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  • PCR & qPCR
    Real-Time Quantitative PCR
    SYBR Green Detection
    Human-based fibrillar nanocomposite hydrogels as bioinstructive matrices to tune stem cell behavior
    Bárbara B. Mendes, Nanoscale - 2018
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    A key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), 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
    Abstract
    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
    Abstract
    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.
    EXERSOMES, METHODS OF PRODUCING AND METHOD OF USING
    Mark Tarnopolsky, EXERKINE CORPORATION (Hamilton, CA) - 2017
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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.
    METHOD FOR MEASURING BONE LOSS RATE - NOBEL BIOCARE SERVICES AG
    Jan Hall, United States Patent - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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.
    THERAPEUTIC AGENTS FOR MODULATING THYMIC FUNCTION AND/OR GROWTH AND/OR TREATING VARIOUS DISORDERS - UCL BUSINESS PLC
    Aimee L. Benjamin, University of Vermont Dissertation - 2016
    Abstract
    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.
    5-HT2A Receptor Binding in the Frontal Cortex of Parkinson’s Disease Patients and Alpha-SynucleinOverexpressing Mice: A Postmortem Study
    Nadja Bredo Rasmussen, Hindawi - 2016
    Abstract
    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
    Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica
    Karin Hjort, Molecular Microbiology - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    CHEMICAL REPROGRAMMING OF HUMAN GLIAL CELLS INTO NEURONS FOR BRAIN AND SPINAL CORD REPAIR - THE PENN STATE RESEARCH FOUNDATION
    Gong Chen, United States Patent - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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.
    METHODS AND COMPOSITIONS INVOLVING LINCRNA AND LEUKEMIA - THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
    Dinesh S Rao, United States patent - 2016
    Abstract
    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
    Abstract
    . 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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    NOTCH INHIBITION IN THE TREATMENT AND PREVENTION OF OBESITY
    Masanori Aikawa, United States Patent - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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.
    NANODIAMOND PARTICLE COMPLEXES - NORTHWESTERN UNIVERSITY
    Ho, Dean, United States Patent - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    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
    COMPOSITIONS AND METHODS FOR cDNA SYNTHESIS - QUANTA BIOSCIENCES
    Rashtchian, Ayoub, United States Patent - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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.
    FIBROBLAST GROWTH FACTOR RECEPTOR INHIBITION FOR THE TREATMENT OF DISEASE
    Faul, Christian, United States Patent - 2015
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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].
    RNA-GUIDED GENE EDITING AND GENE REGULATION - DUKE UNIVERSITY
    Gersbach, Charles A., United States Patent Application - 2016
    Abstract
    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
    Abstract
    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
    Abstract
    * 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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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
    Abstract
    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.
    Probe-based Detection
    Investigation of an outbreak caused by antibiotic-susceptible Klebsiella oxytoca in a neonatal intensive care unit in Norway
    Torunn Gresdal Rønning, Acta Paediatrica - 2018
    Abstract
    Aim Klebsiella spp. have been stated to be the most frequent cause of neonatal intensive care unit (NICU) outbreaks. We report an outbreak of Klebsiella oxytoca in a NICU at a tertiary care hospital in Norway between April 2016 and April 2017. This study describes the outbreak, infection control measures undertaken and the molecular methods developed. Methods The outbreak prompted detailed epidemiological and microbial investigations, where whole-genome sequencing (WGS) was particularly useful for both genotyping and development of two new K. oxytoca-specific real-time PCR assays. Routine screening of patients, as well as sampling from numerous environmental sites, was performed during the outbreak. A bundle of infection control measures was instigated to control the outbreak, among them strict cohort isolation. Results Five neonates had symptomatic infection, and 17 were found to be asymptomatically colonised. Infections varied in severity from conjunctivitis to a fatal case of pneumonia. A source of the outbreak could not be determined. Conclusion This report describes K. oxytoca as a significant pathogen in a NICU outbreak setting and highlights the importance of developing appropriate microbiological screening methods and implementing strict infection control measures to control the outbreak in a setting where the source could not be identified.
    Immunoserology of European seabass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus) as a non-lethal diagnostic tool for viral nervous necrosis
    Koby Tarrab, Aquaculture International - 2018
    Abstract
    Viral nervous necrosis (VNN) is a lethal fish disease that has spread worldwide over the last two decades, causing severe losses in aquaculture. Diagnosis of the infection is generally made by sampling brain tissue, which involves sacrificing often valuable fish. Aiming at developing a non-lethal diagnostic method, the immune responses to an experimental nervous necrosis virus (NNV) infection in sea bass Dicentrarchus labrax and white grouper Epinephelus aeneus, two species most susceptible to the disease, were studied. RT-qPCR revealed presence of NNV in the fish brain within 24 h post-infection, the virus titer remaining high up to 30–35 days post-infection. In D. labrax blood, the virus was detectable within the first 5 days, after which its presence declined rapidly. Mx gene expression correlated to the virus presence in the blood and brain. An indirect ELISA was developed that quantified anti-NNV IgM in the fish blood. In D. labrax, anti-NNV IgM titer increased significantly within 5 days post-infection, and presence of specific IgM was detectable for 180 days. A sandwich ELISA was developed for E. aeneus. In this latter species, anti-NNV IgM titer increased significantly within the first 12 days and was detectable for 208 further days. The sandwich ELISA can be used as a diagnostic tool for detecting NNV exposure in all fish species for which specific antibodies against their IgMs are not yet commercially available. Our immunoserological method can reliably be used for diagnosis of VNN infection and does not require sacrificing the fish.
    Preweaned heifer management on US dairy operations: Part IV. Factors associated with the presence of Escherichia coli O157 in preweaned dairy heifers
    C. Stenkamp-Strahm, Journal of Dairy Science - 2018
    Abstract
    Dairy calves shed pathogenic Escherichia coli O157 (O157) in feces and are a potential route of exposure for human infections. As part of the National Animal Health Monitoring System's (NAHMS) Dairy 2014 study, we evaluated farm, animal, and environmental factors associated with O157 presence in dairy heifer calves. For this O157 study, calves were enrolled from 100 dairy operations in 13 states. Each operation collected data from calves from birth to weaning over an 18-mo period. A single fecal sample was collected from 487 calves in western states and from 871 calves in eastern states (n = 1,358 total), and O157 was detected in 2.5% (n = 34) of fecal samples. Descriptive statistics and univariable screening were used to determine which farm practices, environmental factors, and calf health measures were associated with O157 detection. Multilevel logistic models, controlling for dairy operation, were created using backward elimination of screened variables. The final O157 main effects model included variables for source of colostrum, temperature-humidity index (THI), and serum IgG concentration. Higher serum IgG was associated with lower odds of O157 shedding, whereas calves fed colostrum from their own dam had higher odds of O157 shedding than calves fed colostrum from pooled sources. Interaction models showed that THI level modified the effect of colostrum source on O157 shedding; calves with a THI indicative of heat stress had a significantly increased presence of O157 when fed colostrum from a first-lactation dam. The THI level also modified the effects of serum IgG. Calves with thermoneutral or heat stress THI values had increased presence of O157 with poor (<10 g/L) or adequate (10–15 g/L) serum IgG levels compared with those having excellent (≥15 g/L) serum IgG levels. These results highlight factors that influence the presence of O157 in preweaned dairy heifer calves and may be used to guide practices that mitigate shedding through improved animal husbandry.
    Iron oxide nanoparticles enhance Toll-like receptor-induced cytokines in a particle size- and actin-dependent manner in human blood
    Susann Wolf-Grosse, Nanomedicine - 2018
    Abstract
    Aim: To assess the effects of different-sized iron oxide nanoparticles (IONPs) on inflammatory responses in human whole blood. Materials & methods: Human whole blood with and without 10 and 30 nm IONPs was incubated with Toll-like receptor (TLR) ligands. Cytokine levels, complement activation, reactive oxygen species and viability were determined. Results: The 10 nm IONPs enhanced the TLR2/6, TLR4 and partly TLR8-mediated cytokine production, whereas the 30 nm IONPs partly enhanced TLR2/6 and decreased TLR8-mediated cytokine production. Particle-mediated enhancement of TLR4-induced cytokines could not be explained by complement activation, but was dependent on TLR4/MD2 and CD14, as well as actin polymerization. Conclusion: The IONPs differentially affected the TLR ligand-induced cytokines, which has important implications for biomedical applications of IONPs.
    Phenotypic characterization of genetically distinct Phytophthora cinnamomi isolates from avocado
    Rodger Belisle, Phytopathology - 2018
    Abstract
    Phytophthora cinnamomi, the causal agent of Phytophthora Root Rot (PRR), is the most destructive disease of avocado worldwide. A previous study identified two genetically distinct clades of A2 mating type avocado isolates in California, however the phenotypic variation among them was not assessed. This study described the phenotype of a subset of isolates from these groups regarding growth rate, growth temperature, virulence, and fungicide sensitivity. Isolates corresponding to the A2 clade I group exhibited higher mycelial growth rate and sensitivity to higher temperatures than other isolates. Among the fungicides tested, potassium phosphite had the highest 50% effective concentration for mycelial growth inhibition and oxathiapiprolin had the lowest. Mycelial growth rate and potassium phosphite sensitivity phenotypes correlate with specific group of isolates suggesting that these traits could be a group characteristic. Moreover, isolates that are more virulent in avocado and less sensitive to potassium phosphite were identified. A detached leaf P. cinnamomi inoculation method using Nicotiana benthamiana was developed and validated providing an alternative method for assessing the virulence of a large number of isolates. This information will help avocado PRR management and assist breeding programs for the selection of rootstocks resistant against a more diverse pathogen population.
    M3-subtype muscarinic receptor activation stimulates intracellular calcium oscillations and aldosterone production in human adrenocortical HAC15 cells
    Latha M. Malaiyandi, Molecular and Cellular Endocrinology - 2018
    Abstract
    A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.
    Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil
    Alyssa Burkhardt, Phytopathology - 2018
    Abstract
    Macrophomina phaseolina is a broad-host range fungus that shows some degree of host preference on strawberry, and causes symptoms including crown rot and root rot. Recently, this pathogen has impacted strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan qPCR assay which is able to quantify as little as 2-3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification (RPA) was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%, respectively. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.
    Suitability of group-level oral fluid sampling in ruminant populations for lumpy skin disease virus detection
    K. Dietze, Veterinary Microbiology - 2018
    Abstract
    The geographic expansion of Lumpy skin disease (LSD) from the near East into the European Union highlighted again the need for appropriate disease detection tools applicable to animal host populations where access to individual animals is difficult. This is of particular importance considering that the clinical manifestation of LSD is often mild making early disease detection challenging under the above-mentioned conditions. Building on positive experiences of group-level oral fluid sampling for pathogen detection as it is known to work for swine herds and wild boar, the concept was transferred to ruminants. Two groups of six cattle were infected experimentally with Lumpy skin disease virus (LSDV) under controlled conditions. Blood as well as oropharyngeal and nasal swab samples were collected at regular intervals. Group samples were obtained by placing cotton gauze around a salt lick block provided commonly as dietary supplement. Pieces of the gauze with visible signs of manipulation were tested in parallel to samples obtained from individual animals. Genome load analysis by qPCR technology revealed LSDV detection window starting from day 2 post infection until day 28 post infection, the end of the animal trial. At the individual level, detection periods varied between animals and type of sample and included intermitted detection. The accumulative character of the alternative sampling method makes it suitable to detect LSDV DNA at group-level even at times of the infection where a selective sampling of individuals from a group – as normally done in LSD surveillance – would have most likely failed in the detection.
    Salmonella-mediated inflammation eliminates competitors for fructose-asparagine in the gut.
    Jikang Wu, Infection and Immunity - 2018
    Abstract
    Salmonella enterica elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to Salmonella during infection is dependent upon the pathogenicity islands, SPI1 and SPI2, which in turn are required to provoke inflammation. Here, we determined that F-Asn is present in mouse chow at approximately 400 pmol/mg dry weight. F-Asn is also present in the intestinal tract of germ-free mice at 2700 pmol/mg dry weight, and in the intestinal tract of conventional mice at 15 pmol/mg. These findings suggest that the mouse intestinal microbiota consumes F-Asn. We utilized heavy-labeled precursors of F-Asn to monitor its formation in the intestine, in the presence or absence of inflammation, and none was observed. Finally, we determined that some members of the class Clostridia encode F-Asn utilization pathways and are eliminated from highly inflamed Salmonella-infected mice. Collectively, our studies identify the source of F-Asn as the diet, and that Salmonella-mediated inflammation is required to eliminate competitors and allow the pathogen near-exclusive access to this nutrient.
    Development of a new qPCR method for specific detection and quantification of genetically modified maize MON863
    Patrick Guertler, Food Control - 2018
    Abstract
    Analysis of non-transgenic seed samples for the presence of genetically modified maize MON863 revealed unexpected amplification signals using an official qPCR method. These amplification signals only occured when using other master mix products as in the original validation process. DNA sequence data from an unspecific amplicon could be mapped to mitochondrial maize DNA reference sequence. Oligo sequence analysis revealed that forward primer and probe both can hybridize to the mitochondrial maize DNA leading to unspecific amplification signals in qPCR. Therefore, we designed and validated a new qPCR method for event MON863 with a LOD of 5 copies per reaction. The method shows high specificity as no unspecific amplification signal was detected after analysis of reference material for different genetically modified crops and conventional maize samples. Robustness tests were performed in two different laboratories and no effects on method performance could be observed when using different master mixes and qPCR devices, as well as with variation in oligonucleotide concentrations.
    Effectiveness of 13-pneumococcal conjugate vaccine (PCV13) against invasive pneumococcal disease in children in the Dominican Republic
    Sara Tomczyk, BMC Infectious Diseases - 2018
    Abstract
    Limited data are available on the effectiveness of 13-valent pneumococcal conjugate vaccine (PCV13) in resource-poor settings and PCV naïve populations. The Dominican Republic introduced PCV13 in September 2013 using a 2 + 1 schedule (2, 4, and 12 months) without a catch-up campaign. We evaluated PCV13 effectiveness against vaccine-type (VT) invasive pneumococcal disease (IPD) among children in the Dominican Republic.
    A protocol for quantizing total bacterial 16S rDNA in plasma as a marker of microbial translocation in vivo
    Wei Jiang, Cellular & Molecular Immunology - 2018
    Abstract
    A protocol for quantizing total bacterial 16S rDNA in plasma as a marker of microbial translocation in vivo
    Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator
    Lindsey D. Thiessen, PeerJ - 2018
    Abstract
    Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.
    Climate change favours specific fungal communities in boreal peatlands
    Asma Asemaninejad, Soil Biology and Biochemistry - 2018
    Abstract
    Fungi play a pivotal role in the carbon sequestration potential of boreal peatlands through the process of decomposition. As such, climate-driven changes in the diversity and community composition of peatland fungal communities could have substantial impacts on carbon release from these ecosystems, especially in subsurface peat that represents an important global carbon stock. We used Illumina MiSeq sequencing of rDNA to examine fungal communities after 18 months in intact peatland mesocosms subjected to conditions associated with Canada's future climate, including: warming, elevated atmospheric CO2 and lowered water table. Warming was the main driver of changes in fungal communities across three depths of the peat profile with both Ascomycota- and Basidiomycota-dominated groups becoming more homogenous under warming conditions. Specific changes in fungal functional groups, however, were temperature dependent with potential cellulose decomposers and mycorrhizal root-associated fungi from Basidiomycota dominant under warming of +4 °C, whereas there was a prevalence of potential lignocellulose decomposers and mycorrhizal root-associated fungi from Ascomycota under +8 °C warming. These climate change-induced shifts in the structure of fungal communities in favour of recalcitrant compound decomposers observed across a depth gradient, may reduce long-term carbon storage of boreal peatlands under future climate change scenarios.
    Assessment of Erysiphe necator ascospore release models for use in the Mediterranean climate of Western Oregon
    Lindsey D Thiessen, Plant Disease - 2018
    Abstract
    Predictive models have been developed in several major grape-growing regions to correlate environmental conditions to Erysiphe necator ascospore release; however, these models may not be broadly applicable in regions with different climatic conditions. To assess ascospore release in near-coastal regions of western Oregon, chasmothecia (syn. cleistothecia) were collected prior to leaf drop and placed onto natural and artificial grape trunk segments and overwintered outside. Ascospore release was monitored for three overwintering seasons using custom impaction spore traps from leaf drop (BBCH 97) until the onset of the disease epidemic in the following growing season. Airborne inoculum was concurrently monitored in a naturally infested research vineyard. Weather and ascospore release data were used to assess previously-developed models, and correlate environmental conditions to ascospore release. Ascospore release was predicted by all models prior to bud break (BBCH 08), and was observed from the first rain event following the start of inoculum monitoring until monitoring ceased. Previously developed models over-predicted ascospore release in the Willamette Valley and predicted exhaustion of inoculum prior to bud break. The magnitude of ascospore release could not be correlated to environmental conditions, thus a binary ascospore release model was developed where release is a function of the collective occurrence of the following factors within a 24-hour period: > 6 hours of cumulative leaf wetness during temperatures > 4 °C, precipitation > 2.5 mm, and relative humidity > 80%. The Oregon Model was validated using field collected ascospore datasets, and predicted ascospore release with 66% accuracy (P = 0.02). Extant methods for estimating ascospore release may not be sufficiently accurate to use as predictive models in wet, temperate climatic regions.
    Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions
    Hajeewaka C. Mendis, PLOS ONE - 2018
    Abstract
    Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104–105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.
    Efficacy of hyperbaric oxygen therapy in bacterial biofilm eradication
    Nicholas E. Sanford, Journal of Wound Care - 2018
    Abstract
    Objective: Chronic wounds typically require several concurrent therapies, such as debridement, pressure offloading, and systemic and/or topical antibiotics. The aim of this study was to examine the efficacy of hyperbaric oxygen therapy (HBOT) towards reducing or eliminating bacterial biofilms in vitro and in vivo. Method: Efficacy was determined using in vitro grown biofilms subjected directly to HBOT for 30, 60 and 90 minutes, followed by cell viability determination using propidium monoazide-polymerase chain reaction (PMA-PCR). The efficacy of HBOT in vivo was studied by searching our chronic patient wound database and comparing time-to-healing between patients who did and did not receive HBOT as part of their treatment. Results: In vitro data showed small but significant decreases in cell viability at the 30- and 90-minute time points in the HBOT group. The in vivo data showed reductions in bacterial load for patients who underwent HBOT, and ~1 week shorter treatment durations. Additionally, in patients' chronic wounds there was a considerable emergence of anaerobic bacteria and fungi between intermittent HBOT treatments. Conclusion: The data demonstrate that HBOT does possess a certain degree of biofilm killing capability. Moreover, as an adjuvant to standard treatment, more favourable patient outcomes are achieved through a quicker time-to-healing which reduces the chance of complications. Furthermore, the data provided insights into biofilm adaptations to challenges presented by this treatment strategy which should be kept in mind when treating chronic wounds. Further studies will be necessary to evaluate the benefits and mechanisms of HBOT, not only for patients with chronic wounds but other chronic infections caused by bacterial biofilms.
    Red deer (Cervus elaphus)-specific real-time PCR assay for the detection of food adulteration
    Maria Kaltenbrunner, Food Control - 2018
    Abstract
    We present a red deer-specific real-time PCR assay which, combined with a reference real-time PCR assay published previously, allows the quantification of the red deer content in food products. Thus, it can be applied to detect food adulteration. The primer/probe system of the red deer-specific real-time PCR assay amplifies a 87 bp long fragment of the protein kinase C iota gene. To eliminate cross-reactivity with closely related species, the forward primer was designed to contain one deliberate base mismatch adjacent to one red deer-specific base. The red deer-specific real-time PCR assay did not show cross-reactivity with 23 animal and 50 plant species tested. LOD and LOQ, determined by analyzing a serially diluted DNA extract containing 1% (w/w) red deer DNA in pig DNA, were 0.05% and 0.4%, respectively. The accuracy was validated by analyzing DNA mixtures, meat extract mixtures, meat mixtures and model game sausages with known red deer content. The highest accuracy was obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest. High recoveries were not only obtained for raw samples but also after subjection to thermal treatment, including brewing (15 min at 75–78 °C), boiling (90 min at 100 °C) and microwave treatment (15 s, 40 s or 2 min at 650 W). The deer-specific real-time PCR assay was found to be robust with respect to small deviations in the reaction volume or the annealing temperature and the use of another real-time PCR instrument.
    Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in Nepal
    Sarmila Tandukar, Parasitology Research - 2018
    Abstract
    This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.
    Francisella tularensis in muscle from diseased hares – a risk factor for humans?
    G.Hestvik, Epidemiology & Infection - 2017
    Abstract
    Hestvik
    Assessment of fecal pollution in Lake Pontchartrain, Louisiana
    Jia Xue, Marine Pollution Bulletin - 2017
    Abstract
    Water quality in Lake Pontchartrain was deteriorating and recreational activities along the beach were restricted by the end of the 20th Century. A microbial source tracking (MST) study was conducted to determine the fecal contamination sources at public beach of the lake, so that effective pollution control strategies can be developed. Water samples were collected over an eight-month period at ten locations along the lake in 2016 and 2017. E. coli and Enterococcus were detected in 90.6% (culture) and 97.5% (qPCR), 95.8% (culture) and 91.8% (qPCR) of water samples from all sampling sites, respectively. Significant positive relationship between E. coli and Enterococcus results was observed for both qPCR and culture methods. HF183 marker was detected in 94.3% water samples (149 of 158), with concentrations ranging from 29.0 to 6073.5GC/100ml and from 129.8 to 38,465.6GC/100ml in summer and winter, respectively. The results also indicate that significant rainfall events have the potential to supply considerable loads of fecal bacteria to lake waters. Further research is needed to determine the contribution of other animals to fecal contamination in the region.
    Mucosa-associated lymphoid tissue lymphoma translocation 1 as a novel therapeutic target for rheumatoid arthritis
    Chang Hoon Lee, Scientific Reports - 2017
    Abstract
    Emerging evidence suggests that mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) is a key regulator of inflammatory diseases; however, the pathological role of MALT1 in rheumatoid arthritis (RA) is not well understood. Consequently, this protein has not been therapeutically targeted for the treatment of RA. MALT1 plays a role in the paracaspase pathway, has proteolytic activity and is involved in the regulation of inflammatory responses. In this study, we found that the MALT1-targeting inhibitory small molecule, MALT1 selective inhibitor 2-chloro-N-[4-[5-(3,4-dichlorophenyl)-3-(2-methoxyethoxy)-1H-1,2,4-triazol-1-yl]phenylacetamide (MI-2) strongly suppresses the differentiation of monocytes into osteoclasts in the absence or presence of the inflammatory cytokine tumour necrosis factor α. Furthermore, MI-2 ameliorates pathologic bone erosion and synovitis in an in vivo mouse model of collagen-induced arthritis. Mechanistically, MI-2 blocked expression of the master osteoclast regulator – nuclear factor of activated T cells 1 (NFATc1) – by inhibiting nuclear factor κB (NF-κB), which is a critical regulator of NFATc1. These findings highlight the important regulatory role of MALT1 in the NF-κB–NFATc1-signalling axis during osteoclastogenesis and suggest that targeting MALT1 is a promising treatment option for rheumatoid arthritis.
    Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration - ScienceDirect
    Maria Kaltenbrunner, Food Chemistry - 2017
    Abstract
    The aim of the present study was to develop a real-time PCR assay for the identification and quantification of fallow deer (Dama dama) in food to detect food adulteration. Despite high sequence homology among different deer species, a fallow deer-specific primer/probe system targeting a fragment of the nuclear MC1-R gene was designed. This primer/probe system did not amplify DNA from 19 other animals and 50 edible plant species. Moderate cross-reactivity was observed for sika deer, red deer, roe deer, reindeer and wild boar. The LOD and LOQ of the real-time PCR assay were 0.1% and 0.4%, respectively. To validate the assay, DNA mixtures, meat extract mixtures, meat mixtures and model game sausages were analyzed. Satisfactory quantitative results were obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest.
    Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer
    Cécile Jovelet, PLOS ONE - 2017
    Abstract
    Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results.
