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  • PCR & qPCR
    qPCR Instrumentation
    Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors
    Trang Thi Thu Nguyen, Cancers - 2019
    Abstract
    Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies.
    Real-Time Quantitative PCR
    SYBR Green Detection
    Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathways
    Oscar E. Juarez, PLOS ONE - 2019
    Abstract
    White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptorsignaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females’ WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.
    Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice
    Andrew Wang, PNAS - 2019
    Abstract
    Secondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
    Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant women
    Nsoh Godwin Anabire, PLOS ONE - 2019
    Abstract
    Background The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, it is not known if this co-infection could adversely influence clinical and immunological responses. This study investigated these interactions in pregnant women reporting to antenatal clinics in Ghana. Methods Clinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia, and hepatitis B viremia) and cytokine profiles were assayed and compared across four categories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria+CHB group). Results Women with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM = 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin [P<0.001 for all comparisons]. Similarly, the Malaria+CHB group had significantly elevated pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 [P<0.05 for all comparisons]. In women with Malaria+CHB, correlation analysis showed significant negative association of the pro-inflammatory cytokines responses with malaria parasitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394) and IL-12 (P = 0.011; r = -0.49)]. On the other hand, the pro-inflammatory cytokine levels positively correlated with HBV viremia [TNF-α (P = 0.004; r = 0.549), IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r = 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P = 0.008; r = 0.512) and IL-12 (P<0.001; r = 0.655)]. Also, for women in the Malaria+CHB group, parasitemia was observed to diminish HBV viremia [P = 0.003, r = -0.489]. Conclusion Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine responses and increase susceptibility to liver injury among pregnant women in endemic settings.
    Selection for novel metabolic capabilities in Salmonella enterica
    Omar Warsi, Evolution International Journal of Evolution - 2019
    Abstract
    Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.
    Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes
    Pamela Calvo, Journal of Plant Interactions - 2019
    Abstract
    Plant growth-promoting rhizobacteria (PGPR) enhance plant growth under the influence of multigenic processes, including nitrate (NO−3) and ammonium (NH+4) uptake genes, which could potentially explain the improvement in plant nutrition and plant growth promotion. Studies on the effects of PGPR inoculation on regulation of NO−3 and NH+4 plant uptake genes and nutrient accumulation using soil or soil-like substrates are limited. Here, we tested the hypothesis that the application of PGPR Bacillus mixtures increases overall plant growth, nutrient uptake and the transcript levels of nitrate and ammonium uptake genes in Arabidopsis thaliana. All three PGPR mixtures tested in this study significantly increased plant shoot fresh weight, root fresh, chlorophyll content, nutrient uptake and plant diameter. The transcript levels of five nitrate and four ammonium uptake genes were significantly higher in PGPR-treated plants compared to untreated plants. These results demonstrate that plant growth promotion and enhanced nutrient uptake by select PGPR mixtures.
    The effect of maxillary sinus antrostomy size on the sinus microbiome
    Alexander S. Kim BSE, Allergy and Rhinology - 2019
    Abstract
    Background The optimal maxillary antrostomy size to surgically treat sinusitis is not well known. In this study, we examined clinical metrics of disease severity and symptom scores, measured secreted inflammatory markers, and characterized the sinus microbiome to determine if there were significant differences in outcome between different maxillary ostial sizes. Methods Prospective randomized, single‐blinded clinical trial enrolling 12 individuals diagnosed with recurrent acute or chronic rhinosinusitis. Each patient was blinded and randomized to receive minimal maxillary ostial dilation via balloon sinuplasty on 1 side vs a mega‐antrostomy on the contralateral side. Data collected included symptom scores (20‐item Sino‐Nasal Outcome Test [SNOT‐20]), endoscopy, and radiologic Lund‐Mackay scores. During surgery and at their postoperative visit swabs were obtained from each maxillary sinus, and 16S DNA and inflammatory cytokine levels analyzed. The use of each patient as their own control allowed us to minimize confounding variables. Results There was statistically significant improvement in SNOT‐20 symptom scores postoperatively in all patients. There were no significant differences between maxillary ostial size in postoperative endoscopy scores, cytokine profile, or bacterial burden. There were statistically significant differences in relative postoperative abundance of Staphylococcus, Lactococcus, and Cyanobacteria between the mega‐antrostomy and mini‐antrostomy. Conclusions The method used in surgical maxillary antrostomies had no effect on endoscopy scores or cytokine profiles. Microbiome analysis determined significant differences between the different antrostomy sizes in postoperative Staphylococcus, Lactococcus, and Cyanobacteria abundance. The clinical significance of these changes in the sinus microbiome are not known but may be a result of increased access to postoperative sinonasal irrigations.
