3C or IO. Both I3C and IO decreased basal levels of nuclear factor-kappa B p65- but only I3C suppressed the LPS-induced activity of RelB. Finally- when cultured with naive T cells- bone marrow-derived dendritic cells treated with the dietary AhR ligands increased the frequency of Foxp3+ Tregs in an antigen-specific manner. Taken together- these results indicate that I3C and IO exhibit immunosuppressive and anti-inflammatory effects on DCs. Because I3C and IO are significantly less toxic than TCDD- these natural products may ultimately become useful therapeutics for the treatment of autoimmune and inflammatory diseases.
    Biliverdin Rescues the HO-2 Null Mouse Phenotype of Unresolved Chronic Inflammation Following Corneal Epithelial Injury
    Lars Bellner, Investigative Ophthalmology & Visual Science - 2011
    The authors provide substantial evidence that the injured HO-2 null cornea- which experiences extensive oxidative stress- exaggerated inflammation- and impaired wound healing- can be rescued by the heme oxygenase product biliverdin- further supporting the notion that HO-2 is a critical cytoprotective system in the cornea.
    Targeted suppression of HO-2 gene expression impairs the innate anti-inflammatory and repair responses of the cornea to injury
    Lars Bellner, Molecular Vision - 2011
    Purpose Heme oxygenase (HO)-2 is highly expressed in the corneal epithelium and is a component of the heme oxygenase system that represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration- perforation and neovascularization. In this study- we explore whether a localized corneal suppression of HO-2 is sufficient for disrupting the innate anti-inflammatory and repair capability of the cornea. Methods Silencing hairpin RNA (shRNA) against HO-2 was administered subconjunctivally (100 ng/eye) as well as topically (100 ng/eye) starting one day before corneal epithelial debridement and once daily- thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed by fluorescein staining using a dissecting microscope and image analysis. Inflammatory response was quantified by myeloperoxidase activity. Levels of mRNA were measured by RT-PCR. Results Local injection of HO-2-specific shRNA led to a 50\% reduction in corneal HO-2 mRNA. Administration of HO-2-specific shRNA delayed corneal re-epithelialization when compared with the control shRNA-treated group by 14\%- 20\%- and 12\% at days 3- 4- and 7 after injury- respectively (n=18-24). The observed delay in the wound repair process in HO-2 shRNA treated mice was accompanied by a threefold and 3.5 fold increase in the neovascular response at days 4 and 7 after injury. Further- local knockdown of HO-2 lead to an aberrant chronic inflammatory response- as shown by presence of high numbers of inflammatory cells still present in the cornea at day 7 after injury, 1.04±0.45×106 in HO-2 knockdown mice versus 0.14±0.03×106 inflammatory cells in control mice. Matrix metalloproteinase-2 (MMP-2) but not MMP-9 increased following injury and remained elevated in the injured corneas of the HO-2 shRNA-treated eyes. Conclusions Corneal knockdown of HO-2 via local administration of HO-2-specific shRNA leads to delayed re-epithelialization- increased neovascularization and an aberrant inflammatory response similar to what is observed in the HO-2 null mouse. The elevated MMP-2 expression may contribute to the increase in neovascularization in corneas in which HO-2 expression is suppressed.
    Neurexin-1alpha Contributes to Insulin-containing Secretory Granule Docking
    Merrie Mosedale, Journal of Biological Chemistry - 2012
    Neurexins are a family of transmembrane- synaptic adhesion molecules. In neurons- neurexins bind to both sub-plasma membrane and synaptic vesicle-associated constituents of the secretory machinery- play a key role in the organization and stabilization of the presynaptic active zone- and help mediate docking of synaptic vesicles. We have previously shown that neurexins- like many other protein constituents of the neurotransmitter exocytotic machinery- are expressed in pancreatic Beta cells. We hypothesized that the role of neurexins in Beta cells parallels their role in neurons- with Beta-cell neurexins helping to mediate insulin granule docking and secretion. Here we demonstrate that Beta cells express a more restricted pattern of neurexin transcripts than neurons- with a clear predominance of neurexin-1alpha expressed in isolated islets. Using INS-1E Beta cells- we found that neurexin-1alpha interacts with membrane-bound components of the secretory granule-docking machinery and with the granule-associated protein granuphilin. Decreased expression of neurexin-1alpha- like decreased expression of granuphilin- reduces granule docking at the Beta-cell membrane and improves insulin secretion. Perifusion of neurexin-1alpha KO mouse islets revealed a significant increase in second-phase insulin secretion with a trend toward increased first-phase secretion. Upon glucose stimulation- neurexin-1alpha protein levels decrease. This glucose-induced down-regulation may enhance glucose-stimulated insulin secretion. We conclude that neurexin-1alpha is a component of the Beta-cell secretory machinery and contributes to secretory granule docking- most likely through interactions with granuphilin. Neurexin-1alpha is the only transmembrane component of the docking machinery identified thus far. Our findings provide new insights into the mechanisms of insulin granule docking and exocytosis.
    TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription
    Marta Montes, Molecular and Cellular Biology - 2012
    Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here- we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results- we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore- TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alter