    Distinct activation mechanisms trigger the trypanocidal activity of DNA damaging prodrugs
    Emma Louise Meredith, Molecular Microbiology - 2017
    Abstract
    Quinone-based compounds have been exploited to treat infectious diseases and cancer, with such chemicals often functioning as inhibitors of key metabolic pathways or as prodrugs. Here, we screened an aziridinyl-1,4-benzoquinone (ABQ) library against the causative agents of trypanosomiasis, and cutaneous leishmaniasis, identifying several potent structures that exhibited EC50 values of <100 nM. However, these compounds also displayed significant toxicity towards mammalian cells indicating that they are not suitable therapies for systemic infections. Using anti-T. brucei ABQs as chemical probes, we demonstrated that these exhibit different trypanocidal modes of action. Many functioned as type I nitroreductase (TbNTR) or cytochrome P450 reductase (TbCPR) dependent prodrugs that, following activation, generate metabolites which promote DNA damage, specifically interstrand crosslinks (ICLs). Trypanosomes lacking TbSNM1, a nuclease that specifically repairs ICLs, are hypersensitive to most ABQ prodrugs, a phenotype exacerbated in cells also engineered to express elevated levels of TbNTR or TbCPR. In contrast, ABQs that contain substituent groups on the biologically active aziridine do not function as TbNTR or TbCPR-activated prodrugs and do not promote DNA damage. By unravelling how ABQs mediate their activities, features that facilitate the desired anti-parasitic growth inhibitory effects could be incorporated into new, safer compounds targeting these neglected tropical diseases. This article is protected by copyright. All rights reserved.
    Parvovirus B19 integration into human CD36+ erythroid progenitor cells
    Tyler Janovitz, Virology - 2017
    Abstract
    The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration.
    Detection and Quantification of Acute Myeloid Leukemia-Associated Fusion Transcripts
    Jonathan Schumacher, Acute Myeloid Leukemia - 2017
    Abstract
    Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.
    DNA from dust: comparative genomics of large DNA viruses in field surveillance samples - 5ea6c536d204e515562491e37d3dacaa8380.pdf
    Utsav Pandey, Pennsylvania State University - 2016
    Abstract
    The intensification of the poultry industry over the last sixty years facilitated the evolution of increased virulence and vaccine breaks in Marek’s disease virus (MDV-1). Full genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination, and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown.
    Three-color crystal digital PCR
    J. Madic, Biomolecular Detection and Quantification - 2016
    Abstract
    Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.
    Mitochondrial DNA deletion percentage in sun exposed and non sun exposed skin
    Julia M. Powers, Journal of Photochemistry and Photobiology B: Biology - 2016
    Abstract
    The percentages of mitochondrial genomes carrying the mtDNA3895 and the mtDNA4977 (common) deletion were quantified in sun exposed and non sun exposed skin biopsies, for five cohorts of patients varying either in sun exposure profile, age or skin cancer status. Non-melanoma skin cancer diagnoses are rising in Ireland and worldwide [12] but most risk prediction is based on subjective visual estimations of sun exposure history. A quantitative objective test for pre-neoplastic markers may result in better adherence to sun protective behaviours. Mitochondrial DNA (mtDNA) is known to be subject to the loss of a significant proportion of specific sections of genetic code due to exposure to ultraviolet light in sunlight. Although one such deletion has been deemed more sensitive, another, called the mtDNA4977 or common deletion, has proved to be a more useful indicator of possible risk in this study. Quantitative molecular analysis was carried out to determine the percentage of genomes carrying the deletion using non sun exposed and sun exposed skin biopsies in cohorts of patients with high or low sun exposure profiles and two high exposure groups undergoing treatment for NMSC. Results indicate that mtDNA deletions correlate to sun exposure; in groups with high sun exposure habits a significant increase in deletion number in exposed over non sun exposed skin occurred. An increase in deletion percentage was also seen in older cohorts compared to the younger group. The mtDNA3895 deletion was detected in small amounts in exposed skin of many patients, the mtDNA4977 common deletion, although present to some extent in non sun exposed skin, is suggested to be the more reliable and easily detected marker. In all cohorts except the younger group with relatively lower sun exposure, the mtDNA4977 deletion was more frequent in sun exposed skin samples compared to non-sun exposed skin.
    Ability of device to collect bacteria from cough aerosols generated by adults with cystic fibrosis
    David N. Ku, F1000 Research - 2016
    Abstract
    Background: Identifying lung pathogens and acute spikes in lung counts remain a challenge in the treatment of patients with cystic fibrosis (CF). Bacteria from the deep lung may be sampled from aerosols produced during coughing. Methods: A new device was used to collect and measure bacteria levels from cough aerosols of patients with CF. Sputum and oral specimens were also collected and measured for comparison. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus mitis were detected in specimens using Real-Time Polymerase Chain Reaction (RT-PCR) molecular assays. Results: Twenty adult patients with CF and 10 healthy controls participated. CF related bacteria (CFRB) were detected in 13/20 (65%) cough specimens versus 15/15 (100%) sputum specimens. Commensal S. mitis was present in 0/17 (0%, p=0.0002) cough specimens and 13/14 (93%) sputum samples. In normal controls, no bacteria were collected in cough specimens but 4/10 (40%) oral specimens were positive for CFRB. Conclusions: Non-invasive cough aerosol collection may detect lower respiratory pathogens in CF patients, with similar specificity and sensitivity to rates detected by BAL, without contamination by oral CFRB or commensal bacteria.
    Temperature modulates liver lipid accumulation in Atlantic salmon (Salmo salar L.) fed low dietary levels of long-chain n-3 fatty acids
    N.h. Sissener, Aquaculture Nutrition - 2016
    Abstract
    Atlantic salmon (Salmo salar) were fed five graded levels of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), from 1.4 to 5.2% of total fatty acids (FA, 5–17 mg kg−1 feed), and grew from ~160 g to ~3000 g, with the period from 1450 g onwards conducted both at 6 °C and at 12 °C. All fish appeared healthy, and there were no diet-related differences in haematological or plasma parameters, as well as intestinal histological or gut microbiota analysis. Fish reared at 6 °C had higher accumulation of storage lipids in the liver compared to fish reared at 12 °C. Liver lipids also increased with decreasing dietary EPA + DHA at 6 °C, while there was no such relationship at 12 °C. Gene expression of SREBP1 and 2, LXR, FAS and CPT1 could not explain the differences in liver lipid accumulation. In liver polar lipids, DHA was found to be reduced when dietary EPA + DHA was <2.7% of FAs, while the level of EPA in the membranes was not affected. In conclusion, reducing dietary EPA + DHA from 5.2 to 1.4% of total FAs had a minor impact on fish health. Temperature was the factor that most affected the liver lipid accumulation, but there was also an interaction with dietary components.
    Ability of device to collect bacteria from cough aerosols generated by adults with cystic fibrosis
    David Ku, F1000 Research - 2016
    Abstract
    Background: Identifying lung pathogens and acute spikes in lung counts remain a challenge in the treatment of patients with cystic fibrosis (CF). Bacteria from the deep lung may be sampled from aerosols produced during coughing. Methods: A new device was used to collect and measure bacteria levels from cough aerosols of patients with CF. Sputum and oral specimens were also collected and measured for comparison. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus mitis were detected in specimens using Real-Time Polymerase Chain Reaction (RT-PCR) molecular assays. Results: Twenty adult patients with CF and 10 healthy controls participated. CF related bacteria (CFRB) were detected in 13/20 (65%) cough specimens versus 15/15 (100%) sputum specimens. Commensal S. mitis was present in 0/17 (0%, p=0.0002) cough specimens and 13/14 (93%) sputum samples. In normal controls, no bacteria were collected in cough specimens but 4/10 (40%) oral specimens were positive for CFRB. Conclusions: Non-invasive cough aerosol collection may detect lower respiratory pathogens in CF patients, with similar specificity and sensitivity to rates detected by BAL, without contamination by oral CFRB or commensal bacteria.
    A Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli
    Agatha N.Jassem, Journal of Clinical Microbiology - 2016
    Abstract
    Shiga toxin-producing Escherichia coli (STEC) associated enteric illness is attributed to O157 and non-O157 serotypes, however, traditional culture-based methods under-detect non-O157 STEC. Labor and consumable costs are major barriers for implementation of CDC recommendation of testing all stools for both O157 and non-O157. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening of stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10. The sensitivity was 83% for the pre-enrichment pooling strategy and 100% for the post-enrichment pooling strategy compared with individual NAAT. The difference in cycle threshold between individual and pooled NAAT results for specimens was significantly lower and more consistent for post-enrichment pooling (mean stx1 = 3.90, stx2 = 4.28) compared with pre-enrichment pooling (excluding undetected specimens, mean stx1 = 9.34, stx2 = 8.96) (P≤0.0013). Consumable cost and labor savings of 48-81% and 6-66%, respectively, were estimated for testing of 90 specimens by the post-enrichment pooled NAAT strategy based on an expected 1-2% positivity rate. A 30-day prospective, head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the post-enrichment pooled NAAT strategy. The post-enrichment pooled NAAT strategy described here is suitable for efficient, large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimates of STEC burden and, consequently, appropriate public health interventions.
    N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis
    Thulasi Warrier, Proceedings of the National Academy of Sciences - 2016
    Abstract
    Rapid and accurate identification of Xanthomonas citri subspecies citri by fluorescence in situ hybridisation
    David Waite, Letters in Applied Microbiology - 2016
    Abstract
    Citrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridisation (FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between two and three hours), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR-based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH. This article is protected by copyright. All rights reserved.
    Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay
    Kristen E. Cross, Diagnostic Microbiology and Infectious Disease - 2016
    Abstract
    Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5′-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50 fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease.
    Calcium transcriptionally regulates the biofilm machinery of Xylella fastidiosa to promote continued biofilm development in batch cultures
    Jennifer Parker, Environmental Microbiology - 2016
    Abstract
    The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4 mM Ca supplementation produced denser biofilms that were still developing at 96 h, while biofilm in non-supplemented media had reached the dispersal stage by 72 h. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-h time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in nonsupplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa.
    Calcium transcriptionally regulates the biofilm machinery of Xylella fastidiosa to promote continued biofilm development in batch cultures
    Jennifer Parker, Environmental Microbiology - 2016
    Abstract
    The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4 mM Ca supplementation produced denser biofilms that were still developing at 96 h, while biofilm in non-supplemented media had reached the dispersal stage by 72 h. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-h time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in nonsupplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa.
    Besnoitia besnoiti infections activate primary bovine endothelial cells and promote PMN adhesion and NET formation under physiological flow condition - Springer
    P. Maksimov, Parasitology Research - 2016
    Abstract
    Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle that mainly infects host endothelial cells during acute infection. We here analyzed early innate immune reactions of B. besnoiti-infected primary bovine umbilical vein endothelial cells (BUVEC). B. besnoiti infections significantly activated BUVEC since the gene transcripts of several adhesion molecules (P-selectin, intercellular adhesion molecule 1(ICAM-1)), chemokines (CXCL1, CXCL8, CCL5), and of COX-2 were significantly upregulated during in vitro infection. Overall, the highest upregulation of most transcripts was observed at 24 or 48 h post infection (p.i.). Enhanced adhesion molecule expression in infected host cells was confirmed by PMN adhesion assays being performed under physiological flow conditions revealing a significantly increased PMN adhesion on B. besnoiti-infected BUVEC layers at 24 h p.i. Furthermore, we were able to illustrate neutrophil extracellular traps (NETs) being released by PMN under physiological flow conditions after adhesion to B. besnoiti-infected BUVEC layers. The present study shows that B. besnoiti infections of primary BUVEC induce a cascade of pro-inflammatory reactions and triggers early innate immune responses.
    Inter-Population Variability of Endosymbiont Densities in the Asian Citrus Psyllid (Diaphorina citri Kuwayama)
    Chia-Ching Chu, Microbial Ecology - 2016
    Abstract
    The Asian citrus psyllid (Diaphorina citri Kuwayama) is an insect pest capable of transmitting Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening in North America. D. citri also harbors three endosymbionts, Wolbachia, Candidatus Carsonella ruddii, and Candidatus Profftella armatura, which may influence D. citri physiology and fitness. Although genomic researches on these bacteria have been conducted, much remains unclear regarding their ecology and inter-population variability in D. citri. The present work examined the densities of each endosymbiont in adult D. citri sampled from different populations using quantitative PCR. Under field conditions, the densities of all three endosymbionts positively correlated with each other, and they are associated with D. citri gender and locality. In addition, the infection density of CLas also varied across populations. Although an analysis pooling D. citri from different populations showed that CLas-infected individuals tended to have lower endosymbiont densities compared to uninfected individuals, the difference was not significant when the population was included as a factor in the analysis, suggesting that other population-specific factors may have stronger effects on endosymbiont densities. To determine whether there is a genetic basis to the density differences, endosymbiont densities between aged CLas-negative females of two D. citri populations reared under standardized laboratory conditions were compared. Results suggested that inter-population variability in Wolbachia infection density is associated with the genotypes of the endosymbiont or the host. Findings from this work could facilitate understanding of D. citri-bacterial associations that may benefit the development of approaches for managing citrus greening, such as prevention of CLas transmission.
    Targeting HPV-Infected Cervical Cancer Cells with PEGylated Liposomes Encapsulating siRNA and the Role of siRNA Complexation with Polyethylenimine
    Rachel Levine, Bioengineering & Translational Medicine - 2016
    Abstract
    The greatest obstacle to clinical application of cancer gene therapy is lack of effective delivery tools. Gene delivery vehicles must protect against degradation, avoid immunogenic effects and prevent off target delivery which can cause harmful side effects. PEGylated liposomes have greatly improved tumor localization of small molecule drugs and are a promising tool for nucleic acid delivery as the polyethylene glycol (PEG) coating protects against immune recognition and blood clearance. In this study, small interfering RNA (siRNA) was fully encapsulated within PEGylated liposomes by complexing the siRNA with a cationic polymer, polyethyleneimine (PEI), before encapsulation. Formation methods and material compositions were then investigated for their effects on encapsulation. This technology was translated for protective delivery of siRNA designed for human papillomavirus (HPV) viral gene silencing and cervical cancer treatment. PEGylated liposomes encapsulating siRNA were functionalized with the AG86 targeting peptide-amphiphile which binds to the α6β4 integrin, a cervical cancer biomarker. It was found that both targeting and polymer complexation before encapsulation were critical components to effective transfection. This article is protected by copyright. All rights reserved.
    Identification of low abundance microbiome in clinical samples using whole genome sequencing
    Chao Zhang, BioMed Central - 2015
    Abstract
    Identifying the microbiome composition from primary tissues directly affords an opportunity to study the causative relationships between the host microbiome and disease. However, this is challenging due the low abundance of microbial DNA relative to the host. We present a systematic evaluation of microbiome profiling directly from endoscopic biopsies by whole genome sequencing. We compared our methods with other approaches on datasets with previously identified microbial composition. We applied this approach to identify the microbiome from 27 stomach biopsies, and validated the presence of Helicobacter pylori by quantitative PCR. Finally, we profiled the microbial composition in The Cancer Genome Atlas gastric adenocarcinoma cohort.
    Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters
    Marta Rusiñol, Food and Environmental Virology - 2015
    Abstract
    This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.
    Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor
    Florian A. Lempp, Journal of Hepatology - 2015
    Abstract
    Background & Aims Hepatitis B Virus (HBV) is a major human pathogen restricted to hepatocytes. Expression of the specific receptor human sodium taurocholate co-transporting polypeptide (hNTCP) in mouse hepatocytes renders them susceptible to Hepatitis Delta Virus (HDV), a satellite of HBV; however, HBV remains restricted at an early stage of replication. This study aims at clarifying whether this restriction is caused by the lack of a dependency factor or the activity of a restriction factor. Methods Six hNTCP-expressing mouse and human cell lines were generated and functionally characterized. By fusion with replication-supporting but non-infectable HepG2 cells, we analysed the ability of these heterokaryonic cells to fully support HBV replication by HBcAg expression and HBsAg/HBeAg secretion. Results While hNTCP-expression in three mouse cell lines and the non-hepatic human HeLa cells conferred susceptibility to HDV, HBV replication was still restricted. Upon fusion of refractive cells to HepG2 cells, all heterokaryonic cells supported receptor-mediated infection with HBV. hNTCP was provided by the mouse cells and replication competence came from the HepG2 cell line. Transfection of a covalently closed circular DNA (cccDNA)-like molecule into non-susceptible cells promoted gene expression, indicating that the limiting step is upstream of cccDNA formation. Conclusions In addition to the expression of hNTCP, establishment of HBV infection in mouse and non-hepatocytic human cell lines requires supplementation with a dependency factor and is not limited by a restriction factor. This result opens new avenues for the development of a fully permissive immunocompetent HBV mouse model.
    Evaluation of a triplex real-time PCR system to detect the plant-pathogenic molds Alternaria spp., Fusarium spp. and C. purpurea
    Sabrina Grube, Journal of Microbiological Methods - 2015
    Abstract
    This article describes the development of a triplex real-time PCR system for the simultaneous detection of three major plant-pathogenic mold genera (Alternaria spp., Fusarium spp. and the species Claviceps purpurea). The designed genus-specific primer–probe systems were validated for sensitivity, specificity and amplification in the presence of background DNA.
    Livers provide a reliable matrix for real-time PCR confirmation of avian botulism
    Caroline Le Maréchal, Anaerobe - 2015
    Abstract
    Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis.
    SOX9 modulates the expression of key transcription factors required for heart valve development | Development
    Victoria C. Garside, Development - 2015
    Abstract
    Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor, SOX9, is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and disrupts valve formation. Despite this important role, little is known regarding how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in embryonic day (E) 12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Consequently, we compared regions of putative SOX9 DNA-binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context–independent SOX9 interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9 interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom/Evi1 and Pitx2. Together, our data identifies SOX9 coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation.
    Real-time PCR assays for the detection of Puccinia psidii — Plant Disease
    Dr. Jeyaseelan Baskarathevan, Plant Disease - 2015
    Abstract
    Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family and continues to show an increase in geographic range and considered to be a significant threat to Myrtaceae plants worldwide. In this study we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Dependent on gene target qPCR was able to detect down to 0.011 pg of P. psidii DNA. All qPCR assays specifically detected all P. psidii strains irrespective of their geographical origin and no cross amplifications were observed from other closely related Puccinia species or from plant species in the Myrtaceae family. The most optimum qPCR assay was shown to be highly specific, repeatable and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow for the co-amplification of cytochrome oxidase (Cox) gene sequence from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection before the development of sporulating rust pustules.
    ALLELIC DISCRIMINATION ASSAYS FOR MRSA STRAINS
    Paul Keim, United States Patent Application 20150259727 - 2015
    Abstract
    The present invention provides assays, methods and kits that may be used to detect and differentiate MRSA isolates, e.g., USA100, USA300 and USA600 strains.
    Conventional PCR
    InDel markers for monitoring the introgression of downy mildew resistance from wild relatives into grape varieties
    Serena Foria, Molecular Breeding - 2018
    Abstract
    We identified haplotype-tagging insertion/deletions (InDels) for downy mildew resistance (Rpv3-1) in grapevine and converted them into InDel markers. InDel-25,941 and InDel-26,032 were validated by fragment analysis via capillary electrophoresis in 174 varieties of Vitis vinifera, 50 resistant varieties of the ‘Seibel 4614’ lineage that share Rpv3-1 by descent, and in 83 Vitis accessions. Amplicon sequencing of ancestral and derived alleles revealed that both mutations were caused by deletions. The 25,941-deletion is most likely recent. The derived allele is present only in resistant varieties obtained from ‘Seibel 4614’ and has originated in North American populations through two successive deletions within a predicted multiple stem-loop ssDNA structure, consisting of three nearby short inverted repeats, which shortened the ancestral DNA stepwise. The 26,032-deletion is more ancient. The derived allele is always present in resistant varieties of the ‘Seibel 4614’ lineage, completely absent from V. vinifera, not found in other North American accessions, and rarely present in Asian species. It may have originated in a common ancestral population before the continental disjunction, followed by incomplete lineage sorting, or in either lineage followed by introgression via secondary contacts. Genotyping with these markers does not require special instruments or chemistry for routine screening in breeding practice. Differences in amplicon size between grapes that carry or do not carry Rpv3-1 are detectable via standard agarose gel electrophoresis, or classical melting curve analysis using nonsaturating fluorescent dyes. The recombination rate between each marker and the trait locus is 0.118% for InDel-25,941 and 0.071% for InDel-26,032.
    High-throughput marker assays for FaRPc2-mediated resistance to Phytophthora crown rot in octoploid strawberry
    Young-Hee Noh, Molecular Breeding - 2018
    Abstract
    Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is a destructive disease of the allo-octoploid cultivated strawberry (Fragaria ×ananassa Duch). Many major strawberry cultivars grown worldwide are susceptible to PhCR. Resistance is conferred by the recently-discovered FaRPc2 locus, but high-throughput markers are not yet available for marker-assisted breeding. In the current study, we developed DNA markers for two haplotypes at the FaRPc2 locus associated with resistance, H2 and H3. Marker validation and marker-assisted selection were performed in University of Florida (UF) breeding population. Seven single nucleotide polymorphism-based high resolution melting (HRM) markers linked to H2 and four HRM markers for H3 were developed. One HRM marker, RPCHRM3 linked to H3, was converted to a Kompetitive Allele Specific PCR (KASP) marker. To further examine the utility of the markers, they were screened in University of California Davis cultivars with known phenotypes as well as in 20 diverse accessions with phenotypes that are reported in the literature and that are preserved at the USDA-ARS National Clonal Germplasm Repository, in Corvallis, Oregon. The most informative markers for FaRPc2 resistance are being implemented in the UF strawberry breeding program to improve PhCR resistance.
    Development of High-Throughput SNP Genotyping Assays for Rapid Detection of Strawberry Colletotrichum Species and the G143A Mutation
    Bruna Balen Forcelini, Phytopathology - 2018
    Abstract
    Colletotrichum species cause major diseases of strawberry and disease management depends on the species present. However, species identification based on symptoms and spore morphology is difficult. Therefore, development of molecular techniques for trustworthy and high-throughput identification of Colletotrichum species is vital for the accurate diagnosis. A High-Resolution Melting (HRM) assay was developed for simultaneous identification and differentiation of Colletotrichum species from fungal colonies or from symptomatic strawberry tissue. HRM markers were designed based on the ITS region of C. acutatum and C. gloeosporioides from strawberry, and accurately identified and differentiated the two species. In addition, for the rapid detection of a single nucleotide polymorphism (SNP) in the cytochrome b (cytb) gene of C. acutatum and C. gloeosporioides associated with resistance to quinone-outside inhibitor fungicides, an endpoint SNP genotyping analysis was developed. The HRM and endpoint SNP genotyping assays are useful methods that can be implemented in plant diagnostic clinics for the rapid and accurate identification of Colletotrichum species and detection of the G143A mutation in the cytb gene of C. acutatum and C. gloeosporioides.
    FaRCg1: a quantitative trait locus conferring resistance to Colletotrichum crown rot caused by Colletotrichum gloeosporioides in octoploid strawberry
    Ashlee Anciro, Theoretical and Applied Genetics - 2018
    Abstract
    Colletotrichum crown rot (CCR) is an important disease of strawberry (Fragaria ×ananassa) throughout the Southeastern US and in subtropical climates around the world, where hot and humid conditions facilitate rapid disease development. Yet no resistance loci have been described to date, as genetic studies have been historically difficult in allo-octoploid (2n = 8x = 56) strawberry. In the present study, we investigate the genetic architecture of resistance to CCR. Four population sets from the University of Florida were inoculated in four different seasons from 2013–2014 to 2016–2017. Two large, multiparental discovery population sets were used for QTL discovery, and two validation sets of cultivars and advanced selections representing the parent pool of the breeding program were also assessed. Subgenome-specific single-nucleotide polymorphism (SNP) markers were mapped, and FlexQTL™ software was utilized to perform a Bayesian, pedigree-based QTL analysis. A quantitative trait locus on linkage group 6B, which we name FaRCg1, accounts for most of the genetic variation for resistance in the discovery sets (26.8–29.8% in 2013–2014 and 17% in 2015–2016). High-throughput marker assays were developed for the most significant SNPs which correlated with the mode of the QTL region. The discovery and characterization of the FaRCg1 locus and the molecular tools developed from it will be utilized to achieve increased genetic gains for resistance.