    An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity Against Colorectal Cancer
    Bastian J. Schmied, Cancers - 2019
    Abstract
    The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E—that reportedly increase the affinity to CD16—with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.
    Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stress
    Ramona Dolling, Heliyon - 2019
    Abstract
    Stress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 μmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 μmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 μmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.
    Epigenetic Changes at the Birc5 Promoter Induced by YM155 in Synovial Sarcoma
    Aleksander Mika, Journal of Clinical Medicine - 2019
    Abstract
    YM155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of survivin (gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is a rare soft-tissue malignancy with high protein expression of survivin. We investigated whether YM155 would be a viable therapeutic option to treat synovial sarcoma. YM155 therapy was applied to human synovial sarcoma cell lines and to a genetically engineered mouse model of synovial sarcoma. We discovered that YM155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM155 by looking at the change of lysine modifications of the histone tails that were within 250 base pairs of the Birc5 promoter. Using chromatin immunoprecipitation (ChIP)-qPCR, we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM155 treatment. H3K27me3 and H3K27ac increased, but the net result was decreased Birc5/survivin expression. Furthermore, the combination of molecular events resulted in caspase 3/7/8 upregulation and death of the sarcoma cells.
    Chemokine Receptor Redundancy and Specificity Are Context Dependent
    Douglas P. Dyer, Immunity - 2019
    Abstract
    Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
    Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice
    Carmen J. Reynolds, The Journal of Steroid Biochemistry and Molecular Biology - 2019
    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.
    Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cells
    Zejun Zhou, Scientific Reports - 2019
    Abstract
    Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.
    Chronic liver injury drives non‐traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminase
    L. G. Poole, Journal of Thrombosis and Haemostasis - 2019
    Abstract
    Background Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin‐mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit‐deficient (FXIII−/−) mice. Female wild‐type mice, FXIII−/− mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4) twice weekly for 4 weeks or 6 weeks (1 mL kg−1, intraperitoneal). Results Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin‐catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4‐challenged FXIII−/− mice or FibAEK mice as compared with wild‐type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase‐2 (TGM2)‐deficient (TGM2−/−) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2−/− mice did not affect CCl4‐induced liver fibrosis. Conclusions These results indicate that neither traditional fibrin clots, formed by the thrombin–activated FXIII pathway nor atypical TGM2‐crosslinked fibrin(ogen) contribute to experimental CCl4‐induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.
    Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease [version 1; peer review: 1 approved, 1 approved with reservations]
    Serge Dionne, F1000 Research Open for Science - 2019
    Abstract
    Background: Intestinal fibrosis represents a serious complication of inflammatory bowel diseases (IBD), often necessitating surgical resections. Myofibroblasts are primarily responsible for interstitial matrix accumulation in fibrotic diseases. However intestinal myofibroblasts (IMF) remain inadequately characterized. The aim was to examine fibroblast markers and fibrosis-associated gene expression in IMF isolated from resected intestine from IBD and control patients. As well as determining the effect of the fibrogenic cytokine TGFβ. Methods: Intestinal resections were obtained (n =35) from consenting patients undergoing elective surgery (2014-16). Primary cultures of IMF were isolated using DTT and EDTA and cultured. Viability and phenotypic characterization of IMF was carried out by flow cytometry and fluorescence microscopy. IMF (passages 3-8) were treated for 24 hours. Cytokines were quantified in IMF by real time PCR and in supernatants using the human pro-inflammatory cytokine panel Results: All markers and most fibrosis mediators studied were preferentially expressed by IMF compared to mucosal tissue. Metalloproteinases (MMP) 2 and 3, as well as their inhibitor TIMP1, are highly expressed by IMF. They also highly expressed inflammatory mediators, including IL-6, IL-8, CCL2 and PTGS2. Whereas mucosal expression of pro-inflammatory cytokines such as TNFα and IL-17 is increased in IBD, that of fibrosis mediators was not different. Fibrosis-related gene expression in IMF from IBD patients and controls was similar, but IMF from IBD expressed higher levels of several inflammatory genes. IMF from CD and UC had mostly similar expression profiles. TGFβ induced expression of fibrogenic genes αSMA, COL1A1, CTGF, FN1 and LOX. TGFβ-stimulated IMF released increased levels of IL-6, whereas IL-6, IL-8, as well as small amounts of IFN-γ and IL12p70 were produced following stimulation with IL-1β+IL-23. Conclusions: This study extends knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required.
    Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma
    Lemlem Brook, Journal of Cellular Biochemistry - 2019
    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.