    Rapid viral symbiogenesis via changes in parasitoid wasp genome architecture
    Gaelen R. Burke, Molecular Biology and Evolution - 2018
    Abstract
    Viral genome integration provides a complex route to biological innovation that has rarely but repeatedly occurred in one of the most diverse lineages of organisms on the planet, parasitoid wasps. We describe a novel endogenous virus in braconid wasps derived from pathogenic alphanudiviruses. Limited to a subset of the genus Fopius, this recent acquisition allows an unprecedented opportunity to examine early endogenization events. Massive amounts of virus-like particles (VLPs) are produced in wasp ovaries. Unlike most endogenous viruses of parasitoid wasps, the VLPs do not contain DNA, translating to major differences in parasitism-promoting strategies. Rapid changes include genomic rearrangement, loss of DNA processing proteins, and wasp control of viral gene expression. These events precede the full development of tissue-specific viral gene expression observed in older associations. These data indicate that viral endogenization can rapidly result in functional and evolutionary changes associated with genomic novelty and adaptation in parasitoids.
    Characterization of benthic biogeochemistry and ecology at three methane seep sites on the Northern U.S. Atlantic margin
    D. McVeigh, Deep Sea Research Part II: Topical Studies in Oceanography - 2018
    Abstract
    Several hundred methane seeps were recently discovered along the U.S. Atlantic margin, a passive margin without a major hydrocarbon basin. These seeps represent a compelling opportunity to compare and contrast geochemical parameters, as well as the distribution and composition of associated chemosynthetic ecosystems, across geologically distinct seeps. Specifically, this study characterized the physical, chemical, geological, and biological features at Veatch Canyon, New England, and Shallop Canyon methane seep sites using a suite of state-of-the-art sampling and communication tools: AUV Sentry, HOV Alvin, a video-equipped multicore, and a real-time telepresence connection to the Inner Space Center (ISC) at the University of Rhode Island. Water column backscatter data collected by AUV Sentry confirmed previously detected gas emission and further indicated the presence of 13 actively emitting gas seeps within the Veatch Canyon survey area, 5 within the New England survey area, and 15 within the Shallop Canyon survey area. Complementary high-resolution seafloor bathymetry and backscatter mapping data indicated highly rugose morphology at all surveyed seep sites including the presence of extensive rock outcrops and bivalve beds. Video collected by HOV Alvin provided visual confirmation of gas plumes and the presence of chemosynthetic communities, including microbial mats and mussel beds, as well as large outcrops of authigenic carbonate rock surrounding locations of discrete gas emission. Bathymodiolus was the dominant species observed at the Veatch Canyon methane seep site, while the deep-sea red crab, Chaceon quinquedens, dominated the New England and Shallop Canyon seep sites. Elevated pore water sulfide concentrations suggested stimulation of sulfate-coupled anaerobic oxidation of methane (AOM) and/or degradation of organic matter in seep sediments relative to non-seep sediments; AOM was observed directly within overlying authigenic carbonate rocks. DNA sequencing of the surface sediments revealed diverse sulfide-oxidizing bacteria similar to the taxa described previously at other methane seeps, including members of the Thiotrichales and Campylobacterales. Together, these analyses provide one of the first interdisciplinary descriptions of sites within this massive seep system, setting the stage for more targeted, hypothesis-driven investigations. In this context, telepresence facilitated interdisciplinary research between specialists at the ISC and scientists and engineers at sea, enabling researchers to transfer data and maximize research efforts with sampling tools at sea. Our findings suggest that although the geological setting is distinct, the surficial microbial and macrofaunal communities closely resemble those of previously described methane seeps.
    VKORC1 sequence variants associated with resistance to anticoagulant rodenticides in Irish populations of Rattus norvegicus and Mus musculus domesticus
    Jean Mooney, Scientific Reports - 2018
    Abstract
    While resistance to anticoagulant rodenticides is known to occur in many European populations of Norway rat and house mouse, to-date no data is available on the occurrence in Ireland of such resistance. No genetic evidence for the occurrence of resistance was found in 65 Norway rat samples analysed, indicative of an absence, or low prevalence, of resistance in rats in at least the Eastern region of the island of Ireland. The presence of two of the most commonly found amino acid substitutions Leu128Ser and Tyr139Cys associated with house mouse resistance to anticoagulant rodenticides was confirmed. The occurrence of two such mutations is indicative of the occurrence of resistance to anticoagulant rodenticides in house mice in the Eastern region of the island of Ireland.
    Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection
    Ulrike C. Lange, Virus Research - 2018
    Abstract
    HIV infection is characterized by accumulation of proviral sequences within the human host genome. Integration of viral-derived DNA occurs at preferential loci, suggesting a site-specific crosstalk between viral sequences and human genes. We here describe a genome engineering workflow to generate models for HIV-1 infection that for the first time recapitulate proviral integration at selected genomic loci and provide unique tools to study effects of HIV proviral integration site choice. Using this workflow, we have derived two BACH2–HIV-1 reporter models that mimic largely latent integration in the clinically relevant BACH2 gene locus, which has been associated with recurrent integration and HIV-reservoir maintenance in chronically infected patients.
    Comparison of the oral microbiome in mouthwash and whole saliva samples
    Xiaozhou Fan, PLOS ONE - 2018
    Abstract
    Population-based epidemiologic studies can provide important insight regarding the role of the microbiome in human health and disease. Buccal cells samples using commercial mouthwash have been obtained in large prospective cohorts for the purpose of studying human genomic DNA. We aimed to better understand if these mouthwash samples are also a valid resource for the study of the oral microbiome. We collected one saliva sample and one Scope mouthwash sample from 10 healthy subjects. Bacterial 16S rRNA genes from both types of samples were amplified, sequenced, and assigned to bacterial taxa. We comprehensively compared these paired samples for bacterial community composition and individual taxonomic abundance. We found that mouthwash samples yielded similar amount of bacterial DNA as saliva samples (p from Student’s t-test for paired samples = 0.92). Additionally, the paired samples had similar within sample diversity (p from = 0.33 for richness, and p = 0.51 for Shannon index), and clustered as pairs for diversity when analyzed by unsupervised hierarchical cluster analysis. No significant difference was found in the paired samples with respect to the taxonomic abundance of major bacterial phyla, Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria (FDR adjusted q values from Wilcoxin signed-rank test = 0.15, 0.15, 0.87, 1.00 and 0.15, respectively), and all identified genera, including genus Streptococcus (q = 0.21), Prevotella (q = 0.25), Neisseria (q = 0.37), Veillonella (q = 0.73), Fusobacterium (q = 0.19), and Porphyromonas (q = 0.60). These results show that mouthwash samples perform similarly to saliva samples for analysis of the oral microbiome. Mouthwash samples collected originally for analysis of human DNA are also a resource suitable for human microbiome research.
    The non-motor adaptor HMMR dampens Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation
    Helen Chen, Molecular Biology of the Cell - 2018
    Abstract
    Mitotic spindle assembly and organization require forces generated by motor proteins. The activity of these motors is regulated by non-motor adaptor proteins. However, there are limited studies reporting the functional importance of adaptors on the balance of motor forces and the promotion of faithful and timely cell division. Here, we show that genomic deletion or siRNA silencing of the non-motor adaptor Hmmr/HMMR disturbs spindle microtubule organization and bipolar chromosome-kinetochore attachments with a consequent elevated occurrence of aneuploidy. Rescue experiments show a conserved motif in HMMR is required to generate inter-kinetochore tension and promote anaphase entry. This motif bears high homology with the kinesin Kif15 and is known to interact with TPX2, a spindle assembly factor. We find that HMMR is required to dampen kinesin Eg5-mediated forces through localizing TPX2 and promoting the formation of inhibitory TPX2-Eg5 complexes. In HMMR-silenced cells, K-fiber stability is reduced while the frequency of unattached chromosomes and the time needed for chromosome segregation are both increased. These defects can be alleviated in HMMR-silenced cells with chemical inhibition of Eg5, but not through the silencing of Kif15. Together, our findings indicate that HMMR balances Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation.
    Distribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almonds
    Adeline Picot, International Journal of Food Microbiology - 2017
    Abstract
    To identify predominant isolates for potential use as biocontrol agents, Aspergillus flavus isolates collected soils of almond, pistachio and fig orchard in the Central Valley of California were tested for their membership to 16 atoxigenic vegetative compatibility groups (VCGs), including YV36, the VCG to which AF36, an atoxigenic isolate commercialized in the United States as biopesticide, belongs. A surprisingly large proportion of isolates belonged to YV36 (13.3%, 7.2% and 6.6% of the total almond, pistachio and fig populations, respectively), while the percentage of isolates belonging to the other 15 VCGs ranged from 0% to 2.3%. In order to gain a better insight into the structure and diversity of atoxigenic A. flavus populations and to further identify predominant isolates, seventeen SSR markers were then used to genetically characterize AF36, the 15 type-isolates of the VCGs and 342 atoxigenic isolates of the almond population. There was considerable genetic diversity among isolates with a lack of differentiation among micro-geographical regions or years. Since isolates sharing identical SSR profiles from distinct orchards were rare, we separated them into groups of at least 3 closely-related isolates from distinct orchards that shared identical alleles for at least 15 out of the 17 loci. This led to the identification of 15 groups comprising up to 24 closely-related isolates. The group which contained the largest number of isolates were members of YV36 while five groups were also found to be members of our studied atoxigenic VCGs. These results suggest that these 15 groups, and AF36 in particular, are well adapted to various environmental conditions in California and to tree crops and, as such, are good candidates for use as biocontrol agents.
    Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Alexandre B. Roland, Ecology and Evolution - 2017
    Abstract
    Some South American poison frogs (Dendrobatidae) are chemically defended and use bright aposematic colors to warn potential predators of their unpalatability. Aposematic signals are often frequency-dependent where individuals deviating from a local model are at a higher risk of predation. However, extreme diversity in the aposematic signal has been documented in poison frogs, especially in Oophaga. Here, we explore the phylogeographic pattern among color-divergent populations of the Little Devil poison frog Oophaga sylvatica by analyzing population structure and genetic differentiation to evaluate which processes could account for color diversity within and among populations. With a combination of PCR amplicons (three mitochondrial and three nuclear markers) and genome-wide markers from a double-digested RAD (ddRAD) approach, we characterized the phylogenetic and genetic structure of 199 individuals from 13 populations (12 monomorphic and 1 polymorphic) across the O. sylvatica distribution. Individuals segregated into two main lineages by their northern or southern latitudinal distribution. A high level of genetic and phenotypic polymorphism within the northern lineage suggests ongoing gene flow. In contrast, low levels of genetic differentiation were detected among the southern lineage populations and support recent range expansions from populations in the northern lineage. We propose that a combination of climatic gradients and structured landscapes might be promoting gene flow and phylogenetic diversification. Alternatively, we cannot rule out that the observed phenotypic and genomic variations are the result of genetic drift on near or neutral alleles in a small number of genes.
    Genomic Characterization of Crimean–Congo Hemorrhagic Fever Virus in Hyalomma Tick from Spain, 2014
    Maria N.B. Cajimat, Vector-Borne and Zoonotic Diseases - 2017
    Abstract
    Crimean–Congo hemorrhagic fever (CCHF) is a severe tick-borne disease caused by CCHF virus (CCHFV). Ticks in the genus Hyalomma are the main vectors and reservoirs of CCHFV. In Spain, CCHFV was first detected in Hyalomma ticks from Cáceres in 2010. Subsequently, two autochthonous CCHF cases were reported in August 2016. In this study, we describe the characterization of the CCHFV genome directly from Hyalomma lusitanicum collected in Cáceres in 2014. Phylogenetic analyses reveal a close relationship with clade III strains from West Africa, with an estimated divergence time of 50 years. The results of this work suggest that CCHFV has been circulating in Spain for some time, and most likely originated from West Africa.
    Effect of freshwater mussels on the vertical distribution of anaerobic ammonia oxidizers and other nitrogen-transforming microorganisms in upper Mississippi river sediment [PeerJ]
    Ellen M. Black, PeerJ - 2017
    Abstract
    Targeted qPCR and non-targeted amplicon sequencing of 16S rRNA genes within sediment layers identified the anaerobic ammonium oxidation (anammox) niche and characterized microbial community changes attributable to freshwater mussels. Anammox bacteria were normally distributed (Shapiro-Wilk normality test, W-statistic =0.954, p = 0.773) between 1 and 15 cm depth and were increased by a factor of 2.2 (p < 0.001) at 3 cm below the water-sediment interface when mussels were present. Amplicon sequencing of sediment at depths relevant to mussel burrowing (3 and 5 cm) showed that mussel presence reduced observed species richness (p = 0.005), Chao1 diversity (p = 0.005), and Shannon diversity (p < 0.001), with more pronounced decreases at 5 cm depth. A non-metric, multidimensional scaling model showed that intersample microbial species diversity varied as a function of mussel presence, indicating that sediment below mussels harbored distinct microbial communities. Mussel presence corresponded with a 4-fold decrease in a majority of operational taxonomic units (OTUs) classified in the phyla Gemmatimonadetes, Actinobacteria, Acidobacteria, Plantomycetes, Chloroflexi, Firmicutes, Crenarcheota, and Verrucomicrobia. 38 OTUs in the phylum Nitrospirae were differentially abundant (p < 0.001) with mussels, resulting in an overall increase from 25% to 35%. Nitrogen (N)-cycle OTUs significantly impacted by mussels belonged to anammmox genus Candidatus Brocadia, ammonium oxidizing bacteria family Nitrosomonadaceae, ammonium oxidizing archaea genus Candidatus Nitrososphaera, nitrite oxidizing bacteria in genus Nitrospira, and nitrate- and nitrite-dependent anaerobic methane oxidizing organisms in the archaeal family “ANME-2d” and bacterial phylum “NC10”, respectively. Nitrosomonadaceae (0.9-fold (p < 0.001)) increased with mussels, while NC10 (2.1-fold (p < 0.001)), ANME-2d (1.8-fold (p < 0.001)), and Candidatus Nitrososphaera (1.5-fold (p < 0.001)) decreased with mussels. Co-occurrence of 2-fold increases in Candidatus Brocadia and Nitrospira in shallow sediments suggests that mussels may enhance microbial niches at the interface of oxic–anoxic conditions, presumably through biodeposition and burrowing. Furthermore, it is likely that the niches of Candidatus Nitrososphaera and nitrite- and nitrate-dependent anaerobic methane oxidizers were suppressed by mussel biodeposition and sediment aeration, as these phylotypes require low ammonium concentrations and anoxic conditions, respectively. As far as we know, this is the first study to characterize freshwater mussel impacts on microbial diversity and the vertical distribution of N-cycle microorganisms in upper Mississippi river sediment. These findings advance our understanding of ecosystem services provided by mussels and their impact on aquatic biogeochemical N-cycling.
    KRAS mutations in blood circulating cell-free DNA: a pancreatic cancer case-control study
    Florence Le Calvez-Kelm, Impact Journals - 2016
    Abstract
    The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series.
    Hidden diversity: parasites of stream arthropods
    Daniel S. Grabner, Freshwater Biology - 2016
    Abstract
    * Parasite communities of aquatic macroinvertebrates have so far rarely been studied, even if these host organisms are of high relevance for the ecology of freshwater systems and their parasites likely affect the host populations and communities. Therefore, this study addresses this ‘hidden diversity’ in aquatic arthropods of a stream ecosystem in North Rhine-Westphalia, Germany. * Samples of benthic organisms were taken, and the most abundant host species of major invertebrate groups (amphipods and larvae of chironomids, beetles, caddisflies, mayflies, stoneflies) were tested by PCR for different parasite taxa [microsporidians, acanthocephalans (only amphipods), trematodes, nematodes]. Furthermore, the possible link between prevalence and host feeding type was investigated for each parasite group using a newly developed ‘feeding type score’. * In total, 10 species of aquatic insect larvae and two amphipods of the genus Gammarus spp. were tested and 16 different isolates of microsporidians, three acanthocephalan species, six species of trematodes and one nematode were found. Microsporidians were present in all host species with prevalences ranging from 20 to 100%. Only one presumably specific host–microsporidian association was found for the mayfly Ephemera danica and three isolates were detected exclusively in amphipods. Three acanthocephalan species were detected in the amphipods with prevalences of up to 5.3%. All tested host species were infected with trematodes (prevalences 25–100%), except the caddisfly Sericostoma sp. Nematodes were detected in five host species with prevalences ranging from 14 to 80%. For several positive samples, no sequencing result could be obtained, especially for trematodes and nematodes, therefore, the actual parasite diversity might be even higher. * Active filter feeding showed the highest feeding type prevalence score for microsporidia and nematodes, and passive filter feeders for trematodes, but these results have to be interpreted cautiously as only one active and one passive filter feeder species each were present in the sample. * The impressive number of 26 parasite species (12 microsporidians and 10 helminths) were detected in 12 host species, mostly with surprisingly high prevalences. These results illustrate the ‘hidden diversity’ in aquatic ecosystems and highlight the importance of parasites for aquatic ecological research and the need for studies on the effects of mixed parasite infections on the host populations and communities.
    Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry : Scientific Reports
    Charles Hawkins, Scientific Reports - 2016
    Abstract
    Fragaria vesca is a species of diploid strawberry being developed as a model for the octoploid garden strawberry. This work sequenced and compared the genomes of three F. vesca accessions: ‘Hawaii 4′, ‘Rügen’, and ‘Yellow Wonder’. Genome-scale analyses of shared and distinct SNPs among these three accessions have revealed that ‘Rügen’ and ‘Yellow Wonder’ are more similar to each other than they are to ‘Hawaii 4’. Though all three accessions are inbred seven generations, each accession still possesses extensive heterozygosity, highlighting the inherent differences between individual plants even of the same accession. The identification of the impact of each SNP as well as the large number of Indel markers provides a foundation for locating candidate mutations underlying phenotypic variations among these F. vesca accessions and for mapping new mutations generated through forward genetics screens. Through systematic analysis of SNP variants affecting genes in anthocyanin biosynthesis and regulation, a candidate SNP in FveMYB10 was identified and then functionally confirmed to be responsible for the yellow color fruits made by many F. vesca accessions. As a whole, this study provides further resources for F. vesca and establishes a foundation for linking traits of economic importance to specific genes and variants.
    HETEROZYGOUS MODIFICATIONS OF TUMOR SUPPRESSOR GENES - Recombinetics, Inc.
    Adrienne Leigh Biggar, United States Patent - 2016
    Abstract
    Animals genomically modified to have heterozygous modifications of one or more tumor suppressor genes are disclosed.
    The 5-HTTLPR and BDNF polymorphisms moderate the association between uncinate fasciculus connectivity and antidepressants treatment response in major depression
    Erica L. Tatham, European Archives of Psychiatry and Clinical Neuroscience - 2016
    Abstract
    Symptom improvement in depression due to antidepressant treatment is highly variable and clinically unpredictable. Linking neuronal connectivity and genetic risk factors in predicting antidepressant response has clinical implications. Our investigation assessed whether indices of white matter integrity, serotonin transporter-linked polymorphism (5-HTTLPR) and brain-derived neurotrophic factor (BDNF) val66met polymorphism predicted magnitude of depression symptom change following antidepressant treatment. Fractional anisotropy (FA) was used as an indicator of white matter integrity and was assessed in the uncinate fasciculus and superior longitudinal fasciculus using tract-based spatial statistics (TBSS) and probabilistic tractography. Forty-six medication-free patients with major depressive disorder participated in a diffusion tensor imaging scan prior to completing an 8-week treatment regime with citalopram or quetiapine XR. Indexed improvements in Hamilton Depression Rating Scale score from baseline to 8-week endpoint were used as an indicator of depression improvement. Carriers of the BDNF met allele exhibited lower FA values in the left uncinate fasciculus relative to val/val individuals [F(1, 40) = 7.314, p = 0.009]. Probabilistic tractography identified that higher FA in the left uncinate fasciculus predicted percent change in depression severity, with BDNF moderating this association [F(3, 30) = 3.923, p = 0.018]. An interaction between FA in the right uncinate fasciculus and 5-HTTLPR also predicted percent change in depression severity [F(5, 25) = 5.315, p = 0.002]. Uncorrected TBSS results revealed significantly higher FA in hippocampal portions of the cingulum bundle in responders compared to non-responders (p = 0.016). The predictive value of prefrontal and amygdala/hippocampal WM connectivity on antidepressant treatment response may be influenced by 5-HTTLPR and BDNF polymorphisms in MDD.
    A comprehensive survey of larval digenean trematodes and their snail hosts in central Alberta, Canada
    Michelle A. Gordy, Parasitology Research - 2016
    Abstract
    Digenean trematode distributions, compatibility profiles with their snail hosts, and complete life cycles remain mysteries in many parts of the world. Surveys of digenean biology and ecology provide further insight and perspective into just how incredibly diverse and important helminth parasites are in shaping local ecosystems. Past surveys have provided substantial characterizations of adult digeneans within their definitive hosts, and many now have contributed toward furthering our understanding of larval digeneans within their intermediate host communities. However, much information about the diversity of digeneans and their relationships with their snail intermediate hosts are lacking in many locations across the globe. This is certainly true in Canada, where few records related to digenean-snail relationships exist. Currently, there is a need for more information about the presence and distribution of digeneans across Canada, and how this compares to other parts of North America and beyond. To address this diversity gap in Western Canada, six lakes within central Alberta were surveyed for the presence of snails and larval digenean species and their associations. This investigation into the diversity of digeneans utilized a combined approach of morphological and molecular tools to identify 39 digenean species among five snail host species, from biweekly collections, taken over the course of 2 years (2013–2014). Here, digenean-snail combinations, presence, and distribution across sampling sites and lakes are reported. Overall, this survey contributes new information toward digenean-snail compatibility, life cycles, and distribution in Northern lake ecosystems within North America.
    Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay
    Andries Blokzijl, PLOS ONE - 2016
    Abstract
    Background The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder. Objectives To investigate SOX10 as a potential biomarker for melanoma and vitiligo. Methods In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes. Results The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically. Conclusions We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.
    Expression profile of androgen-modulated microRNAs in the fetal murine lung
    Wafae Bouhaddioui, Biology of Sex Differences - 2016
    Abstract
    BackgroundAndrogens are known to delay lung development. As a consequence, the incidence and morbidity of respiratory distress syndrome of the neonate are higher for male than for female premature infants. We previously reported that many genes were expressed with a sex difference in the mouse developing lung and that several genes were under the control of androgens in the male fetal lung. microRNAs are small non-coding RNAs known to negatively regulate the expression of specific genes. In this study, we examined whether murine miRNAs are under the control of androgens in the male developing lung.MethodsExpression profiling of microRNAs was performed by microarrays using RNA extracted from male fetal lungs isolated on gestational day (GD) 17.0 and GD 18.0 after daily injection of pregnant mice from GD 10.0 with the antiandrogen flutamide or vehicle only. To identify putative miRNA target genes, the data obtained here were combined with gene profiling data reported previously using the same RNA preparations. qPCR was used to confirm microarray data with fetal lungs from other litters than those used in microarrays.ResultsFlutamide induced downregulation and upregulation of several miRNAs on GD 17.0 and GD 18.0. Of the 43 mature miRNAs modulated by flutamide on GD 17.0, 60 % were downregulated, whereas this proportion was only of 34 % for the 35 mature miRNAs modulated on GD 18.0. For 29 and 26 flutamide-responsive miRNAs, we found a corresponding target inversely regulated by androgens on GD 17.0 and 18.0, respectively. The androgen-regulated target genes were involved in several biological processes (lipid metabolism, cell proliferation, and lung development) and molecular functions, mainly transcription factor binding.ConclusionsRegulation of male lung development involves several miRNAs that are under androgen modulation in vivo.
    Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase - fpd.2015.2088
    Jean Guard,, FOODBORNE PATHOGENS AND DISEASE - 2016
    Abstract
    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB -linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase ( cyaA ). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer ex- tension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a * 89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition.
    The influence of 5-HTTLPR and Val66Met polymorphisms on cortical thickness and volume in limbic and paralimbic regions in depression: a preliminary study
    Natalia Jaworska, BMC Psychiatry - 2016
    Abstract
    Structural brain abnormalities have been investigated in multi-genetic and complex disorders such as major depressive disorder (MDD). Among the various candidate genes implicated in MDD, the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and 5-HT transporter gene linked polymorphism (5-HTTLPR) have garnered the most attention due to their putative roles in neural plasticity and antidepressant response. However, relatively few studies have assessed the influence of these polymorphysims on cortical thickness or brain volume in para-limbic and limbic regions in MDD, which was the aim of this study.
    Growth release of stunted black spruce (Picea mariana) in Kalmia heath: the role of ectomycorrhizal fungi and near-ground microclimate
    Philippe St. Martin, Canadian Journal of Forest Research - 2016
    Abstract
    Naturally regenerating and planted black spruce (Picea mariana (Mill.) B.S.P.) in post-fire landscapes in eastern Canada often exhibit stunted growth in the presence of ericaceous shrubs such as Kalmia angustifolia L. After a period of stunted growth, some seedlings experience a growth release, exhibiting growth rates closer to normally growing seedlings. We hypothesized that an increase in colonization of root tips by ectomycorrhizal (ECM) fungi is responsible for this release and that the percentage of root tips colonized by ECM fungi would be higher on seedlings that had a released or normally growing neighbour within close proximity. We quantified ECM fungi diversity and abundance from 255 soil cores from stunted, released, and normally growing black spruce seedlings sampled in two Kalmia-dominated sites in Newfoundland. Growth and microsite characteristics around each seedling were also measured. We found that normal and released seedlings had significantly higher proportions of ECM fungi root tips t..., L’épinette noire (Picea mariana (Mill.) B.S.P.) qui se régénère naturellement ou qui est plantée après feu dans les paysages de l’est du Canada a une croissance retardée en présence d’éricacées arbustives telles que Kalmia angustifolia L. Après une période de croissance retardée, certains semis connaissent une reprise de croissance dont le taux se rapproche du taux de croissance normal des semis. Nous avons émis l’hypothèse qu’une augmentation de la colonisation des apex racinaires par des champignons ectomycorhiziens (ECM) serait responsable de cette reprise de croissance et que le pourcentage d’apex racinaires colonisés par des champignons ECM serait plus élevé chez les semis ayant un voisin rapproché qui a connu une reprise de croissance ou croissait normalement. Nous avons quantifié la diversité et l’abondance des champignons ECM dans 255 carottes de sol associées à des semis rabougris, de semis ayant connu une reprise de croissance et des semis croissant normalement échantillonnés dans deux stations ...
    The utility of whole genome sequencing in characterizing Acinetobacter epidemiology and analyzing hospital outbreaks
    Margaret Fitzpatrick, Journal of Clinical Microbiology - 2015
    Abstract
    Acinetobacter baumannii frequently causes nosocomial infections and outbreaks. Whole genome sequencing (WGS) is a promising technique for strain typing and outbreak investigation. We compared the performance of conventional methods with WGS for strain typing clinical Acinetobacter isolates and for analyzing a carbapenem-resistant A. baumannii (CRAB) outbreak. We performed two band-based typing techniques (pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR) as well as multi-locus sequence type (MLST) analysis and WGS on 148 Acinetobacter calcoaceticus-Acinetobacter baumannii complex bloodstream isolates collected from a single hospital from 2005-2012. Phylogenetic trees inferred from core genome single nucleotide polymorphisms (SNPs) confirmed three Acinetobacter species within this collection. Four major A. baumannii clonal lineages (as defined by MLST) circulated during the study, three of which are globally distributed and one of which is novel. WGS indicated that a threshold of 2,500 core SNPs accurately distinguished A. baumannii isolates from different clonal lineages. The band-based techniques performed poorly in assigning isolates to clonal lineages and exhibited little agreement with sequence-based techniques. After applying WGS to a CRAB outbreak during the study, we identified a threshold of 2.5 core SNPs that distinguished non-outbreak from outbreak strains. WGS was more discriminatory than the band-based techniques and was used to construct a more accurate transmission map that resolved many of the plausible transmission routes suggested by epidemiologic links. Our study demonstrates that WGS is superior to conventional techniques for A. baumannii strain typing and outbreak analysis. These findings support incorporating WGS into healthcare infection prevention efforts.
    Expression of small intestinal nutrient transporters in embryonic and posthatch turkeys
    M. L. Weintraut, Poultry Science - 2015
    Abstract
    Nutrients are absorbed in the small intestine through a variety of transporter proteins, which have not been as well characterized in turkeys as in chickens. The objective of this study was to profile the mRNA expression of amino acid and monosaccharide transporters in the small intestine of male and female turkeys. Jejunum was collected during embryonic development (embryonic d 21 and 24, and d of hatch (DOH)) and duodenum, jejunum, and ileum were collected in a separate experiment during posthatch development (DOH, d 7, 14, 21, and 28). Real-time PCR was used to determine expression of aminopeptidase N (APN), one peptide (PepT1), 6 amino acid (ASCT1, bo,+AT, CAT1, EAAT3, LAT1, y+LAT2) and 3 monosaccharide (GLUT2, GLUT5, SGLT1) transporters. Data were analyzed by ANOVA using JMP Pro 11.0. APN, bo,+AT, PepT1, y+LAT2, GLUT5, and SGLT1 showed increased expression from embryonic d 21 and 24 to DOH. During posthatch, all genes except GLUT2 and SGLT1 were expressed greater in females than males. GLUT2 was expressed the same in males as females and SGLT1 was expressed greater in males than females. All basolateral membrane transporters were expressed greater during early development then decreased with age, while the brush border membrane transporters EAAT3, GLUT5, and SGLT1 showed increased expression later in development. Because turkeys showed high-level expression of the anionic amino acid transporter EAAT3, a direct comparison of tissue-specific expression of EAAT3 between chicken and turkey was conducted. The anionic amino acid transporter EAAT3 showed 6-fold greater expression in the ileum of turkeys at d 14 compared to chickens. This new knowledge can be used not only to better formulate turkey diets to accommodate increased glutamate transport, but also to optimize nutrition for both sexes.
    Comparative Genomics of cpn60-Defined Enterococcus hirae Ecotypes and Relationship of Gene Content Differences to Competitive Fitness
    Isha Katyal, Microbial Ecology - 2015
    Abstract
    Natural microbial communities undergo selection-driven succession with changes in environmental conditions and available nutrients. In a previous study of the pig faecal Enterococcus community, we demonstrated that cpn60 universal target (UT) sequences could resolve phenotypically and genotypically distinct ecotypes of Enterococcus spp. that emerged over time in the faecal microbiome of growing pigs. In this study, we characterized genomic diversity in the identified Enterococcus hirae ecotypes in order to define further the nature and degree of genome content differences between taxa resolved by cpn60 UT sequences. Genome sequences for six representative isolates (two from each of three ecotypes) were compared. Differences in phosphotransferase systems and amino acid metabolism pathways for glutamine, proline and selenocysteine were observed. Differences in the lac family phosphotransferase system corresponded to lactose utilization phenotypes of the isolates. Competitive fitness of the E. hirae ecotypes was evaluated by in vitro growth competition assays in pig faecal extract medium. Isolates from E. hirae-1 and E. hirae-2 ecotypes were able to out-compete isolates from the E. hirae-3 ecotype, consistent with the relatively low abundance of E. hirae-3 relative to E. hirae-1 and E. hirae-2 previously observed in the pig faecal microbiome, and with observed differences between the ecotypes in gene content related to biosynthetic capacity. Results of this study provide a genomic basis for the definition of ecotypes within E. hirae and confirm the utility of the cpn60 UT sequence for high-resolution profiling of complex microbial communities.
    Rare Circulating Cells in Familial Waldenström Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization
    Maroulio Pertesi, PLOS ONE - 2015
    Abstract
    The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10−7). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV.
  • Next Generation Sequencing (NGS)
    Mixed transmission modes and dynamic genome evolution in an obligate animal-bacterialsymbiosis
    Russell SL, ISME Journal - 2017
    Abstract
    Reliable transmission of symbionts between host generations facilitates the evolution of beneficial and pathogenic associations. Although transmission mode is typically characterized as either vertical or horizontal, the prevalence of intermediate transmission modes, and their impact on symbiont genome evolution, are understudied. Here, we use population genomics to explore mixed transmission modes of chemosynthetic bacterial symbionts in the bivalve Solemya velum. Despite strong evidence for symbiont inheritance through host oocytes, whole-genome analyses revealed signatures of frequent horizontal transmission, including discordant mitochondrial-symbiont genealogies, widespread recombination and a dynamic symbiont genome structure consistent with evolutionary patterns of horizontally transmitted associations. Population-level analyses thus provide a tractable means of ascertaining the fidelity of vertical versus horizontal transmission. Our data support the strong influence horizontal transmission can have on symbiont genome evolution, and shed light on the dynamic evolutionary pressures shaping symbiotic bacterial genomes.
    Association of the gut microbiota mobilome with hospital location and birth weight in preterm infants
    Ravi A, Pediatric Research - 2017
    Abstract
    BackgroundThe preterm infant gut microbiota is vulnerable to different biotic and abiotic factors. Although the development of this microbiota has been extensively studied, the mobilome-i.e. the mobile genetic elements (MGEs) in the gut microbiota-has not been considered. Therefore, the aim of this study was to investigate the association of the mobilome with birth weight and hospital location in the preterm infant gut microbiota.MethodsThe data set consists of fecal samples from 62 preterm infants with and without necrotizing enterocolitis (NEC) from three different hospitals. We analyzed the gut microbiome by using 16S rRNA amplicon sequencing, shot-gun metagenome sequencing, and quantitative PCR. Predictive models and other data analyses were performed using MATLAB and QIIME.ResultSThe microbiota composition was significantly different between NEC-positive and NEC-negative infants and significantly different between hospitals. An operational taxanomic unit (OTU) showed strong positive and negative correlation with NEC and birth weight, respectively, whereas none showed significance for mode of delivery. Metagenome analyses revealed high levels of conjugative plasmids with MGEs and virulence genes. Results from quantitative PCR showed that the plasmid signature genes were significantly different between hospitals and in NEC-positive infants.ConclusionOur results point toward an association of the mobilome with hospital location in preterm infants.
    Abundance of antibiotics, antibiotic resistance genes and bacterial community composition in wastewater effluents from different Romanian hospitals
    Szekeres E, Environmental Pollution - 2017
    Abstract
    Antimicrobial resistance represents a growing and significant public health threat, which requires a global response to develop effective strategies and mitigate the emergence and spread of this phenomenon in clinical and environmental settings. We investigated, therefore, the occurrence and abundance of several antibiotics and antibiotic resistance genes (ARGs), as well as bacterial community composition in wastewater effluents from different hospitals located in the Cluj County, Romania. Antibiotic concentrations ranged between 3.67 and 53.05 μg L-1, and the most abundant antibiotic classes were β-lactams, glycopeptides, and trimethoprim. Among the ARGs detected, 14 genes confer resistance to β-lactams, aminoglycosides, chloramphenicol, macrolide-lincosamide-streptogramin B (MLSB) antibiotics, sulfonamides, and tetracyclines. Genes encoding quaternary ammonium resistance and a transposon-related element were also detected. The sulI and qacEΔ1 genes, which confer resistance to sulfonamides and quaternary ammonium, had the highest relative abundance with values ranging from 5.33 × 10-2 to 1.94 × 10-1 and 1.94 × 10-2 to 4.89 × 10-2 copies/16 rRNA gene copies, respectively. The dominant phyla detected in the hospital wastewater samples were Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Among selected hospitals, one of them applied an activated sludge and chlorine disinfection process before releasing the effluent to the municipal collector. This conventional wastewater treatment showed moderate removal efficiency of the studied pollutants, with a 55-81% decrease in antibiotic concentrations, 1-3 order of magnitude lower relative abundance of ARGs, but with a slight increase of some potentially pathogenic bacteria. Given this, hospital wastewaters (raw or treated) may contribute to the spread of these emerging pollutants in the receiving environments. To the best of our knowledge, this study quantified for the first time the abundance of antibiotics and ARGs in wastewater effluents from different Romanian hospitals.
    Microbial diversity of consumption milk during processing and storage
    Porcellato D, International Journal of Food Microbiology - 2018
    Abstract
    Bovine milk contains a complex microbial community that affects the quality and safety of the product. Detailed knowledge of this microbiota is, therefore, of importance for the dairy industry. In this study, the bacterial composition of consumption milk was assessed during different stages in the production line and throughout the storage in cartons by using culturing techniques and 16S rRNA marker gene sequencing. Monthly samples from two dairies were analyzed to capture the seasonal variations in the milk microbiota. Although there was a core microbiota present in milk samples from both dairies, the composition of the bacterial communities were significantly influenced by sampling month, processing stage and storage temperature. Overall, a higher abundance of operational taxonomic units (OTUs) within the order Bacillales was detected in samples of raw and pasteurized milk from the spring and summer months, while Pseudomonadales and Lactobacillales OTUs were predominant in the winter months. OTUs belonging to the order Lactobacillales, Pseudomonadales, Clostridiales and Bacillales were significantly more abundant in milk samples taken immediately after pasteurization compared to raw milk samples. During storage of milk in cartons at 4°C, the bacterial composition remained stable throughout the product shelf life, while storage at 8°C significantly increased the abundance of OTUs belonging to the genus Bacillus and the plate count levels of presumptive Bacillus cereus. The knowledge obtained in this work will be useful to the dairy industry during their quality assurance work and risk assessment practices.
    Application of a novel amplicon-based sequencing approach reveals the diversity of the Bacillus cereus group in stored raw and pasteurized milk
    Davide Porcellato, Food Microbiology - 2018
    Abstract
    Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products.
    RNA‐seq: Applications and Best Practices
    Michele Araújo Pereira, Intech - 2017
    Abstract
    RNA‐sequencing (RNA‐seq) is the state‐of‐the‐art technique for transcriptome analysis that takes advantage of high‐throughput next‐generation sequencing. Although being a powerful approach, RNA‐seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA‐seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high‐quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well‐known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments.
    A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice
    Chrstine Bäuerl, JoVE (Journal of Visualized Experiments) - 2017
    Abstract
    Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.
    Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine
    René Pagel, The FASEB Journal - 2017
    Abstract
    Endogenous circadian clocks regulate 24 h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse–chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA–transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.—Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.
    Microbial Composition and Diversity Patterns in Deep Hyperthermal Aquifers from the Western Plain of Romania
    Cecilia M. Chiriac, Microbial Ecology - 2017
    Abstract
    A limited number of studies have investigated the biodiversity in deep continental hyperthermal aquifers and its influencing factors. Here, we present the first description of microbial communities inhabiting the Pannonian and Triassic hyperthermal aquifers from the Western Plain of Romania, the first one being considered a deposit of “fossilized waters,” while the latter is embedded in the hydrological cycle due to natural refilling. The 11 investigated drillings have an open interval between 952 and 3432 m below the surface, with collected water temperatures ranging between 47 and 104 °C, these being the first microbial communities characterized in deep continental water deposits with outflow temperatures exceeding 80 °C. The abundances of bacterial 16S rRNA genes varied from approximately 105–106 mL−1 in the Pannonian to about 102–104 mL−1 in the Triassic aquifer. A 16S rRNA gene metabarcoding analysis revealed distinct microbial communities in the two water deposits, especially in the rare taxa composition. The Pannonian aquifer was dominated by the bacterial genera Hydrogenophilus and Thermodesulfobacterium, together with archaeal methanogens from the Methanosaeta and Methanothermobacter groups. Firmicutes was prevalent in the Triassic deposit with a large number of OTUs affiliated to Thermoanaerobacteriaceae, Thermacetogenium, and Desulfotomaculum. Species richness, evenness, and phylogenetic diversity increased alongside with the abundance of mesophiles, their presence in the Triassic aquifer being most probably caused by the refilling with large quantities of meteoric water in the Carpathian Mountains. Altogether, our results show that the particular physico-cheminal characteristics of each aquifer, together with the water refilling possibilities, seem to determine the microbial community structure.
    The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health
    Özgün C. O. Umu, PLOS ONE - 2016
    Abstract
    Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning.
    Transition from infant- to adult-like gut microbiota
    E. Avershina, Environmental Microbiology - 2016
    Abstract
    Transition from an infant to an adult associated gut microbiota with age through establishment of strict anaerobic bacteria remains one of the key unresolved questions in gut microbial ecology. Here a comprehensive comparative analysis of stool microbiota in a large cohort of mothers and their children sampled longitudinally up until 2 years of age using sequencing analysis tool was presented that allows realistic microbial diversity estimates. In this work, evidence for the switch from children to adult associated microbial profile between 1 and 2 years of age was provided, suggestively driven by Bifidobacterium breve. An Operational Taxonomic Unit (OTU) belonging to B. breve was highly prevalent in the population throughout the first year of life, and was negatively associated with detection of a range of adult-like OTUs. Although an adult profile was not fully established by 2 years of age, it was demonstrated that with regards to the most prevalent OTUs, their prevalence in the child population by then already resembled that of the adult population. Taken together, it was proposed that late-colonizing OTUs were recruited at a later stage and were not acquired at birth with the recruitment being controlled by gatekeeping OTUs until the age of 1 year.
    Microbial biogeography of a university campus
    Ashley A. Ross, Microbiome - 2015
    Abstract
    Microorganisms are distributed on surfaces within homes, workplaces, and schools, with the potential to impact human health and disease. University campuses represent a unique opportunity to explore the distribution of microorganisms within built environments because of high human population densities, throughput, and variable building usage. For example, the main campus of the University of Waterloo spans four square kilometres, hosts over 40,000 individuals daily, and is comprised of a variety of buildings, including lecture halls, gyms, restaurants, residences, and a daycare.
  • Reverse Transcription
    Quantitative RT-qPCR
    The ubiquitin-protein ligase E6AP/UBE3A supports early encephalomyocarditis virus replication
    Marybeth Carmody, Virus Research - 2018
    Abstract
    Many viruses make use of, and even direct, the ubiquitin-proteasome system to facilitate the generation of a cellular environment favorable for virus replication, while host cells use selected protein ubiquitylation pathways for antiviral defense. Relatively little information has been acquired, however, regarding the extent to which protein ubiquitylation determines the replication success of picornaviruses. Here we report that the ubiquitin-protein ligase E6AP/UBE3A, recently shown to be a participant in encephalomyocarditis virus (EMCV) 3C protease concentration regulation, also facilitates the early stages of EMCV replication, probably by a mechanism that does not involve 3C protease ubiquitylation. Using stably transfected E6 AP knockdown cells, we found that reduced E6AP concentration extends the time required for infected cells to undergo the morphological changes caused by virally induced pathogenesis and to begin the production of infectious virions. This lag in virion production is accompanied by a corresponding delay in the appearance of detectable levels of viral proteins and RNA. We also found, by using both immunofluorescence microscopy and cell fractionation, that E6AP is partially redistributed from the nucleus to the cytoplasm in EMCV-infected cells, thereby increasing its availability to participate in cytoplasmic virus replication processes.
    Xenobiotic Nuclear Receptor Signaling Determines Molecular Pathogenesis of Progressive Familial Intrahepatic Cholestasis
    Kang Ho Kim, Endocrinology - 2018
    Abstract
    Abstract. Progressive familial intrahepatic cholestasis (PFIC) is a genetically heterogeneous disorder of bile flow disruption due to abnormal canalicular tran
    Synthesis and antiviral evaluation of novel peptidomimetics as Norovirus protease inhibitors
    Franck Amblard, Bioorganic & Medicinal Chemistry Letters - 2018
    Abstract
    A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.
    Control of Citrus Huanglongbing (HLB) via Trunk Injection of Plant Activators and Antibiotics
    Jiahuai Hu, Phytopathology - 2017
    Abstract
    Citrus Huanglongbing (HLB) or greening is a devastating disease of citrus worldwide and no effective control measure is currently available. Plant activators represent environment friendly compounds capable of inducing resistance against many plant pathogens. Earlier studies showed that foliar spray of plant defense inducers could slow down HLB disease progress. In this study, eight plant activators and three antibiotics were evaluated in 3 field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that 4 trunk injections of several activators including salicylic acid, oxalic acid, acibenzolar-S-methyl and potassium phosphate provided significant control of HLB by suppressing Las titer and disease progress. Trunk injection of penicillin, streptomycin and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of Las titer and HLB symptom expressions than plant activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance (SAR) under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision-making regarding developing an effective HLB management program.
    The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification - viruses-09-00207-v2.pdf
    Leanne M. Bilawchuk, viruses - 2017
    Abstract
    Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition ( p=0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p=0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition;p=0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses.
    Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method
    Irit Davidson, Avian Pathology - 2016
    Abstract
    The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, dropping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA was now upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the env and the NS5 genes, increasing the sensitivity by 10-100 fold compared to the previously existing assays. Based on the recent complete sequences of 5 TMEV isolates we now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009-2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least, in experimentally-infected turkeys and in 3 cases of disease encountered in commercial flocks.
    Regulation of Cytochrome P450 2B10 (CYP2B10) Expression in Liver by Peroxisome Proliferator-Activated Receptor-β/δ Modulation of SP1 Promoter Occupancy
    Takayuki Koga, Journal of Biological Chemistry - 2016
    Abstract
    Alcoholic liver disease is a pathological condition caused by over-consumption of alcohol. Due to the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Since peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression in Pparβ/δ+/+ mice, but not in Pparβ/δ-/- mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. Peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), a co-activator of both CAR and PPARβ/δ, was up-regulated in Pparβ/δ+/+ liver following ethanol exposure, but not in Pparβ/δ-/- liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARβ/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARβ/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PGC1α expression and/or activity independent of CAR activity. Ligand activation of PPARβ/δ attenuates ethanol-induced Cyp2b10 expression in Pparβ/δ+/+ liver, but not in Pparβ/δ-/- liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARβ/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARβ/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.
    Targeting NF-κB RelA/p65 phosphorylation overcomes RITA resistance
    Yiwen Bu, Cancer Letters - 2016
    Abstract
    Inactivation of p53 occurs frequently in various cancers. RITA is a promising anticancer small molecule that dissociates p53-MDM2 interaction, reactivates p53 and induces exclusive apoptosis in cancer cells, but acquired RITA resistance remains a major drawback. This study found that the site-differential phosphorylation of nuclear factor-κB (NF-κB) RelA/p65 creates a barcode for RITA chemosensitivity in cancer cells. In naïve MCF7 and HCT116 cells where RITA triggered vast apoptosis, phosphorylation of RelA/p65 increased at Ser536, but decreased at Ser276 and Ser468; oppositely, in RITA-resistant cells, RelA/p65 phosphorylation decreased at Ser536, but increased at Ser276 and Ser468. A phosphomimetic mutation at Ser536 (p65/S536D) or silencing of endogenous RelA/p65 resensitized the RITA-resistant cells to RITA while the phosphomimetic mutant at Ser276 (p65/S276D) led to RITA resistance of naïve cells. In mouse xenografts, intratumoral delivery of the phosphomimetic p65/S536D mutant increased the antitumor activity of RITA. Furthermore, in the RITA-resistant cells ATP-binding cassette transporter ABCC6 was upregulated, and silencing of ABCC6 expression in these cells restored RITA sensitivity. In the naïve cells, ABCC6 delivery led to RITA resistance and blockage of p65/S536D mutant-induced RITA sensitivity. Taken together, these data suggest that the site-differential phosphorylation of RelA/p65 modulates RITA sensitivity in cancer cells, which may provide an avenue to manipulate RITA resistance.
    Low β 2 -adrenergic receptor level may promote development of castration resistant prostate cancer and altered steroid metabolism
    Peder Rustøen Braadland, Oncotarget - 2015
    Abstract
    he underlying mechanisms responsible for the development of castration- resistant prostate cancer (CRPC) in patients who have undergone androgen deprivation therapy are not fully understood. This is the first study to address whether β2-adrenergic receptor (ADRB2)- mediated signaling may affect CRPC progression in vivo. By immunohistochemical analyses, we observed that low levels of ADRB2 is associated with a more rapid development of CRPC in a Norwegian patient cohort. To elucidate mechanisms by which ADRB2 may affect CRPC development, we stably transfected LNCaP cells with shRNAs to mimic low and high expression of ADRB2. Two UDP-glucuronosyltransferases, UGT2B15 and UGT2B17, involved in phase II metabolism of androgens, were strongly downregulated in two LNCaP shADRB2 cell lines. The low-ADRB2 LNCaP cell lines displayed lowered glucuronidation activities towards androgens than high-ADRB2 cells. Furthermore, increased levels of testosterone and enhanced androgen responsiveness were observed in LNCaP cells expressing low level of ADRB2. Interestingly, these cells grew faster than high- ADRB2 LNCaP cells, and sustained their low glucuronidation activity in castrated NOD/Oncotarget2 www.impactjournals.com/oncotarget INTRODUCTIONAndrogen deprivation therapy (ADT) is the first line of treatment for patients with advanced or metastatic prostate cancer [1]. ADT is initially effective in controlling tumor growth and symptoms, but most tumors eventually develop resistance to ADT and become castration resistant prostate cancers (CRPC). Over the last years, it has become evident that the androgen signaling axis plays a pivotal role in the development of CRPC [2]. The multiple molecular mechanisms by which the androgen receptor (AR) contributes to disease progression despite castration levels of androgens in prostate cancer have been thoroughly reviewed [3-6]. Several new targets in the AR activation pathway have emerged in recent years [7, 8]. The steroidogenic pathway has received increasing attention, as drugs targeting this pathway, such as abiraterone (an inhibitor of cytochrome P450, family 17, subfamily A,polypeptide 1 (CYP17)) improve the life expectancy of patients with CRPC, despite the assumed androgen-independence of these cancer cases [8]. No curative options for CRPC are, however, available today. Increased knowledge of the mechanisms by which the cancer cells progress to CRPC is hence needed. Recently, targeting the androgen extrahepatic phase-II metabolic pathways has arisen as a potential tool to help maintain androgen-deprived conditions during ADT [9]. The UDP-glucuronosyltransferases 2B15 (UGT2B15) and 2B17 (UGT2B17) are of special interest, as they are expressed in prostate tissue and cell lines, and they exhibit specificity for androgen metabolites [10].The β2-adrenergic receptor (ADRB2) and its downstream effectors cyclic AMP (cAMP) and cAMP-dependent protein kinase A (PKA) have been implicated in prostate cancer progression and AR signaling [11]. In particular, sympathetic stimulation of ADRB2 has been shown to potentially sensitize AR in cell lines under androgen depleted conditions [12], suggesting that ADRB2 might play a role in the development of CRPC. Furthermore, a number of target genes are common for the androgen and the PKA signaling cascades [13], and in steroidogenic cells both cAMP and PKA have been shown to regulate transcription of steroidogenic genes such as CYP17 and STAR [14-16], as well as to modulate their activity at the protein level [17].While most pre-clinical evidence points towards a tumor promoting role of β-adrenergic signaling [18, 19], a previous study by Yu et al. reported an inverse correlation between ADRB2 expression levels and prostate cancer progression [20]. Low levels of ADRB2 in prostate cancer tissue were found to correlate with biochemical recurrence measured as increasing prostate-specific antigen (PSA) levels, or metastatic disease after radical prostatectomy. Conversely, our group has recently reported an association between the use of β-blockers (ADRB antagonists) and improved prostate cancer specific survival both for patients who have undergone ADT [21] and for patients with high risk or metastatic disease [22].Our knowledge about the potential role of the ADRB2 in prostate cancer and CRPC development is still limited. Therefore, in this study, we have addressed this topic by performing immunohistochemical analyses and investigated the potential role of ADRB2 in development of CRPC in ADRB2 knockdown cell lines. RESULTS Low ADRB2 expression level in tumor tissue is associated with poor prognosis after androgen deprivation therapyTissue from 45 prostate cancer patients who had received hormonal therapy and had been treated with transurethral resection of the prostate (TUR-P) at Oslo University Hospital, Aker (the Oslo ADT cohort) were included in a tissue micro-array study. Five patients were excluded due to lack of cancerous tissue following staining with anti-ADRB2 antibody. The mean follow-up from initiation of ADT for the 40 patients included in the survival analyses was 71 months. For prostate cancer- specific mortality the mean follow-up was 70 months, as we lacked information on the cause of death for four patients. Patient and tumor characteristics at time of diagnosis are shown in Supplementary Table 2. Examples of negative and strong ADRB2 staining of two specimens with Gleason score 9 are shown in Figure 1a and 1b. Kaplan-Meier plots showing time to CRPC development and prostate cancer- specific mortality in patients stratified according to staining intensity above and below mean are shown in Figure 1c and 1d. Competing risk regression modelling showed that increasing staining intensity was associated with increased time to CRPC development, with an adjusted SHR of 0.67 (95% CI 0.46-0.97, p-value 0.035; adjusted for age at initiation of ADT and Gleason score) (Table 1). For prostate cancer- specific mortality, the association was not statistically significant (adjusted SCID mice. ADRB2 immunohistochemical staining intensity correlated with UGT2B15 staining intensity in independent TMA studies and with UGT2B17 in one TMA study. Similar to ADRB2, we show that low levels of UGT2B15 are associated with a more rapid CRPC progression. We propose a novel mechanism by which ADRB2 may affect the development of CRPC through downregulation of UGT2B15 and UGT2B17.
    Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606
    Daryl L. Richie, PLOS ONE - 2016
    Abstract
    The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl β- d -1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3- O -[( R )-3-OH-C 14 ]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A . baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth.
    Impaired function of trophoblast cells derived from translocated hESCs may explain pregnancy loss in women with balanced translocation (11;22)
    Alina Shpiz, Journal of Assisted Reproduction and Genetics - 2016
    Abstract
    PurposeThe aim of the study was to study whether the trophoblasts carrying unbalanced translocation 11,22 [t(11;12)] display abnormal expression of trophoblastic genes and impaired functional properties that may explain implantation failure.Methodst(11;22) hESCs and control hESCs were differentiated in vitro into trophoblast cells in the presence of BMP4, and trophoblast vesicles (TBVs) were created in suspension. The expression pattern of extravillous trophoblast (EVT) genes was compared between translocated and control TBVs. The functional properties of the TBVs were evaluated by their attachment to endometrium cells (ECC1) and invasion through trans-well inserts.ResultsTBVs derived from control hESCs expressed EVT genes from functioning trophoblast cells. In contrast, TBVs differentiated from the translocated hESC line displayed impaired expression of EVT genes. Moreover, the number of TBVs that were attached to endometrium cells was significantly lower compared to the controls. Correspondingly, invasiveness of trophoblast-differentiated translocated cells was also significantly lower than that of the control cells.ConclusionsThese results may explain the reason for implantation failure in couple carriers of t(11;22). They also demonstrate that translocated hESCs comprise a valuable in vitro human model for studying the mechanisms underlying implantation failure.
    Prevalence of influenza A virus in live-captured North Atlantic gray seals: a possible wild reservoir
    Wendy Blay Puryear, Emerging Microbes & Infections - 2016
    Abstract
    Influenza A virus (IAV) has been associated with multiple unusual mortality events (UMEs) in North Atlantic pinnipeds, frequently attributed to spillover of virus from wild-bird reservoirs. To determine if endemic infection persists outside of UMEs, we undertook a multiyear investigation of IAV in healthy, live-captured Northwest Atlantic gray seals (Halichoerus grypus). From 2013 to 2015, we sampled 345 pups and 57 adults from Cape Cod, MA, USA and Nova Scotia, Canada consistently detecting IAV infection across all groups. There was an overall viral prevalence of 9.0% (95% confidence interval (CI): 6.4%–12.5%) in weaned pups and 5.3% (CI: 1.2%–14.6%) in adults, with seroprevalences of 19.3% (CI: 15.0%–24.5%) and 50% (CI: 33.7%–66.4%), respectively. Positive sera showed a broad reactivity to diverse influenza subtypes. IAV status did not correlate with measures of animal health nor impact animal movement or foraging. This study demonstrated that Northwest Atlantic gray seals are both permissive to and tolerant of diverse IAV, possibly representing an endemically infected wild reservoir population.
    Replication of human norovirus RNA in mammalian cells reveals a lack of interferon response
    Lin Qu, Journal of Virology - 2016
    Abstract
    Human noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (α/β) and III (IL-29) IFN treatment. However, in cells capable of robust IFN response to Sendai virus (SeV) and poly(I:C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is neither enhanced by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R, nor by shRNA knockdown of MAVS or IRF3 in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells and a SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce IFN response, suggesting that the IFN response may play a limited role in host restriction of HuNoV replication. IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small animal model, little is known about the innate host defense to these viruses. Studies on murine norovirus (MNV) have shown the importance of an interferon (IFN) response in host control of MNV replication, but this remains unclear for HuNoVs. Here we investigated the IFN response to HuNoV RNA replication in mammalian cells using Norwalk virus stool RNA transfection, a reverse genetics system, IFN neutralization reagents, and shRNA knockdown methods. Our results show that HuNoV RNA replication in mammalian cells does not induce an IFN response, nor can it be enhanced by blocking the IFN response. These results suggest a limited role of the IFN response in host control of HuNoV RNA replication, providing important insights into our understanding of host defense to HuNoVs that differs from MNV.
    RALFL34 regulates formative cell divisions in Arabidopsis pericycle during lateral root initiation
    Evan Murphy, Journal of Experimental Botany - 2016
    Abstract
    In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation.
    Engagement of distinct epitopes on CD43 induces different co-stimulatory pathways in human T cells
    Madhura Modak, Immunology - 2016
    Abstract
    Co-receptors, being either co-stimulatory or co-inhibitory, play a pivotal role in T-cell immunity. Several studies have indicated that CD43, one of the abundant T-cell surface glycoproteins, acts not only as a potent co-receptor but also as a negative regulator for T-cell activation. Here we demonstrate that co-stimulation of human peripheral blood (PB) T cells through two distinct CD43 epitopes recognized by monoclonal antibodies (mAb) CD43-6E5 (T6E5-act) and CD43-10G7 (T10G7-act) potently induced T-cell proliferation. However, T-cell co-stimulation through two CD43 epitopes differentially regulated activation of nuclear factor of activated T cells (NFAT) and nuclear factor-κB (NF-κB) transcription factors, T-cell cytokine production and effector function. T6E5-act produced high levels of interleukin-22 (IL-22) and interferon-γ (IFN-γ) similar to T cells activated via CD28 (TCD28-act), whereas T10G7-act produced low levels of inflammatory cytokines but higher levels of regulatory cytokines transforming growth factor-β (TGF-β) and interleukin-35 (IL-35). Compared with T6E5-act or to TCD28-act, T10G7-act performed poorly in response to re-stimulation and further acquired a T-cell suppressive function. T10G7-act did not directly inhibit proliferation of responder T cells, but formed stable heterotypic clusters with dendritic cells (DC) via CD2 to constrain activation of responder T cells. Together, our data demonstrate that CD43 is a unique and polarizing regulator of T-cell function.
    Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways
    Martha Gómez-Marroquín, MDPI - 2016
    Abstract
    In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu+ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu+ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome. Keywords:
    Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis
    Rushita A. Bagchi, Cell and Tissue Research - 2016
    Abstract
    The glycoprotein fibronectin is a key component of the extracellular matrix. By interacting with numerous matrix and cell surface proteins, fibronectin plays important roles in cell adhesion, migration and intracellular signaling. Up-regulation of fibronectin occurs in tissue fibrosis, and previous studies have identified the pro-fibrotic factor TGFβ as an inducer of fibronectin expression, although the mechanism responsible remains unknown. We have previously shown that a key downstream effector of TGFβ signaling in cardiac fibroblasts is the transcription factor scleraxis, which in turn regulates the expression of a wide variety of extracellular matrix genes. We noted that fibronectin expression tracked closely with scleraxis expression, but it was unclear whether scleraxis directly regulated the fibronectin gene. Here, we report that scleraxis acts via two E-box binding sites in the proximal human fibronectin promoter to govern fibronectin expression, with the second E-box being both sufficient and necessary for scleraxis-mediated fibronectin expression to occur. A combination of electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that scleraxis interacted to a greater degree with the second E-box. Over-expression or knockdown of scleraxis resulted in increased or decreased fibronectin expression, respectively, and scleraxis null mice presented with dramatically decreased immunolabeling for fibronectin in cardiac tissue sections compared to wild-type controls. Furthermore, scleraxis was required for TGFβ-induced fibronectin expression: TGFβ lost its ability to induce fibronectin expression following scleraxis knockdown. Together, these results demonstrate a novel and required role for scleraxis in the regulation of cardiac fibroblast fibronectin gene expression basally or in response to TGFβ.
    The impact of the long-distance transport of a BEL1-Like Messenger RNA on Development
    Tian Lin, Original Research - 2016
    Abstract
    BEL1- and KNOTTED1-type proteins are transcription factors from the three-amino-loop-extension superclass that interact in atandem complex to regulate the expression of target genes. In potato (Solanum tuberosum), StBEL5 and its Knox protein partner regulate tuberization by targeting genes that control growth. RNA movement assays demonstrated that StBEL5 transcripts move through the phloem to stolon tips, the site of tuber induction.StBEL5 messenger RNA originates in the leaf, and its movement to stolons is induced by a short-day photoperiod. Here, we report the movement of StBEL5 RNA to roots correlated with increased growth, changes in morphology, and accumulation of GA2-oxidase1,YUCCA1a, and ISOPENTENYL TRANSFERASE transcripts. Transcription of tBEL5 n leaves is induced by light but insensitive to photoperiod, whereas in stolon tips growing in the dark, romoter activity is enhanced by short days. The heterodimer of StBEL5 and POTH1, a KNOTTED1-type transcription factor,binds to a tandem TTGAC-TTGAC motif that is essential for regulating transcription. The discovery of an inverted tandem motifin the tBEL5 romoter with TTGAC motifs on opposite strands may explain the induction of tBEL5 romoter activity in stolon ips under short days. Using transgenic potato lines, deletion of one of the TTGAC motifs from the tBEL5 romoter results in he reduction of GUS activity in new tubers and roots. Gel-shift assays demonstrate BEL5/POTH1 binding specificity to the otifs present in the tBEL5 promoter and a double tandem motif present in the StGA2-oxidase1 promoter. These results suggest that, in addition to tuberization, the movement of StBEL5 messenger RNA regulates other aspects of vegetative development.
    The liver-specific microRNA-122*, the complementary strand of microRNA-122, acts as a tumor suppressor by modulating the p53/mouse double minute 2 homolog circuitry
    Alina Simerzin, Hepatology - 2016
    Abstract
    The tumor suppressor p53 is a central regulator of signaling pathways that controls the cell cycle and maintains the integrity of the human genome. p53 level is regulated by mouse double minute 2 homolog (Mdm2), which marks p53 for proteasomal degradation. The p53-Mdm2 circuitry is subjected to complex regulation by a variety of mechanisms, including microRNAs (miRNAs). We found a novel effector of this regulatory circuit, namely, miR-122*, the passenger strand of the abundantly expressed liver-specific miR-122. Here, we demonstrate that miR-122* levels are reduced in human hepatocellular carcinoma (HCC). We found that miR-122* targets Mdm2, thus participating as an important player in the p53-Mdm2 circuitry. Moreover, we observed significant negative correlation between levels of miR-122* and Mdm2 in a large set of human HCC samples. In vivo tumorigenicity assays demonstrate that miR-122* is capable of inhibiting tumor growth, emphasizing the tumor-suppressor characteristics of this miRNA. Furthermore, we show that blocking miR-122 in murine livers with an antagomiR-122 (miRNA inhibitor) results in miR-122* accumulation, leading to Mdm2 repression followed by elevated p53 protein levels. Conclusion: We show that miR-122*, the passenger strand of miR-122, regulates the activity of p53 by targeting Mdm2. Importantly, similarly to miR-122, miR-122* is significantly down-regulated in human HCC. We therefore propose that miR-122* is an important contributor to the tumor suppression activity previously attributed solely to miR-122. (Hepatology 2016)
    High Prevalence of Highly Variable Atypical Porcine Pestiviruses Found in Germany
    M. Beer, Transboundary and Emerging Diseases - 2016
    Abstract
    Recently, a novel atypical porcine pestivirus (APPV) with significant distribution was described in the USA. Subsequent screening of the German pig sector showed a high prevalence of APPV with high variability among strains. First indication of a cell culture isolate is provided which will allow further investigations like pathogenesis studies.
    Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells : Scientific Reports
    Marie-Claude Gaudreau, Scientific Reports - 2016
    Abstract
    The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.
    Reliable quantification of rhinovirus species C using real-time PCR
    Chisha T. Sikazwe, Research Gate - 2016
    Abstract
    Background: Rhinovirus C (RV-C) is an important respiratory pathogen of children, but little is known about its contribution to disease severity, though viral load appears to be important. Difficulty in RV-C cultivation and target sequence variation has precluded the development of a PCR based quantification method. Objective: The aim of this study was to develop and validate reverse transcription quantitative PCR (RT-qPCR) assays for a broad range of circulating RV-C genotypes in nasopharyngeal aspirates (NPAs). Study design: Four assays were designed to quantify a 296bp region located within the 5' untranslated region (UTR) of RV-C types. These assays were based on in silico analysis of available RV-C sequences. Probes were designed to provide 100% homology to the corresponding RV-C genotypes. Results: The linear dynamic range of each of the four assays spanned eight orders of magnitude (10(4)-10(11) copies/mL). The limit of detection for assays 1-4 was estimated to be 1147 copies/mL, 765 copies/mL, 1138 copies/mL and 1470 copies/mL respectively. Each assay demonstrated a strong linear relationship (r(2)=>0.995) and amplification efficiency greater than 95%. Repeatability and reproducibility of the method were shown to be high, with coefficients of variations lower than 8% and 15% respectively. Discover the world's research
    DENGUE VIRUS (DV) POLYPEPTIDE SEQUENCES, T CELL EPITOPES AND METHODS AND USES THEREOF - LA JOLLA INSTITUTE FOR ALLERGY AND IMMUNOLOGY
    Sujan Shresta, United States Patent - 2016
    Abstract
    Dengue virus (DV) peptides, including T cell epitopes, structural and non-structural (NS) polypeptide sequences, subsequences and modifications thereof, nucleotide sequences encoding such peptides, and compositions including such peptides and encoding nucleotide sequences, and cells expressing such peptides, are provided. Such DV peptides, nucleotide sequences and compositions, can be used to elicit, stimulate, induce, promote, increase, enhance or activate an anti-DV CD8+ T cell response or an anti-DV CD4+ T cell response. Such peptides, nucleotide sequences and compositions can also be used for and in methods of vaccination/immunization of a subject against Dengue virus (DV) (e.g., to provide protection against DV infection and/or pathology), and for treatment of a subject in need thereof, for example, treatment of the subject for a Dengue virus (DV) infection or pathology.
    Overwintering of Rabies Virus in Silver Haired Bats ( Lasionycteris noctivagans )
    April Davis, PLOS ONE - 2016
    Abstract
    Silver-haired bats, ( Lasionycteris noctivagans ) are semi-colonial, migratory tree bats that have infrequent contact with humans. Despite the species rarity, the L . noctivagans rabies variant is the most commonly reported rabies virus variant (RABV) in domestically acquired human rabies cases in the US. Unlike big brown bats ( Eptesicus fuscus ) and little brown bats ( Myotis lucifugus ), L . noctivagans are not considered true hibernators. It is unknown if RABV can overwinter in hibernating L . noctivagans or is only maintained in members of this taxa that migrate to warmer climates. To better understand RABV overwintering in this species, L . noctivagans were inoculated intramuscularly with either a homologous RABV ( L . noctivagans Virus 1) or one of two heterologous RABV ( Eptesicus fuscus Virus 2 and Myotis lucifugus Virus 1). Five days following inoculation, L . noctivagans were placed in a hibernation chamber for 6 weeks. Our results demonstrate that rabies virus can overwinter in L . noctivagans yet the incubation period was extended 6 weeks when compared to bats maintained at ambient temperatures. Additionally, we found that the longer the incubation period, the greater the viral dissemination to the salivary glands. Similar to our previous studies, L . noctivagans were most susceptible to a homologous variant. In summary, we found that RABV incubation is extended following a subcutaneous exposure or maintenance in hibernation and longer incubation times increase dissemination and potential for transmission.
    Reliable quantification of rhinovirus species C using real-time PCR
    Chisha T. Sikazwe, Journal of Virological Methods - 2016
    Abstract
    Background Rhinovirus C (RV-C) is an important respiratory pathogen of children, but little is known about its contribution to disease severity, though viral load appears to be important. Difficulty in RV-C cultivation and target sequence variation has precluded the development of a PCR based quantification method. Objective The aim of this study was to develop and validate reverse transcription quantitative PCR (RT-qPCR) assays for a broad range of circulating RV-C genotypes in nasopharyngeal aspirates (NPAs). Study design Four assays were designed to quantify a 296 bp region located within the 5′ untranslated region (UTR) of RV-C types. These assays were based on in silico analysis of available RV-C sequences. Probes were designed to provide 100% homology to the corresponding RV-C genotypes. Results The linear dynamic range of each of the four assays spanned eight orders of magnitude (104–1011 copies/mL). The limit of detection for assays 1–4 was estimated to be 1147 copies/mL, 765 copies/mL, 1138 copies/mL and 1470 copies/mL respectively. Each assay demonstrated a strong linear relationship (r2 = >0.995) and amplification efficiency greater than 95%. Repeatability and reproducibility of the method were shown to be high, with coefficients of variations lower than 8% and 15% respectively.
    Bosentan reverses the hypoxia-induced downregulation of the bone morphogenetic protein signaling in pulmonary artery smooth muscle cells
    Hidekazu Maruyama, Life Sciences - 2016
    Abstract
    Aims Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases. Bone morphogenetic protein (BMP) and endothelin-1 signaling pathways have been shown to be altered in hypoxic PH and to play crucial roles in the associated pulmonary artery remodeling. We, therefore, aimed to study the potential link between hypoxia and the alteration of BMP and endothelin-1 signaling observed in pulmonary artery smooth muscle cells (PA-SMCs) in hypoxic PH. Materials and Methods Human PA-SMCs were treated with hypoxia-mimetic agent cobalt chloride (CoCl2; 100 μM), with or without pretreatment with a dual endothelin receptor antagonist bosentan (10 μM). Expressions of preproendothelin-1 (PPET1), BMP type 2 receptor (BMPR-2), and one BMP signaling target gene, the inhibitor of DNA binding 1 (ID1) were evaluated by real time quantitative polymerase chain reaction. BMP2-treated PA-SMCs were assessed for Smad1/5/8 signaling activation by Western Blotting. Key findings Treatment of PA-SMCs with CoCl2 increased PPET1 gene expression, while it did not alter expressions of endothelin converting enzyme, endothelin receptor type A or type B. Hypoxia-mimetic agent CoCl2 decreased the expressions of BMPR-2 and ID1 maximally after 3- and 6-hour treatment respectively, while CoCl2 treatment progressively increased noggin expression. Bosentan pretreatment restored expressions of BMPR-2 and ID1, as well as the activation (by phosphorylation) of Smad1/5/8 signaling induced by BMP2. Significance Hypoxia induces the downregulation of the BMP signaling in PA-SMCs, at least, partly through the endothelin system. In hypoxic PH, increased endothelin-1 production might therefore contribute to the altered BMP signaling and subsequent PA-SMC hyperplasia.
    Soluble Fn14 Is Detected and Elevated in Mouse and Human Kidney Disease
    M. Nusrat Sharif, PLOS ONE - 2016
    Abstract
    The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Fas lpr/lpr ), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in urine are significantly increased in DN patients and correlated with proteinuria and MCP-1 levels. Thus our data not only confirm the up-regulation of Fn14/TWEAK pathway in kidney diseases, but also suggest a novel mechanism for its regulation by the generation of sFn14. The correlation of sFn14 levels and disease severity suggest that sFn14 may serve as a potential biomarker for both acute and chronic kidney diseases.
    Molecular evolution of H9N2 avian influenza viruses in Israel
    Irit Davidson, International Journal of Vaccine Research - 2016
    Abstract
    Avian influenza viruses, H9N2 subtype, are endemic in Asia and the Middle East. The Israeli H9N2 (G1 lineage) consists of five phylogenetic clades that were detected in the country since the year 2000. The influence of the inter-clade genetic differences on vaccine efficacy was evaluated by vaccination-challenge trials in specific pathogen free (SPF) chickens. Experimental vaccines utilizing isolates belonging to Israeli H9 clades IV and V were prepared with Montanide ISA 70VG adjuvant. When challenged with H9 AIV strains from different genetic clades and antigenic groups provided similar protection, indicating the existence of cross-genetic clade protection (i.e. reduction in amount of virus shed and number of chickens shedding virus). Notably, the protection conferred by both experimental vaccines was highly dependent on the infection dose of the challenge virus
    Cotargeting MNK and MEK kinases induces the regression of NF1-mutant cancers
    rebecca Lock, Journal of Clinical Investigation - 2016
    Abstract
    Neurofibromin 1–mutant (NF1-mutant) cancers are driven by excessive Ras signaling; however, there are currently no effective therapies for these or other Ras-dependent tumors. While combined MEK and mTORC1 suppression causes regression of NF1-deficient malignancies in animal models, the potential toxicity of cotargeting these 2 major signaling pathways in humans may necessitate the identification of more refined, cancer-specific signaling nodes. Here, we have provided evidence that MAPK-interacting kinases (MNKs), which converge on the mTORC1 effector eIF4E, are therapeutic targets in NF1-deficient malignancies. Specifically, we evaluated primary human NF1-deficient peripheral nervous system tumors and found that MNKs are activated in the majority of tumors tested. Genetic and chemical suppression of MNKs in NF1-deficient murine tumor models and human cell lines potently cooperated with MEK inhibitors to kill these cancers through effects on eIF4E. We also demonstrated that MNK kinases are important and direct targets of cabozantinib. Accordingly, coadministration of cabozantinib and MEK inhibitors triggered dramatic regression in an aggressive genetically engineered tumor model. The cytotoxicity of this combination required the suppression of MNK-induced eIF4E phosphorylation and was not recapitulated by suppressing other cabozantinib targets. Collectively, these studies demonstrate that combined MNK and MEK suppression represents a promising therapeutic strategy for these incurable Ras-driven tumors and highlight the utility of developing selective MNK inhibitors for these and possibly other malignancies.
    3-3-Di-Substituted-Oxindoles as Inhibitors of Translation Initiation
    Halperin, José A, US Patent - 2016
    Abstract
    Compositions and methods for inhibiting translation are provided. Compositions, methods and kits for treating (1) cellular proliferative disorders, (2) non-proliferative, degenerative disorders, (3) viral infections, and/or (4) disorders associated with viral infections, using diaryloxindole compounds are described.
    CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos
    Evguenia Kouranova, Human Gene Therapy - 2016
    Abstract
    The rapid development of CRISPR technology greatly impacts the field of genetic engineering. The simplicity in design and generation of highly efficient CRISPR reagents allows more and more researchers to take on genome editing in different model systems in their own labs, even for those who found it daunting before. An active CRISPR complex contains a protein component (Cas9) and an RNA component (small guide RNA [sgRNA]), which can be delivered into cells in various formats. Cas9 can be introduced as a DNA expression plasmid, in vitro transcripts, or as a recombinant protein bound to the RNA portion in a ribonucleoprotein particle (RNP), whereas the sgRNA can be delivered either expressed as a DNA plasmid or as an in vitro transcript. Here we compared the different delivery methods in cultured cell lines as well as mouse and rat single-cell embryos and view the RNPs as the most convenient and efficient to use. We also report the detection of limited off-targeting in cells and embryos and discuss approaches to lower that chance. We hope that researchers new to CRISPR find our results helpful to their adaptation of the technology for optimal gene editing.
    Cardiac hypertrophy and decreased high-density lipoprotein cholesterol in Lrig3-deficient mice
    Martin Hellstrom, American Journal of Physiology - Regulatory, Integrative and Comparative Physiology - 2016
    Abstract
    Genetic factors confer risk for cardiovascular disease. Recently, large genome-wide population studies have shown associations between genomic loci close to LRIG3 and heart failure and plasma high-density lipoprotein (HDL) cholesterol level. Here, we ablated Lrig3 in mice and investigated the importance of Lrig3 for heart function and plasma lipid levels. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to analyze Lrig3 expression in the hearts of wild-type and Lrig3-deficient mice. In addition, molecular, physiological, and functional parameters such as organ weights, heart rate, blood pressure, heart structure and function, gene expression in the heart, and plasma insulin, glucose and lipid levels were evaluated. The Lrig3-deficient mice were smaller than the wild-type mice but otherwise appeared grossly normal. Lrig3 was expressed at detectable but relatively low levels in adult mouse hearts. At nine months of age, ad libitum fed Lrig3-deficient mice had lower insulin levels than wild-type mice. At 12 months of age, Lrig3-deficient mice exhibited increased blood pressure, and the Lrig3-deficient female mice displayed signs of cardiac hypertrophy as assessed by echocardiography, heart-to-body-weight ratio, and expression of the cardiac hypertrophy marker gene Nppa. Additionally, Lrig3-deficient mice had reduced plasma HDL-cholesterol and free glycerol. These findings in mice complement the human epidemiological results and suggest that Lrig3 may influence heart function and plasma lipid levels in mice and humans.
    MAPK Activation Is Essential for Waddlia chondrophila Induced CXCL8 Expression in Human Epithelial Cells
    Skye Storrie, PLOS ONE - 2016
    Abstract
    Background Waddlia chondrophila ( W . chondrophila ) is an emerging agent of respiratory and reproductive disease in humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial agents, such as Chlamydia abortus ( C . abortus ). The current study investigated the growth characteristics and innate immune responses of human and ruminant epithelial cells in response to infection with W . chondrophila . Methods Human epithelial cells (HEp2) were infected with W . chondrophila for 24h. CXCL8 release was significantly elevated in each of the cell lines by active-infection with live W . chondrophila , but not by exposure to UV-killed organisms. Inhibition of either p38 or p42/44 MAPK significantly inhibited the stimulation of CXCL8 release in each of the cell lines. To determine the pattern recognition receptor through which CXCL8 release was stimulated, wild-type HEK293 cells which express no TLR2, TLR4, NOD2 and only negligible NOD1 were infected with live organisms. A significant increase in CXCL8 was observed. Conclusions/Significance W . chondrophila actively infects and replicates within both human and ruminant epithelial cells stimulating CXCL8 release. Release of CXCL8 is significantly inhibited by inhibition of either p38 or p42/44 MAPK indicating a role for this pathway in the innate immune response to W . chondrophila infection. W . chondrophila stimulation of CXCL8 secretion in HEK293 cells indicates that TLR2, TLR4, NOD2 and NOD1 receptors are not essential to the innate immune response to infection.
    The transcription factor scleraxis is a critical regulator of cardiac fibroblast phenotype
    Rushita A. Bagchi, BMC Biology - 2016
    Abstract
    Resident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis.
    RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation
    Jayanth Kumar Palanichamy, Journal of Clinical Investigation - 2016
    Abstract
    Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia–rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3′ untranslated regions (3′UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.
    Oxysterol-related-binding-protein related Protein-2 (ORP2) regulates cortisol biosynthesis and cholesterol homeostasis
    Tamara Escajadillo, Molecular and Cellular Endocrinology - 2016
    Abstract
    Oxysterol binding protein-related protein 2 (ORP2) is a lipid binding protein that has been implicated in various cellular processes, including lipid sensing, cholesterol efflux, and endocytosis. We recently identified ORP2 as a member of a protein complex that regulates glucocorticoid biosynthesis. Herein, we examine the effect of silencing ORP2 on adrenocortical function and show that the ORP2 knockdown cells exhibit reduced amounts of multiple steroid metabolites, including progesterone, 11-deoxycortisol, and cortisol, but have increased concentrations of androgens, and estrogens. Moreover, silencing ORP2 suppresses the expression of most proteins required for cortisol production and reduces the expression of steroidogenic factor 1 (SF1). ORP2 silencing also increases cellular cholesterol, concomitant with decreased amounts of 22-hydroxycholesterol and 7-ketocholesterol, two molecules that have been shown to bind to ORP2. Further, we show that ORP2 binds to liver X receptor (LXR) and is required for nuclear LXR expression. LXR and ORP2 are recruited to the CYP11B1 promoter in response to cAMP signaling. Additionally, ORP2 is required for the expression of other LXR target genes, including ABCA1 and the LDL receptor (LDLR). In summary, we establish a novel role for ORP2 in regulating steroidogenic capacity and cholesterol homeostasis in the adrenal cortex.
    ANTIBODIES THAT BIND TO TL1A AND METHODS OF TREATING INFLAMMATORY OR AUTOIMMUNE DISEASE COMPRISING ADMINISTERING SUCH ANTIBODIES - THE USA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES
    Siegel, Richard M, United States Patent - 2016
    Abstract
    Methods and compositions for treating inflammatory or autoimmune diseases in a subject comprising blocking the interaction between DR3 and TL1A. In the methods of treating inflammatory or autoimmune disease, the inflammatory or autoimmune disease can be an autoimmune disease with a T cell component, including asthma, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, graft versus host disease or inflammatory bowel disease.
    LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus
    F. Stumpf, The Royal Society of Chemistry 2016 - 2015
    Abstract
    Portable point-of-care devices for pathogen detection require easy, minimal and user-friendly handling steps and need to have the same diagnostic performance compared to centralized laboratories. In this work we present a fully automated sample-to-answer detection of influenza A H3N2 virus in a centrifugal LabDisk with complete prestorage of reagents. Thus, the initial supply of the sample remains the only manual handling step. The self-contained LabDisk automates by centrifugal microfluidics all necessary process chains for PCR-based pathogen detection: pathogen lysis, magnetic bead based nucleic acid extraction, aliquoting of the eluate into 8 reaction cavities, and real-time reverse transcription polymerase chain reaction (RT-PCR). Prestored reagents comprise air dried specific primers and fluorescence probes, lyophilized RT-PCR mastermix and stick-packaged liquid reagents for nucleic acid extraction. Employing two different release frequencies for the stick-packaged liquid reagents enables on-demand release of highly wetting extraction buffers, such as sequential release of lysis and binding buffer. Microfluidic process-flow was successful in 54 out of 55 tested LabDisks. We demonstrate successful detection of the respiratory pathogen influenza A H3N2 virus in a total of 18 LabDisks with sample concentrations down to 2.39 × 104 viral RNA copies per ml, which is in the range of clinical relevance. Furthermore, we detected RNA bacteriophage MS2 acting as internal control in 3 LabDisks with a sample concentration down to 75 plaque forming units (pfu) per ml. All experiments were applied in a 2 kg portable, laptop controlled point-of-care device. The turnaround time of the complete analysis from sample-to-answer was less than 3.5 hours.
    Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
    Robin E. Pearce, Drug Metabolism and Disposition - 2015
    Abstract
    Although CYP2B6 catalyzes the biotransformation of many drugs used clinically in children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs) and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes so that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to post-natal samples and all of the fetal HLMs failed to catalyze bupropion hydroxylation, but fetal CYP2B6 protein levels were not significantly different from post-natal levels. Considerable inter-individual variation in CYP2B6 mRNA expression, protein levels and activity was observed in post-natal HLMs (mRNA, ~40,000-fold; protein, ~300-fold; activity, ~600-fold). The extremely wide range of inter-individual variability in CYP2B6 expression and activity was significantly associated with age (ρ<0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 inter-individual variability was not significantly associated with genetic variation in CYP2B6, nor with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.
    Evaluation of the maturation of individual Dengue virions with flow virometry
    Sonia Zicari, Virology - 2016
    Abstract
    High-throughput techniques are needed to analyze individual virions to understand how viral heterogeneity translates into pathogenesis since in bulk analysis the individual characteristics of virions are lost. Individual Dengue virions (DENV) undergo a maturation that involves a proteolytic cleavage of prM precursor into virion-associated M protein. Here, using a new nanoparticle-based technology, “flow virometry”, we compared the maturation of individual DENV produced by BHK-21 and LoVo cells. The latter lacks the furin-protease that mediates prM cleavage. We found that prM is present on about 50% of DENV particles produced in BHK-21 cells and about 85% of DENV virions produced in LoVo, indicating an increase in the fraction of not fully matured virions. Flow virometry allows us to quantify the number of fully mature particles in DENV preparations and proves to be a useful method for studying heterogeneity of the surface proteins of various viruses.
    Human Intestinal Enteroids: A New Model to Study Human Rotavirus Infection, Host Restriction and Pathophysiology
    Kapil Saxena, Journal of Virology - 2015
    Abstract
    Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes, goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these non-transformed, 3D HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs as they generally replicate poorly in transformed cell lines and host range restriction prevents their replication in many animal models whereas many animal RVs (ARV) exhibit a broader host range and replicate in mice. Using HRVs, including the RV1 Rotarix™ vaccine strain and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than ARV. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused a physiologic lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model, which will allow the study of HRV biology including host restriction, cell-type restriction, and viral-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology, pathophysiology and the epithelial response, including host restriction to gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤ age five. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, diverse cellular and physiologically relevant epithelial cultures, which recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions, host and cell-type restriction, viral-induced fluid secretion, cell-cell communication within the epithelium, and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures.
    Spatiotemporal analysis of zebrafish hox gene regulation by Cdx4
    Albert G. Hayward, Developmental Dynamics - 2015
    Abstract
    Background: Cdx factors expressed in caudal regions of vertebrate embryos regulate hox patterning gene transcription. While loss of Cdx function is known to shift hox spatial expression domains posteriorly, the mechanism underlying the shift is not understood. We addressed this problem by analyzing the spatiotemporal expression profile of all 49 zebrafish hox genes in wild-type and Cdx4-deficient embryos. Results: Loss of Cdx4 had distinct effects on hox spatial expression in a paralogous group-dependent manner: in the head, group 4 expression was expanded posteriorly; in the trunk, group 5–10 expression was shifted posteriorly; and in the tail, group 11–13 genes were expressed in the tail bud but not in more differentiated tissues. In the trunk neural tissue, loss of Cdx4 severely delayed both transcriptional activation of hox genes during the initiation phase, and the anterior-ward expansion of hox expression domains during the establishment phase. In contrast, in the trunk mesoderm, loss of Cdx4 only delayed the hox initiation phase. Conclusions: These results indicate that Cdx4 differentially regulates the transcription of head, trunk and tail hox genes. In the trunk, Cdx4 conveys spatial positional information to axial tissues primarily by regulating the time of hox gene transcriptional activation during the initiation phase. Developmental Dynamics, 2015. © 2015 Wiley Periodicals, Inc.
    First-Strand cDNA Synthesis
    Beta-2 Adrenergic and Glucocorticoid Receptor Agonists Modulate Ozone-Induced Pulmonary Protein Leakage and Inflammation in Healthy and Adrenalectomized Rats
    Andres R. Henriquez, Toxicological Sciences - 2018
    Abstract
    We have shown that acute ozone inhalation activates sympathetic-adrenal-medullary and hypothalamus-pituitary-adrenal stress axes, and adrenalectomy (AD) inhibits ozone-induced lung injury and inflammation. Therefore, we hypothesized that stress hormone receptor agonists (β2 adrenergic-β2AR and glucocorticoid-GR) will restore the ozone injury phenotype in AD, while exacerbating effects in sham-surgery (SH) rats. Male Wistar Kyoto rats that underwent SH or AD were treated with vehicles (saline + corn oil) or β2AR agonist clenbuterol (CLEN, 0.2 mg/kg, i.p.) + GR agonist dexamethasone (DEX, 2 mg/kg, s.c.) for 1 day and immediately prior to each day of exposure to filtered air or ozone (0.8 ppm, 4 h/day for 1 or 2 days). Ozone-induced increases in PenH and peak-expiratory flow were exacerbated in CLEN+DEX-treated SH and AD rats. CLEN+DEX affected breath waveform in all rats. Ozone exposure in vehicle-treated SH rats increased bronchoalveolar lavage fluid (BALF) protein, N-acetyl glucosaminidase activity (macrophage activation), neutrophils, and lung cytokine expression while reducing circulating lymphocyte subpopulations. AD reduced these ozone effects in vehicle-treated rats. At the doses used herein, CLEN+DEX treatment reversed the protection offered by AD and exacerbated most ozone-induced lung effects while diminishing circulating lymphocytes. CLEN+DEX in air-exposed SH rats also induced marked protein leakage and reduced circulating lymphocytes but did not increase BALF neutrophils. In conclusion, circulating stress hormones and their receptors mediate ozone-induced vascular leakage and inflammatory cell trafficking to the lung. Those receiving β2AR and GR agonists for chronic pulmonary diseases, or with increased circulating stress hormones due to psychosocial stresses, might have altered sensitivity to air pollution.
    Atlantic salmon post-smolts adapted for a longer time to seawater develop an effective humoral and cellular immune response against Salmonid alphavirus
    N. Nuñez-Ortiz, Fish & Shellfish Immunology - 2018
    Abstract
    Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection.
    Neutrophils Promote Amphiregulin Production in Intestinal Epithelial Cells through TGF-β and Contribute to Intestinal Homeostasis
    Feidi Chen, The Journal of Immunology - 2018
    Abstract
    Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-β promoted AREG production by IECs. Mechanistically, TGF-β activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-β–induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.
    Reduced expression of the IL7Ra signaling pathway in Neuromyelitis optica
    Livnat Brill, Journal of Neuroimmunology - 2018
    Abstract
    Neuromyelitis optica (NMO) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system that most commonly affects the optic nerves and spinal cord. To characterize the immunological pathways involved in NMO, whole blood RNA expression array was performed using Nanostring nCounter technology. Two major clusters of genes were found associated with NMO: T cell-associated genes and the TNF/NF-kB signaling pathway. Analysis of the genes within the first cluster confirmed significantly reduced expression of IL7Ra (CD127) in the peripheral blood of NMO patients vs that in healthy controls. IL7Ra upstream transcription factors and its downstream survival signaling pathway were also markedly reduced. In line with the essential role of IL7Ra in T cell maturation and survival, a significantly lower number of naïve T cells, and reduced T cell survival signaling mediated by increased BID (BH3-interacting domain death agonist) expression and increased apoptosis was observed. Cumulatively, these findings indicate that the IL7Ra signaling pathway may play a role in the autoimmune process in NMO.
    Variation in the responsiveness of induced resistance against Pseudomonas syringae pv. tomato by Solanum lycopersicum treated with para-aminobenzoic acid
    P. H. Goodwin, Physiological and Molecular Plant Pathology - 2018
    Abstract
    Para-aminobenzoic acid (PABA) induced resistance against Pseudomonas syringae pv. tomato in Solanum lycopersicum. Fertilizer application was necessary for induced resistance and only two out of eight breeding lines showed PABA-induced resistance. A comparison of gene expression between a PABA-responsive and PABA-non-responsive line showed that either the responsive line had a faster and stronger increase in expression or the responsive line showed increased expression due to PABA. Although PABA affected SA-related gene expression, changes in gene expression were not always directly related to the responsiveness to PABA. The response to PABA by SA, JA and ET-related mutants showed SA dependence. Factors, such as fertilization practices and host genotype, are important considerations in the development of PABA as a disease management product.
    Small adipose stores in cystic fibrosis mice are characterized by reduced cell volume, not cell number
    Ilya R. Bederman, American Journal of Physiology-Gastrointestinal and Liver Physiology - 2018
    Abstract
    Cystic fibrosis (CF) is a lethal genetic disorder that affects many organ systems of the body, including various endocrine and exocrine tissues. Health and survival positively associate with body mass and as a consequence, CF clinical care includes high-fat, high-calorie diets to maintain and increase adipose tissue stores. Such strategies have been implemented without a clear understanding of the cause and effect relationship between body mass and patients' health. Here, we use CF mouse models, which display small adipose stores, to begin examining body fat as a prelude into mechanistic studies of low body growth in CF, so that optimal therapeutic strategies can be developed. We reasoned that low adiposity must result from reduced number and/or volume of adipocytes. To determine relative contribution of either mechanism, we quantified volume of intraperitoneal and subcutaneous adipocytes. We found smaller, but not fewer, adipocytes in CF compared to wildtype (WT) animals. Specifically, intraperitoneal CF adipocytes were half the volume of WT cells, whereas subcutaneous cells were less affected by the Cftr genotype. No differences were found in cell types between CF and WT adipose tissues. Adipose tissue CFTR mRNA was detected and we found greater CFTR expression in intraperitoneal depots as compared with subcutaneous samples. RNA sequencing revealed that CF adipose tissue exhibited lower expression of several key genes of adipocyte function (Lep, Pck1, Fas, Jun), consistent with low triglyceride storage. The data indicate that CF adipocytes contain less triglycerides than WT cells and a role for CFTR in these cells is proposed.
    Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission
    C. A. Cobine, The Journal of Physiology - 2018
    Abstract
    Key points summary Inhibitory NMT was compared in the IAS and rectum of the Cynomolgus monkey; an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+/VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were similar. SK3 channel activation and inhibition caused greater/faster changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in the IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. Abstract Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey; an animal with high gene sequence identity to humans. Electrical field stimulation produced NOS-dependent contractile inhibition in both muscles whereas P2Y1-dependent purinergic NMT was restricted to rectum. An additional NOS-independent, α-Chymotrypsin-sensitive component was identified in the IAS consistent with VIPergic NMT. Microelectrode recordings revealed slow NOS-dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1-dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+/SK3+ cells were closely aligned with nNOS+/VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2-14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was 5 times faster in rectum than the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. This article is protected by copyright. All rights reserved
    CETP Inhibition Improves HDL Function but Leads to Fatty Liver and Insulin Resistance in CETP-Expressing Transgenic Mice on a High-Fat Diet
    Lin Zhu, Diabetes - 2018
    Abstract
    In clinical trials inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but doesn’t robustly improve cardiovascular outcomes. About 2/3 of trial participants were obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define if CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP-transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality including reverse cholesterol transport and HDL’s anti-oxidative capacity in HFD-fed mice better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD-fed vs. chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver, and demonstrate a context-selectivity of CETP inhibition that might be important in obese subjects.
    TIMP Loss Activates Metalloproteinase-TNFα-DKK1 Axis To Compromise Wnt Signaling and Bone Mass
    Yan Chen, Journal of Bone and Mineral Research - 2018
    Abstract
    Deregulated proteolysis invariably underlies most human diseases including bone pathologies. Metalloproteinases constitute the largest of the five protease families, and the metzincin metalloproteinases are inhibited by the four tissue inhibitors of metalloproteinase called TIMPs. We hypothesized that Timp genes are essential for skeletal homeostasis. We bred individual Timp knockout mice to generate unique mouse models, the quadruple Timp null strain (QT) as well as mice harboring only a single Timp3 allele (QT3+/–). QT mice are grossly smaller and exhibit a dramatic reduction of trabeculae in long bones by μCT imaging with a corresponding increase in metalloproteinase activity. At the cellular level, Timp deficiency compromised differentiation markers, matrix deposition and mineralization in neonatal osteoblasts from calvariae, as well as the fibroblastic colony-forming unit (CFU-F) capacity of bone marrow–derived stromal cells. In contrast, we observed that osteoclasts were overactive in the Timp null state, consistent with the noted excessive bone resorption of QT bones. Immunohistochemistry (IHC) and immunofluorescence (IF) analyses of bone sections revealed higher Cathepsin K and RANKL signals upon Timp loss. Seeking the molecular mechanism, we identified abnormal TNFα bioactivity to be a central event in Timp-deficient mice. Specifically, TNFα triggered induction of the Wnt signaling inhibitor Dkk1 in the osteoblasts at the mRNA and protein levels, with a simultaneous increase in RANKL. Neutralizing TNFα antibody was capable of rescuing the induction of Dkk1 as well as RANKL. Therefore, the generation of novel Timp-deficient systems allowed us to uncover the essential and collective function of TIMP proteins in mammalian long-bone homeostasis. Moreover, our study discovers a functional TIMP/metalloproteinase-TNFα-Dkk1/RANKL nexus for optimal control of the bone microenvironment, which dictates coexistence of the osteoblast and osteoclast lineages. © 2018 American Society for Bone and Mineral Research.
    Genetics and expression of anthocyanin pathway genes in the major skin-pigmented Portuguese cultivar ‘Vinhão’ developing berries
    Vanessa Ferreira, Scientia Horticulturae - 2018
    Abstract
    ‘Vinhão’ is an autochthonous Portuguese cultivar with an intense black-bluish skin color, highly appreciated due to this feature. This study aimed to give the first insights into the genetic background that may be responsible for the skin color properties of cv. ‘Vinhão’. For this purpose, the allelic composition of MYBA1 and MYBA2 genes was investigated, along with quantification of the expression levels of structural and regulatory genes involved in the anthocyanin biosynthetic pathway via qRT-PCR. The molecular characterization of MYBA1 and MYBA2 loci revealed that cv. ‘Vinhão’ is homozygous for the functional allele in both genes, corresponding to the most ancestral haplotype, which is consistent with the high colored phenotype that characterizes this cultivar. There were no differences in the DNA sequence of the MYBA1 promoter region between cv. ‘Vinhão’ and the grapevine reference genome Pinot Noir. The expression patterns of genes playing key functional roles in anthocyanin biosynthesis was analyzed in four developmental stages. The dynamics occurring throughout grape berry development revealed the involvement of these genes in the progression of key development events, mainly from veraison to mature berries. These findings provide the first molecular characterization focused on the skin color feature of cv. ‘Vinhão’ to improve our understanding of the genetics behind its intense skin pigmentation.
    Induction of early growth response gene 1 (EGR1) by endoplasmic reticulum stress is mediated by the extracellular regulated kinase (ERK) arm of the MAPK pathways
    Jixiu Shan, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research - 2018
    Abstract
    Endoplasmic reticulum (ER) stress activates three principal signaling pathways, collectively known as the unfolded protein response, leading to translational and transcriptional control mechanisms that dictate the cell's response as adaptive or apoptotic. The present study illustrates that for HepG2 human hepatocellular carcinoma cells the signaling pathways triggered by ER stress extend beyond the three principal pathways to include mitogen-activated protein kinase (MAPK) signaling, leading to activation of transcription from the early growth response 1 (EGR1) gene. Analysis provided evidence for a SRC-RAS-RAF-MEK-ERK cascade mechanism that leads to enhanced phosphorylation of the transcription factor ELK1. ELK1 and serum response factor (SRF) are constitutively bound to the EGR1 promoter and are phosphorylated by nuclear localized ERK. The promoter abundance of both phospho-SRF and phopsho-ELK1 was increased by ER stress, but the SRF phosphorylation was transient. Knockdown of ELK1 had little effect on the basal EGR1 mRNA content, but completely blocked the increase in response to ER stress. Conversely, knockdown of SRF suppressed basal EGR1 mRNA content, but had only a small effect on the induction by ER stress. This research highlights the importance of MAPK signaling in response to ER stress and identifies ELK1 as a transcriptional mediator and the EGR1 gene as a target.
    The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D
    Grant P. Parnell, Human Molecular Genetics - 2018
    Abstract
    Epidemiological, molecular and genetic studies have indicated high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The Vitamin D Receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude-dependence than in risk genes for other diseases. The transcription factor genes ZMIZ1 and IRF8 - risk genes for many of these diseases - have VDR binding peaks co-incident with the risk SNPs. We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.
    Treatment with Lecinoxoids Attenuates Focal and Segmental Glomerulosclerosis Development in Nephrectomized Rats
    Niva Yacov, Basic & Clinical Pharmacology & Toxicology - 2018
    Abstract
    Focal segmental glomerulosclerosis (FSGS) is a scarring process associated with chronic low-grade inflammation ascribed to toll-like receptor (TLR) activation and monocyte migration. We developed synthetic, small-molecule lecinoxoids, VB-201 and VB-703, that differentially inhibit TLR-2- and TLR-4-mediated activation and monocyte migration. The efficacy of anti-inflammatory lecinoxoid treatment on FSGS development was explored using a 5/6 nephrectomy rat model. Five-sixths nephrectomized rats were treated with lecinoxoids VB-201, VB-703 or PBS, for 7 weeks. Upon sacrifice, albumin/creatinine ratio, glomerulosclerosis, fibrosis-related gene expression, and the number of glomerular and interstitial monocyte were evaluated. Treatment of nephrectomized rats with lecinoxoids ameliorated glomerulosclerosis. The percent of damaged glomeruli, glomerular sclerosis and glomeruli fibrotic score were significantly reduced following VB-201 and VB-703 treatment. VB-703 attenuated the expression of fibrosis hallmark genes collagen, fibronectin (FN), and transforming growth factor β (TGF-β) in kidneys and improved albumin/creatinine ratio with higher efficacy than did VB-201, but only VB-201 significantly reduced the number of glomerular and interstitial monocytes. These results indicate that treatment with TLR-2, and more prominently, TLR-4 antagonizing lecinoxioids, is sufficient to significantly inhibit FSGS. Moreover, inhibiting monocyte migration can also contribute to treatment of FSGS. Our data demonstrate that targeting TLR2-4 and/or monocyte migration, directly affects the priming phase of fibrosis and may consequently perturb disease parthogenesis. This article is protected by copyright. All rights reserved.
    Myeloperoxidase deficiency attenuates systemic and dietary Iron-induced adverse effects
    Xia Xiao, The Journal of Nutritional Biochemistry - 2018
    Abstract
    Iron deficiency is routinely treated with oral or systemic iron supplements, which is highly reactive and could induce oxidative stress via augmenting the activity of proinflammatory enzyme, myeloperoxidase (MPO). To investigate the extent to which MPO is involved in iron-induced toxicity, acute (24 h) iron toxicity was induced by intraperitoneal administration of FeSO4 (25 mg/kg body weight) to MPO deficient (MpoKO) mice and their WT littermates. Acute iron-toxicity was also assessed in WT mice pretreated with a MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH). Systemic iron administration upregulated circulating MPO, neutrophil elastase and elevated systemic inflammatory and organ damage markers in WT mice. However, genetic deletion of MPO or its inhibition significantly reduced iron-induced organ damage and systemic inflammatory responses. In contrast to the acute model, 8 weeks of 2% carbonyl iron diet feeding to WT mice did not change the levels of circulating MPO and neutrophil elastase but promoted their accumulation in the liver. Even though both MpoKO and WT mice displayed similar levels of diet-induced hyperferremia, MpoKO mice showed significantly reduced inflammatory response and oxidative stress than the WT mice. In addition, WT bone marrow-derived neutrophils (BMDN) generated more reactive oxygen species than MPO deficient BMDN upon iron stimulation. Altogether, genetic deficiency or pharmacologic inhibition of MPO substantially attenuated acute and chronic iron-induced toxicity. Our results suggest that targeting MPO during iron supplementation is a promising approach to reduce iron-induced toxicity/side effects in vulnerable population.
    Vamorolone Treatment Improves Skeletal Muscle Outcome in a Critical Illness Myopathy Rat Model
    Hazem Akkad, Acta Physiologica - 2018
    Abstract
    Aim Critical illness myopathy (CIM) is a consequence of modern critical care, leading to skeletal muscle atrophy/paralysis with negative consequences for mortality/morbidity and health care costs. Glucocorticoids (GCs) have been proposed to trigger CIM. Here, we compare outcomes of two GCs, the commonly used prednisolone and the newly developed dissociative vamorolone in response to the intensive care unit (ICU) condition for 5 days, i.e., sedation, immobilization, and mechanical ventilation. Methods Rats were divided into a 0-day sham-operated control group, and three groups exposed to 5 days ICU condition during treatment with prednisolone (PRED) or vamorolone (VAM) or none of these GCs (ICU-group). Survival, body and muscle weights, cytokine concentrations, regulation of muscle contraction in single fast- and slow-twitch muscle fibers, myofibrillar protein expression and protein degradation pathways were studied. Results CIM geno- and phenotypes were confirmed in the ICU group. However, VAM and PRED groups showed reduced atrophy/weakness than the ICU group, and muscle specific differences with more severe negative effects on fast-twitch muscle fibers in the PRED than the other groups. Conclusion These results show that vamorolone provides a GC intervention superior to typical GCs in improving CIM outcomes. Further, the findings do not support the notion that moderate-dose GC treatment represents a factor triggering CIM. This article is protected by copyright. All rights reserved.
    Humoral and cellular immune response of European sea bass (Dicentrarchus labrax(vaccinated with heat-killed Mycobacterium marinum (iipA::kan mutant)
    N. Ziklo, Journal of Aquatic Animal Health - 2018
    Abstract
    No vaccine is yet commercially available against Mycobacterium marinum, the etiological agent of “fish tuberculosis”. The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to moderate M. marinum pathology in zebrafish. Two doses of heat-killed, wild type virulent M. marinum and two doses of a heat-killed avirulent M. marinum iipA::kan mutant strain were used in parallel to vaccinate sea bass (Dicentrarchus labrax). The fish were then challenged with live virulent M. marinum and the pathogenesis of the infection was monitored. High specific IgM response and increase in cytokine TNF-α mRNA expression levels were observed in all vaccinated fish. One month post-challenge, TNF-α expression levels increased in spleen tissues of fish vaccinated with the virulent type and of unvaccinated fish, whereas in the head-kidney expression up-regulated only in unvaccinated fish. The expression then decreased and two months post-challenge appeared similar in all vaccination types. Highest survival rate (75%) was recorded in the group of fish vaccinated with high dose of iipA::kan avirulent mutant. The iipA::kan mutant induced a strong immune response accompanied by only modest tissue disruption. Coupled with an effective program of booster treatments, the iipA::kan mutant vaccine may be developed into a powerful preventive measure against fish mycobacteriosis. This article is protected by copyright. All rights reserved.
    Peripubertal stress increases play fighting at adolescence and modulates nucleus accumbens CB1 receptor expression and mitochondrial function in the amygdala
    Aurélie Papilloud, Translational Psychiatry - 2018
    Abstract
    Play fighting is a highly rewarding behavior that helps individuals to develop social skills. Early-life stress has been shown to alter play fighting in rats and hamsters as well as to increase aggressive behaviors at adulthood. However, it is not known whether individual differences in stress-induced play fighting are related to differential developmental trajectories towards adult aggression. To address this question, we used a rat model of peripubertal stress (PPS)-induced psychopathology that involves increased aggression at adulthood. We report that, indeed, PPS leads to enhanced play fighting at adolescence. Using a stratification approach, we identify individuals with heightened levels of play fighting as the ones that show abnormal forms of aggression at adulthood. These animals showed as well a rapid habituation of their corticosterone responsiveness to repeated stressor exposure at peripuberty. They also showed a striking increase in mitochondrial function in the amygdala—but not nucleus accumbens—when tested ex vivo. Conversely, low, but not high players, displayed increased expression of the CB1 cannabinoid receptor in the nucleus accumbens shell. Our results highlight adolescence as a potential critical period in which aberrant play fighting is linked to the emergence of adult aggression. They also point at brain energy metabolism during adolescence as a possible target to prevent adult aggression.
    Sesamin and sesamolin reduce amyloid-β toxicity in a transgenic Caenorhabditis elegans
    Roongpetch Keowkase, Biomedicine & Pharmacotherapy - 2018
    Abstract
    Alzheimer’s disease (AD) is a devastating neurodegenerative disease characterized by β-amyloid (Aβ) plaques in the brain. At the present, there is no approved drug with a proven disease-modifying effect. Sesame seed (Sesame indicum) has long been known as a healthy food in Southeast Asian countries. Sesame lignans obtained from sesame seed possess antioxidant property that exhibit a variety of beneficial effects in various models. The objective of this study was to investigate the protective effects of sesame lignans including sesamin, sesamolin, and sesamol against Aβ toxicity in Caenorhabditis elegans (C. elegans) model of Aβ toxicity and to address whether these sesame lignans have a positive effect on lifespan extension. A transgenic C. elegans expressing human Aβ was used to investigate protective effects of sesame lignans against Aβ toxicity. Sesamin and sesamolin significantly alleviated Aβ-induced paralysis. The real-time PCR revealed that both sesamin and sesamolin did not affect the expression of Aβ transgene. However, we found that only sesamin inhibited Aβ oligomerization. These findings demonstrated that, among three sesame lignans tested, sesamin protected against Aβ toxicity by reducing toxic Aβ oligomers. Sesamin and sesamolin also significantly improved Aβ-induced defect in chemotaxis behavior and reversed the defect to normal. Moreover, sesamin prolonged median and mean lifespan of the wild type worm. On the other hand, sesamolin and sesamol failed to extend lifespan. These results offer valuable evidence for the future use of sesamin in the development of agents for the treatment of AD. It is also worth investigating the structure-activity relationship of lignan-related structures and their anti-Aβ toxicity activities in the future.
    Extracellular dsRNA induces a type I interferon response mediated via class A scavenger receptors in a novel Chinook salmon derived spleen cell line
    S. L. Semple, Developmental & Comparative Immunology - 2018
    Abstract
    Despite increased global interest in Chinook salmon aquaculture, little is known of their viral immune defenses. This study describes the establishment and characterization of a continuous cell line derived from Chinook salmon spleen, CHSS, and its use in innate immune studies. Optimal growth was seen at 14–18 °C when grown in Leibovitz's L-15 media with 20% fetal bovine serum. DNA analyses confirmed that CHSS was Chinook salmon and genetically different from the only other available Chinook salmon cell line, CHSE-214. Unlike CHSE-214, CHSS could bind extracellular dsRNA, resulting in the rapid and robust expression of antiviral genes. Receptor/ligand blocking assays confirmed that class A scavenger receptors (SR-A) facilitated dsRNA binding and subsequent gene expression. Although both cell lines expressed three SR-A genes: SCARA3, SCARA4, and SCARA5, only CHSS appeared to have functional cell-surface SR-As for dsRNA. Collectively, CHSS is an excellent cell model to study dsRNA-mediated innate immunity in Chinook salmon.
    Alzheimer’s disease neuropathology may not predict functional impairment in HIV: a report of two individuals
    Susan Morgello, Journal of NeuroVirology - 2018
    Abstract
    With aging of HIV populations, there is concern that Alzheimer’s disease (AD) may become prevalent and difficult to distinguish from HIV-associated neurocognitive disorders. To date, there are no reports documenting histologically verified Alzheimer’s neuropathology in individuals with HIV and dementia. Herein, we report two antiretroviral-treated, virally suppressed, HIV-infected individuals autopsied by the Manhattan HIV Brain Bank. Subject A presented to study at 52 years, already dependent in instrumental activities of daily living (ADLs), with severe cognitive impairment inclusive of learning and memory dysfunction. Her history was significant for educational disability and head trauma. She had rapid cognitive decline and, by death at age 59 years, was bed-bound, incontinent, and non-communicative. At autopsy, she exhibited severe AD neuropathologic change (NIA-AA score A3B3C3) and age-related tau astrogliopathy (ARTAG). She was homozygous for APOE ε3/ε3. No HIV DNA was detected in frontal lobe by nested polymerase chain reaction. Subject B was a community dwelling 81-year-old woman who experienced sudden death by pulmonary embolus. Prior to death, she was fully functional, living independently, and managing all ADLs. At autopsy, she displayed moderate amyloid and severe tau AD neuropathologic changes (A2B3C2), ARTAG, and cerebral congophilic angiopathy. She was an APOE ε3/ε4 heterozygote, and HIV DNA, but not RNA, was detected in frontal lobe, despite 20 years of therapy-induced viral suppression. We conclude that in the setting of HIV, AD neuropathology may occur with or without symptomatic cognitive dysfunction; as with seronegative individuals, there are likely to be complex factors in the generation of clinically relevant impairments.
    EZH1 is an antipsychotic-sensitive epigenetic modulator of social and motivational behavior that is dysregulated in schizophrenia
    Andrea L. Johnstone, Neurobiology of Disease - 2018
    Abstract
    Background With the capacity to modulate gene networks in an environmentally-sensitive manner, the role of epigenetic systems in mental disorders has come under intense investigation. Dysregulation of epigenetic effectors, including microRNAs and histone-modifying enzymes, may better explain the role of environmental risk factors and the observed heritability rate that cannot be fully attributed to known genetic risk alleles. Here, we aimed to identify novel epigenetic targets of the schizophrenia-associated microRNA 132 (miR-132). Methods Histone modifications were quantified by immunodetection in response to viral-mediated overexpression of miR-132 while a luminescent reporter system was used to validate targets of miR-132 in vitro. Genome-wide profiling, quantitative PCR and NanoSting were used to quantify gene expression in post-mortem human brains, neuronal cultures and prefrontal cortex (PFC) of mice chronically exposed to antipsychotics. Following viral-mediated depletion of Enhancer of Zeste 1 (EZH1) in the murine PFC, behaviors including sociability and motivation were assessed using a 3-chambered apparatus and forced-swim test, respectively. Results Overexpression of miR-132 decreased global histone 3 lysine 27 tri-methylation (H3K27me3), a repressive epigenetic mark. Moreover, the polycomb-associated H3K27 methyltransferase, EZH1, is regulated by miR-132 and upregulated in the PFC of schizophrenics. Unlike its homolog EZH2, expression of EZH1 in the murine PFC decreased following chronic exposure to antipsychotics. Viral-mediated depletion of EZH1 in the mouse PFC attenuated sociability, enhanced motivational behaviors, and affected gene expression pathways related to neurotransmission and behavioral phenotypes. Conclusions EZH1 is dysregulated in schizophrenia, sensitive to antipsychotic medications, and a brain-enriched miR-132 target that controls neurobehavioral phenotypes.
    A piRNA utilizes HILI and HIWI2 mediated pathway to down-regulate ferritin heavy chain 1 mRNA in human somatic cells
    Sumirtha Balaratnam, Nucleic Acids Research - 2018
    Abstract
    The piwi interacting RNAs (piRNAs) are small non-coding RNAs that specifically bind to the PIWI proteins, a functional requirement. The piRNAs regulate germline development, transposons control, and gene expression. However, piRNA-mediated post-transcriptional gene regulation in human somatic cells is not well understood. We discovered a human piRNA (piR-FTH1) which has a complementary sequence in the ferritin heavy chain 1 (Fth1) mRNA. We demonstrated that expression of piR-FTH1 and Fth1 are inversely correlated in the tested tumor cell lines. We found that piR-FTH1 negatively regulates the Fth1 expression at post-transcriptional level in triple negative breast cancer (TNBC) cells. Additionally, we confirmed that transfected piR-FTH1 knocks down the Fth1 mRNA via the HIWI2 and HILI mediated mechanism. piR-FTH1 mediated Fth1 repression also increased doxorubicin sensitivity by a remarkable 20-fold in TNBC cells. Since the current piRNA-mediated knockdowns of target mRNA are mostly reported in germ line cells, piRNA-mediated post-transcriptional gene regulation in somatic cells is rather unique in its application and mechanistically uses an alternative pathway to siRNA and miRNA. This work begins to lay the groundwork with a broader impact on treatment of various diseases that are linked to elevated levels of specific mRNAs which have a piRNA target.
    Resident brain neural precursor cells develop age-dependent loss of therapeutic functions in Alzheimer's mice
    Nina Fainstein, Neurobiology of Aging - 2018
    Abstract
    There is vast knowledge on pathogenic mechanisms in Alzheimer's disease, but very little on means by which the brain protects itself from disease. A major candidate in providing neuroprotection is the resident brain neural stem/precursor cell (NPC) pool. Transplanted NPCs possess powerful immune-modulatory and trophic properties in vivo and in vitro, underscoring the question whether resident brain NPCs have any role in regulating disease pathology in Alzheimer's disease, and particularly whether they fail to protect the brain from degeneration. To evaluate brain NPC function in relation to disease pathology, we first characterized the pathological properties of 5xFAD transgenic mouse model of Alzheimer's disease at different ages. We found that age seven months is a critical time point of heavy amyloid deposition and gliosis but prior to neurodegeneration and a normal basal rate of NPC turnover in the subventricular zone (SVZ) of 5xFAD mice as compared to wild type mice. Analysis of NPC functional properties showed that despite preserved rate of turnover, there was substantial SVZ NPC dysfunction as indicated by both ex vivo and in vivo assays. Freshly isolated NPCs from seven months old 5xFAD mice exhibited reduced expansion rate, and diminished immune-modulatory and trophic properties. Moreover, there was slowed recovery of SVZ NPCs after Cytosine-arabinoside insult and markedly reduced migratory response following a Lysolecithine-induced lesion in the Corpus-Callosum in vivo. Importantly, these functions were fully preserved in two-months old 5xFAD mice, a time-point prior to Alzheimer's-specific pathological changes. There was reduced expression of key genes involved in NPC proliferative and migratory response in NPCs derived from seven months old 5xFAD mice. The dysfunctional properties and down-regulation of gene expression were reversible in NPCs derived from seven-months old 5xFAD mice following in-vitro expansion and were reproduced in wild type NPC by addition of amyloid beta peptide. Thus, there is age-dependent acquired NPC dysfunction, with loss of immune-modulatory and neurotrophic properties, which is induced by the pathological Alzheimer's brain environment at a critical time point prior to neurodegeneration. We suggest that failure of resident NPC to provide tissue support may be involved in promoting neurodegeneration.
    Gja1 expression is regulated by cooperation between SOX8/SOX9 and cJUN transcription factors in TM4 and 15P-1 Sertoli cell lines
    Firas Ghouili, Molecular Reproduction and Development - 2018
    Abstract
    Within the seminiferous tubules of the testis, Gja1-encoded connexin43 plays a critical role in intercellular communication between Sertoli cells. These cells nurture, protect and stimulate the developing germ cells and spermatids. SOX transcription factors are known to play an important role in male fertility and sex determination; however, their physiological function and the identity of their target genes in postnatal Sertoli cells remain to be defined. Members of the AP-1 family have been shown to regulate Gja1 expression in myometrial and testicular cells and to physically interact with SOX members, suggesting that these transcription factors may regulate its expression within the testis. Hence, we performed co-transfections of expression plasmids encoding SOX4, SOX8, SOX9 and cJUN with different mouse Gja1 promoter/luciferase reporter constructs within TM4 and 15P-1 Sertoli cells. We showed that a functional cooperation between cJUN and SOX8 or SOX9 regulates Gja1 expression and may involve DNA regulatory elements located between -132 and -26 bp. Such synergy relies on the recruitment of cJUN to the -47 bp AP-1 DNA regulatory element of the mouse Gja1 promoter. Hence, SOX and AP-1 members cooperate to regulate Gja1 within testicular Sertoli cells. This article is protected by copyright. All rights reserved.
    CSDC2, a cold shock domain RNA-binding protein in decidualization
    Griselda Vallejo, Journal of Cellular Physiology - 2018
    Abstract
    RNA-binding proteins (RBPs) have been described for cancer cell progression and differentiation, although there is still much to learn about their mechanisms. Here, using in vivo decidualization as a model, we describe the role of RBP cold shock domain containing C2 (CSDC2) in the endometrium. Csdc2 messenger RNA expression was differentially regulated depending on time and areas of decidua development, with the most variation in antimesometrium (AM) and, to a lesser degree, in the junctional zone (JZ). Immunohistochemistry of CSDC2 showed a preferentially cytoplasmic localization at AM and JZ, and nuclear localization in underneath myometrium and mesometrium (M). Cytoplasmic localization coincided with differentiated, DESMIN-marked areas, while nuclear localization coincides with proliferative zones. Uterine suppression of CSDC2 through intrauterine-injected-specific small interfering RNA (siRNA) led to abnormal decidualization in early pregnancy, with more extended antimesometrial area and with poor M development if compared with control siRNA-injected animals. These results suggest that CSDC2 could be a regulator during decidua development.
    Induction of brain Nrf2-HO-1 pathway and antinociception after different physical training paradigms in mice
    Abdulkarim Tutakhail, Life Sciences - 2018
    Abstract
    Aim Activation of the Nrf2-antioxidant response element signaling pathway is a major mechanism in the cellular defense against oxidative or electrophilic stress through conjugative reactions and by enhancing cellular antioxidant capacity. Although exercise training up-regulates antioxidant defenses system, while information regarding the intensity levels of physical exercise that acts on the cellular protection systems is limited. Main methods The present study evaluated the effects of different durations and intensities of physical exercise on the hippocampus, cortex and hypothalamus Nrf2 and HO-1 gene expression and related protein content and the nociception thresholds in adult C57Bl male mice. Exercise training consisted of daily running on a 10-lane rodent motor-driven treadmill for either 3 or 7 weeks at three different intensities. Pain responses were evaluated after exercise and in untrained mice by Von Frey hair test and cold plate test. Key findings This study confirmed that only vigorous and longer duration aerobic exercise increased Nrf2 protein level in the hippocampus and HO-1 protein level in the cortex and reduced pain perception. Mechanical and thermal hypoalgesia were only observed in exercise groups after 7 weeks of physical training. Significance The overall findings in this study confirm that only the long duration intensive forced exercise reduced inflammatory pain by induction of Nrf2/HO-1 antioxidant signaling pathway.
    Deletion of Axin1 in condylar chondrocytes leads to osteoarthritis-like phenotype in temporomandibular joint via activation of β-catenin and FGF signaling
    Yachuan Zhou, Journal of Cellular Physiology - 2018
    Abstract
    Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease in the adult, which is characterized by the pathological degeneration of condylar cartilage. Axin1 plays a critical role in the regulation of cartilage development and homeostasis. To determine the role of Axin1 in TMJ tissue at the adult stage, we generated Axin1Agc1ER mice, in which Axin1 was deleted in aggrecan-expressing chondrocytes at 2 months of age. Histology, histomorphometry, and immunostaining analyses were performed using TMJ tissues harvested from 4- and 6-month-old mice after tamoxifen administration. Total RNA isolated from TMJ cartilage of 6-month-old mice was used for gene expression analysis. Progressive OA-like degeneration was observed in condylar cartilage in Axin1 knockout (KO) mice with loss of surface continuity and the formation of vertical fissures. In addition, reduced alcian blue staining in condylar cartilage was also found in Axin1 KO mice. Immunostaining and reverse transcription quantitative polymerase chain reaction (qRT-PCR) assays revealed disturbed homeostasis in condylar cartilage with increased expressions of MMP13 and Adamts5 and decreased lubricin expression in Axin1-deficient chondrocytes. Less proliferative cells with increased hypertrophic and apoptotic activities were presented in the condylar cartilage of Axin1Agc1ER KO mice. As a scaffolding protein, the deletion of Axin1 stimulated not only the β-catenin but also the fibroblast growth factor (FGF) signaling via extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. The qRT-PCR results showed an increased expression of Fgfr1 in Axin1 KO cartilage. Overall, the deletion of Axin1 in condylar chondrocytes altered the β-catenin and FGF/ERK1/2 signaling pathways, thus cooperatively contribute to the cartilage degeneration.
    Cultivation of Purified Primary Purkinje Cells from Rat Cerebella
    Jonas Tjaden, Cellular and Molecular Neurobiology - 2018
    Abstract
    Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.
    Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas
    Enrica Saponara, The Journal of Pathology - 2018
    Abstract
    Pancreatic ductal adenocarcinoma (PDAC), the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical to develop alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacologic approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up serotonin in a transport-mediated manner. Serotonin uptake promoted the activation of the small GTPase Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1), which is required for trans-differentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the serotonin transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of acinar-to-ductal metaplasia with the potential to stall initiation and progression of pancreatic carcinogenesis. This article is protected by copyright. All rights reserved.
    Assessment of Sep1virus interaction with stationary cultures by transcriptional and flow cytometry studies
    Luís D. R. Melo, FEMS Microbiology Ecology - 2018
    Abstract
    The majority of phage infection studies are performed in bacteria that are growing exponentially, although in nature, phages usually interact also with non-replicating cells. These stationary-phase cells differ from exponential cells morphologically, physiologically and metabolically. The interaction of a Sep1virus with Staphylococcus epidermidis stationary and exponential phase cells was explored. Phage SEP1 efficiently infected both cell culture states, without the addition of any fresh nutrients to stationary cultures. Phage-host interactions, analysed by flow cytometry, showed stationary-phase cells response to phage immediately after SEP1 addition. Quantitative PCR experiments corroborate that phage genes are expressed within 5 min of contact with stationary phase cells. The increase of host RNA polymerase transcripts in stationary cells, suggests that SEP1 infection leads to upregulation of host machinery fundamental for completion of its lytic life cycle. SEP1 infection and replication process highlights its potential clinical interest targeting stationary phase cells.
    Loss of IGF1R in human astrocytes alters complex I activity and support for neurons
    Laura E. Ratcliffe, Neuroscience - 2018
    Abstract
    The insulin/insulin-like growth factor 1 (IGF1) signalling pathways are implicated in longevity and in progression of Alzheimer’s disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signalling transcripts are reduced in astrocytes in human brain with progression of Alzheimer’s neuropathology and developed a model of IGF1 signalling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signalling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.
    Differential gene expression to an LPS challenge in relation to exogenous corticosterone in the invasive cane toad (Rhinella marina)
    Steven Gardner, Developmental & Comparative Immunology - 2018
    Abstract
    The cane toad (Rhinella marina) is an invasive amphibian in several parts of the world. Much of the research performed on assessing the dispersal potential of invasive species has focused immunity. Invaders are predicted to rely less on pro-inflammatory immunity, allowing them to allocate energy to dispersal. Elevated stress may play a role in regulation of immune responses used by invasive species. RNA sequencing of spleen tissue from cane toads subjected to an acute LPS challenge revealed genes coding for cytokines involved in typical innate responses such as phagocytic cell recruitment, extravasation, inflammation, and lymphocyte differentiation were significantly upregulated, while toads receiving transdermal application of corticosterone in addition to an LPS injection showed downregulation of genes involved with cell mediated immunity. These results indicate hormonal changes associated with acute stress may alter investment into mounting cell-mediated or humoral responses while allowing for prolonged phagocytic innate responses in this invasive species.
    NLRP3 inflammasome mediates oxidative stress-induced pancreatic islet dysfunction.
    Marina Sokolova, American Journal of Physiology-Endocrinology and Metabolism - 2018
    Abstract
    Inflammasomes are multi-protein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1β and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and based on the central role of IL-1β in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT), NLRP3-/- and ASC-/- mice after exposing islets to short-term hypoxia or alloxan induced islet damage. NLRP3 deficient islets compared to WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Further, NLRP3 and ASC deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low dose alloxan administration, and this was associated with reduced β-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated β-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate which causes macrophage depletion prior to induction of a diabetic phenotype by alloxan treatment resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on β-cell preservation.
    https://www.nature.com/articles/s41401-018-0068-9
    Xiao Zhu, Acta Pharmacologica Sinica - 2018
    Abstract
    Perivascular adipose tissue (PVAT), a special type of adipose tissue, closely surrounds vascular adventitia and produces numerous bioactive substances to maintain vascular homeostasis. PVAT dysfunction has a crucial role in regulating vascular remodeling, but the exact mechanisms remain unclear. In this study, we investigated whether and how obesity-induced PVAT dysfunction affected adventitia remodeling in early vascular injury stages. Mini pigs were fed a high sugar and fat diet for 6 months to induce metabolic syndrome and obesity. In the mini pigs, left carotid vascular injury was then generated using balloon dilation. Compared with normal mini pigs, obese mini pigs displayed significantly enhanced vascular injury-induced adventitial responses, evidenced by adventitia fibroblast (AF) proliferation and differentiation, and adventitia fibrosis, as well as exacerbated PVAT dysfunction characterized by increased accumulation of resident macrophages, particularly the M1 pro-inflammatory phenotype, increased expression of leptin and decreased expression of adiponectin, and production of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Primary AFs cultured in PVAT-conditioned medium from obese mini pigs also showed significantly increased proliferation and differentiation. We further revealed that activated nod-like receptor protein 3 (NLRP3) inflammasome and its downstream products, i.e., IL-1 family members such as IL-1β and IL-18 were upregulated in the PVAT of obese mini pigs; PVAT dysfunction was also demonstrated in preadipocytes treated with palmitic acid. Finally, we showed that pretreatment with IL-1 receptor (IL-1R) antagonist or IL-1R knockdown blocked AF proliferation and differentiation in AFs cultured in PVAT-conditioned medium. These results demonstrate that obesity-induced PVAT dysfunction aggravates adventitial remodeling after early vascular injury with elevated AF proliferation and differentiation via activating the NLRP3/IL-1 signaling pathway.
    MED31 involved in regulating self-renewal and adipogenesis of human mesenchymal stem cells
    Erik P. Beadle, Molecular Biology Reports - 2018
    Abstract
    Regulation of gene expression is critical for the maintenance of cell state and homeostasis. Aberrant regulation of genes can lead to unwanted cell proliferation or misdirected differentiation. Here we investigate the role of MED31, a highly conserved subunit of the Mediator complex, to determine the role this subunit plays in the maintenance of human mesenchymal stem cell (hMSC) state. Using siRNA-mediated knockdown of MED31 we demonstrate a decrease in self-renewal based on cell assays and monitoring of gene expression. In addition, in the absence of MED31, hMSCs also displayed a reduction in adipogenesis as evidenced by diminished lipid vesicle formation and expression of specific adipogenic markers. These data present evidence for a significant role for MED31 in maintaining adult stem cell homeostasis, thereby introducing potential novel targets for future investigation and use in better understanding stem cell behavior and adipogenesis.
    Viral RNA load and histological changes in tissues following experimental infection with an arterivirus of possums (wobbly possum disease virus)
    Julia Giles, Virology - 2018
    Abstract
    Tissues from Australian brushtail possums (Trichosurus vulpecula) that had been experimentally infected with wobbly possum disease (WPD) virus (WPDV) were examined to elucidate pathogenesis of WPDV infection. Mononuclear inflammatory cell infiltrates were present in livers, kidneys, salivary glands and brains of WPD-affected possums. Specific staining was detected by immunohistochemistry within macrophages in the livers and kidneys, and undefined cell types in the brains. The highest viral RNA load was found in macrophage-rich tissues. The detection of viral RNA in the salivary gland, serum, kidney, bladder and urine is compatible with transmission via close physical contact during encounters such as fighting or grooming, or by contact with an environment that has been contaminated with saliva or urine. Levels of viral RNA remained high in all tissues tested throughout the study, suggesting that on-going virus replication and evasion of the immune responses may be important in the pathogenesis of disease.
    MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss
    Guney Bademci, Human Genetics - 2018
    Abstract
    While recent studies have revealed a substantial portion of the genes underlying human hearing loss, the extensive genetic landscape has not been completely explored. Here, we report a loss-of-function variant (c.72delA) in MPZL2 in three unrelated multiplex families from Turkey and Iran with autosomal recessive nonsyndromic hearing loss. The variant co-segregates with moderate sensorineural hearing loss in all three families. We show a shared haplotype flanking the variant in our families implicating a single founder. While rare in other populations, the allele frequency of the variant is ~ 0.004 in Ashkenazi Jews, suggesting that it may be an important cause of moderate hearing loss in that population. We show that Mpzl2 is expressed in mouse inner ear, and the protein localizes in the auditory inner and outer hair cells, with an asymmetric subcellular localization. We thus present MPZL2 as a novel gene associated with sensorineural hearing loss.
    The search for proteins involved in the formation of crustacean cuticular structures
    Shai Abehsera, Hydrobiologia - 2018
    Abstract
    Crustacean cuticular structures are key features formed during a molt cycle. These structures are complex biomaterials comprising chitin and different mineral forms in distinct scaffold organizations. The formation of these complex biomaterials is controlled by the organic extracellular matrix including structural proteins. Since cuticular structures are formed de novo during each molt cycle, the spatial and temporal expression patterns of structural proteins are tightly linked to molt cycle events. As a model scenario, we demonstrate the molt-related pattern of expression of the gene encoding GAP65, a core structural protein involved in the formation of the cuticular structures of Cherax quadricarinatus. Based on this typical pattern of expression and using a binary-patterning approach, which is a specialized tool for the study of molt-related proteins, we revealed and characterized additional candidate proteins involved in the formation of crustacean cuticular structures. We propose that our approach be applied as a framework in the search for proteins involved in the formation of the crustacean cuticle. To stimulate research on this important aspect of structural biology, we put forward a schematic representation of the extracellular matrix and its proteins in three cuticular structures of C. quadricarinatus, the gastroliths, the mandibles, and the mineralized cuticle.
    Lactobacillus casei BL23 modulates the innate immune response in Staphylococcus aureus-stimulated bovine mammary epithelial cells
    R.f.s. Souza, Beneficial Microbes - 2018
    Abstract
    Probiotics have been adopted to treat and prevent various diseases in humans and animals. They were notably shown to be a promising alternative to prevent mastitis in dairy cattle. This inflammation of the mammary gland is generally of infectious origin and generates extensive economic losses worldwide. In a previous study, we found that Lactobacillus casei BL23 was able to inhibit the internalisation of Staphylococcus aureus, one of the major pathogens involved in mastitis, into bovine mammary epithelial cells (bMEC). In this study, we further explored the capacity of this strain to modulate the innate immune response of bovine mammary epithelial cells during S. aureus infection. L. casei BL23 was able to decrease the expression of several pro-inflammatory cytokines, including interleukins 6, 8, 1α and 1β and tumour necrosis factor alpha, in S. aureus-stimulated bMEC, 8 h post-infection. On the other hand, L. casei did not impair the induction of defensins, such as lingual antimicrobial peptide and defensin β1 in the presence of S. aureus, and even slightly increased the induction of tracheal antimicrobial peptide during S. aureus infection. Finally, this strain did not alter the expression of the pattern recognition receptor nucleotide-binding oligomerisation domain proteins (NOD2). This study demonstrates that L. casei BL23 displayed anti-inflammatory properties on S. aureus-stimulated bMEC. These results open the way to further characterisation of the BL23 probiotic potential in a bovine mammary gland context and to a better understanding of how all these beneficial properties combine in vivo to combat mastitis pathogens.
    The von Hippel Lindau tumour suppressor gene is a novel target of E2F4-mediated transcriptional repression in preeclampsia
    Sruthi Alahari, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease - 2018
    Abstract
    The von Hippel Lindau tumour suppressor (VHL) protein is essential for proper placental development and is downregulated in preeclampsia (PE), a devastating disorder of pregnancy typified by chronic hypoxia. To date, knowledge on VHL genetic and epigenetic regulation is restricted to inactivating mutations and loss-of-heterozygosity in renal cell carcinomas. Herein, we sought to examine whether VHL DNA is subject to differential methylation, and if so, whether it is altered in early-onset PE (E-PE). Sodium bisulfite modification and methylation-specific PCR analysis revealed that VHL is subject to extensive methylation in a CpG-rich region within its promoter in the human placenta. Notably, we detected significant differences in methylation in E-PE placentae relative to normotensive age-matched controls at key transcription factor binding sites, including that of the transcriptional repressor E2F4. Treatment of JEG3 cells with 5-Aza-2′-deoxycytidine, revealed that loss of DNA methylation promoted VHL transcription by attenuating VHL association with E2F4. RNAi knockdown of E2F4 in vitro confirmed its function on VHL repression. Exposure of JEG3 cells to transforming growth factor beta (TGFβ) downregulated VHL mRNA. In line with elevated levels of TGFβ3 in E-PE, chromatin immunoprecipitation assays revealed that E2F4-VHL association was enhanced upon TGFβ3 treatment, indicative of VHL transcriptional inhibition. In line with decreased VHL expression in E-PE, augmented E2F4-VHL association was also observed in E-PE placental tissue relative to controls. In conclusion, we demonstrate for the first time that hypomethylation of VHL DNA at a key transcription factor binding site has significant consequences for its transcriptional repression in early-onset preeclampsia.
    Microphysiological flux balance platform unravels the dynamics of drug induced steatosis
    Avner Ehrlich, Lab on a Chip - 2018
    Abstract
    Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15–20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of β-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.
    A STAT4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B
    R. El Sharkawy, Alimentary Pharmacology & Therapeutics - 2018
    Abstract
    Background Host genetic modifiers of the natural history of chronic hepatitis B (CHB) remain poorly understood. Recently, a genome-wide association study (GWAS)-identified polymorphism in the STAT4 gene that contributes to the risk for hepatocellular carcinoma (HCC) was shown to be associated with the full spectrum of hepatitis B virus (HBV) outcomes in Asian patients. However, the functional mechanisms for this effect are unknown and the role of the variant in modulating HBV disease in Caucasians has not been investigated. Aims To determine whether STAT4 genetic variation is associated with liver injury in Caucasian patients with CHB and to investigate potential mechanisms mediating this effect. Methods STAT4 rs7574865 was genotyped in 1085 subjects (830 with CHB and 255 healthy controls). STAT4 expression in liver, PBMCs and NK cells, STAT4 phosphorylation and secretion of interferon-gamma (IFN-γ) according to STAT4 genetic variation was examined. Results STAT4 rs7574865 genotype was independently associated with hepatic inflammation (OR: 1.42, 95% CI: 1.07-2.06, P = 0.02) and advanced fibrosis (OR: 1.83, 95% CI: 1.19-2.83, P = 0.006). The minor allele frequency of rs7574865 was significantly lower than that in healthy controls. rs7574865 GG risk carriers expressed lower levels of STAT4 in liver, PBMCs and in NK cells, while NK cells from patients with the risk genotype had impaired STAT4 phosphorylation following stimulation with IL-12/IL-18 and a reduction in secretion of IFN-γ. Conclusion Genetic susceptibility to HBV persistence, hepatic inflammation and fibrosis in Caucasians associates with STAT4 rs7574865 variant. Downstream effects on NK cell function through STAT4 phosphorylation-dependent IFN-γ production likely contribute to these effects.
    Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity
    Michael Michaelides, Molecular Psychiatry - 2018
    Abstract
    Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.
    Prenatal stress disrupts social behavior, cortical neurobiology and commensal microbes in adult male offspring
    Tamar L. Gur, Behavioural Brain Research - 2018
    Abstract
    In utero and early neonatal exposure to maternal stress is linked with psychiatric disorders, and the underlying mechanisms are currently being elucidated. We used a prenatal stressor in pregnant mice to examine novel relationships between prenatal stress exposure, changes in the gut microbiome, and social behavior. Here, we show that males exposed to prenatal stress had a significant reduction in social behavior in adulthood, with increased corticosterone release following social interaction. Male offspring exposed to prenatal stress also had neuroinflammation, decreased oxytocin receptor, and decreased serotonin metabolism in their cortex in adulthood, which are linked to decreased social behavior. Finally, we found a significant difference in commensal microbes, including decreases in Bacteroides and Parabacteroides, in adult male offspring exposed to prenatal stress when compared to non-stressed controls. Our findings indicate that gestation is a critical window where maternal stress contributes to the development of aberrant social behaviors and alterations in cortical neurobiology, and that prenatal stress is sufficient to disrupt the male gut-brain axis into adulthood.
    Development of Escherichia coli based gene expression profiling of sewage sludge leachates
    Manish Goswami, Journal of Applied Microbiology - 2018
    Abstract
    Aims The impact of municipal waste on pathogenic microorganisms released into the environment is a public health concern. The present study aims to evaluate the effects of sewage sludge and antibiotic contaminants on stress response, virulence and antibiotic resistance in a pathogenic Escherichia coli. Methods and Results The effects of sewage sludge leachates on uropathogenic E. coli CFT073 were determined by monitoring the expression of 45 genes associated with antibiotic/metal resistance, stress response and virulence using RT-qPCR. The E. coli gene expression was validated using sub-inhibitory concentrations of tetracycline and ciprofloxacin. E. coli exposed to sewage sludge or sewage sludge-fly ash leachates altered the expression of 5 antibiotic and metal resistance, 3 stress response and 2 virulence associated genes. When antibiotics were combined with sludge or sludge-fly ash the antibiotic-associated gene expression was altered. Conclusions E. coli treated with two sludge leachates had distinct gene expression patterns that were altered when the sludge leachates were combined with tetracycline, although to a lesser extent with ciprofloxacin. Significance and Impact of Study The E. coli multigene expression analysis is a potential new tool for assessing the effects of pollutants on pathogenic microbes in environmental waters for improved risk assessment. This article is protected by copyright. All rights reserved.
    High MUC2 Mucin Biosynthesis in Goblet Cells Impedes Restitution and Wound Healing by Elevating Endoplasmic Reticulum Stress and Altered Production of Growth Factors
    Adelaide Tawiah, The American Journal of Pathology - 2018
    Abstract
    Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing factors and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of fibroblast growth factor (FGF) 1, FGF2, vascular endothelial growth factor-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-22. FGF2- and IL-22–induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium–induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the mid and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of growth factors critical in wound repair.
    PTCH1 isoform 1b is the major transcript in the development of basal cell nevus syndrome
    Robbert-Jan C. A. M. Gielen, Journal of Human Genetics - 2018
    Abstract
    Basal cell nevus syndrome (BCNS) is an autosomal dominant disorder most commonly caused by a germline mutation in the PTCH1 gene. PTCH1 is known to have different isoforms with different functional properties and expression patterns among tissues. We detected a novel, pathogenic de novo mutation in PTCH1 isoform 1b (c.114delG) in a BCNS patient. Furthermore, we elucidated the specific expression pattern of PTCH1 isoforms in normal skin, BCC and peripheral blood by studying expression of different PTCH1 isoforms. Human skin showed expression of isoforms 1b and 1d, while peripheral blood additionally showed 1a and 1e expression. BCCs showed expression of all isoforms. Here we report a patient with a novel, isoform 1b specific mutation in PTCH1 and thereby distinguish PTCH1 isoform 1b as the major transcript in the development of BCNS.
    Roflumilast promotes memory recovery and attenuates white matter injury in aged rats subjected to chronic cerebral hypoperfusion
    Amanda Santiago, Neuropharmacology - 2018
    Abstract
    Chronic cerebral hypoperfusion (CCH) has been associated with aging-related vascular dementia, including Alzheimer's disease. It can be induced by the four-vessel occlusion/internal carotid artery (4VO/ICA) model in aged rats, resulting in persistent memory deficits, white matter injury, and significant neuronal loss in the hippocampus and cerebral cortex. The phosphodiesterase type 4 inhibitor (PDE4-I) roflumilast has been reported to have pro-cognitive effects in several behavioral paradigms. The present study evaluated the effects of repeated roflumilast treatment in aged rats that were subjected to CCH. After surgery, roflumilast (0.003 and 0.01 mg/kg) was administered intraperitoneally once per day for 29 days. Memory performance was assessed in the aversive radial maze (AvRM) 7, 14, and 21 days after CCH. The effects of roflumilast on hippocampal neurodegeneration and white matter injury were investigated using Nissl and Kluver-Barrera staining, respectively. Western blot and RT-qPCR were used to explore microglial polarization using M1 (Iba-1 and iNOS) and M2 (Arginase-1) markers. Chronic cerebral hypoperfusion caused persistent memory deficits, hippocampal neurodegeneration, and vacuolization and fiber disarrangement in white matter. Repeated roflumilast treatment restored CCH-induced cognitive impairments in aged rats but in the absence of the rescue of hippocampal neurons. Attenuation of white matter injury was detected in the optic tract in aged CCH rats that were treated with roflumilast. In vitro, roflumilast increased Arg-1 gene expression in myelin-laden primary microglia. The present data suggest that roflumilast might be useful for the treatment of cognitive sequelae associated with CCH.
    The origins of global invasions of the German wasp (Vespula germanica) and its infection with four honey bee viruses
    Evan C. Brenton-Rule, Biological Invasions - 2018
    Abstract
    A successful control or eradication programme using biological control or genetically-mediated methods requires knowledge of the origin and the extent of wasp genetic diversity. Mitochondrial DNA variation in the native and invaded range of the social wasp Vespula germanica was used to examine intra-specific genetic variation and invasive source populations. We also examined wasps for the presence of four viruses found in honey bees: Acute bee paralysis virus, Deformed wing virus, Israeli acute paralysis virus and Kashmir bee virus. German wasps showed reduced genetic diversity in the invaded range compared to that of their native range. Populations in the introduced range are likely to have arrived from different source populations. All four viral honey bee pathogens were found in V. germanica, although they varied in their distribution and strain. Multiple introductions of German wasps have occurred for most invaded regions, though some populations are genetically homogenous. The differing locations of origin will guide researchers searching for biocontrol agents and the reduced genetic diversity may make these wasps a potentially viable target for control via gene drives.
    Novel key roles for Structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development
    Uros Midic, Molecular Reproduction and Development - 2018
    Abstract
    Structural maintenance of chromosome flexible domain containing 1 (Smchd1) is a chromatin regulatory gene for which mutations are associated with facioscapulohumeral muscular dystrophy and arhinia. The contribution of oocyte- and zygote-expressed SMCHD1 to early development was examined in mice (Mus musculus) using an siRNA knockdown approach. Smchd1 knockdown compromised long-term embryo viability, with reduced embryo nuclear volumes at the morula stage, reduced blastocyst cell number, formation and hatching, and reduced viability to term. RNAseq analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of a small number of trophectoderm-related genes and reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 (Skp2). Smchd1 expression was elevated in embryos deficient for Caudal type homeobox transcription factor 2 (Cdx2, a key regulator of trophectoderm specification), indicating that Smchd1 is normally repressed by CDX2. These results indicate that Smchd1 plays an important role in the preimplantation embryo, regulating early gene expression and contributing to long-term embryo viability. These results extend the known functions of SMCHD1 to the preimplantation period and highlight important function for