    Neuroendocrine Whiplash: Slamming the Breaks on Anabolic-Androgenic Steroids Following Repetitive Mild Traumatic Brain Injury in Rats May Worsen Outcomes
    Jason Tabor, Frontiers in Neurology - 2019
    Abstract
    Sport-related concussion is an increasingly common injury among adolescents, with repetitive mild traumatic brain injuries (RmTBI) being a significant risk factor for long-term neurobiological and psychological consequences. It is not uncommon for younger professional athletes to consume anabolic-androgenic steroids (AAS) in an attempt to enhance their performance, subjecting their hormonally sensitive brains to potential impairment during neurodevelopment. Furthermore, RmTBI produces acute neuroendocrine dysfunction, specifically in the anterior pituitary, disrupting the hypothalamic-pituitary adrenal axis, lowering cortisol secretion that is needed to appropriately respond to injury. Some AAS users exhibit worse symptoms post-RmTBI if they quit their steroid regime. We sought to examine the pathophysiological outcomes associated with the abrupt cessation of the commonly abused AAS, Metandienone (Met) on RmTBI outcomes in rats. Prior to injury, adolescent male rats received either Met or placebo, and exercise. Rats were then administered RmTBIs or sham injuries, followed by steroid and exercise cessation (SEC) or continued treatment. A behavioral battery was conducted to measure outcomes consistent with clinical representations of post-concussion syndrome and chronic AAS exposure, followed by analysis of serum hormone levels, and qRT-PCR for mRNA expression and telomere length. RmTBI increased loss of consciousness and anxiety-like behavior, while also impairing balance and short-term working memory. SEC induced hyperactivity while Met treatment alone increased depressive-like behavior. There were cumulative effects whereby RmTBI and SEC exacerbated anxiety and short-term memory outcomes. mRNA expression in the prefrontal cortex, amygdala, hippocampus, and pituitary were modified in response to Met and SEC. Analysis of telomere length revealed the negative impact of SEC while Met and SEC produced changes in serum levels of testosterone and corticosterone. We identified robust changes in mRNA to serotonergic circuitry, neuroinflammation, and an enhanced stress response. Interestingly, Met treatment promoted glucocorticoid secretion after injury, suggesting that maintained AAS may be more beneficial than abstaining after mTBI.
    MiRNA-27a sensitizes breast cancer cells to treatment with Selective Estrogen Receptor Modulators
    Bojan Ljepoja, The Breast - 2019
    Abstract
    Background MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest. Methods Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment. Results Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression. Conclusion MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.
    Fine-scale spatial and temporal dynamics of kdr haplotypes in Aedes aegypti from Mexico
    Marissa K. Grossman, BMC - 2019
    Abstract
    Background As resistance to insecticides increases in disease vectors, it has become exceedingly important to monitor populations for susceptibility. Most studies of field populations of Aedes aegypti have largely characterized resistance patterns at the spatial scale of the city or country, which may not be completely informative given that insecticide application occurs at the scale of the house or city block. Phenotypic resistance to pyrethroids dominates in Ae. aegypti, and it has been partially explained by mutations in the voltage-gated sodium channel gene. Here, we assess community-level patterns of four knockdown resistance (kdr) haplotypes (C1534/I1016, F1534/I1016, C1534/V1016 and F1534/V1016) in Ae. aegypti in 24 randomly chosen city blocks from a city in Yucatán State, Mexico, during both the dry and wet season and over two years. Results Three of the four haplotypes, C1534/I1016, C1534/V1016 and F1534/V1016 were heterogeneous between city blocks at all four sampling time points, and the double mutant haplotype, C1534/I1016, showed a significant increase following the wet season. The F1534/I1016 haplotype was rarely detected, similar to other studies. However, when haplotype frequencies were aggregated to a coarser spatial scale, the differences in space and time were obscured. Conclusions Our results provide empirical evidence that the selection of kdr alleles is occurring at fine spatial scales, indicating that future studies should include this scale to better understand evolutionary processes of resistance in natural populations.
    Loss-of-function mutations in caspase recruitment domain-containing protein 14 (CARD14) are associated with a severe variant of atopic dermatitis
    Alon Peled BMedSci, Science Direct - 2019
    Abstract
    Background Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. Objective We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. Methods Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. Results In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. Conclusions Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.
    Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
    Vianey Gonzalez-Villasana, Hindawi - 2019
    Abstract
    miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.
    Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis
    Mette Pernille Myklebust, Frontiers in Genetics - 2019
    Abstract
    Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.
    Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes
    Tamara A. Potapova, Journal of Cell Biology - 2019
    Abstract
    The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization
    Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies
    Sarah Irmscher, Nature Communications - 2019
    Abstract
    Persistent inflammation is a hallmark of many human diseases, including anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis. Here, we describe a dominant trigger of inflammation: human serum factor H-related protein FHR1. In vitro, this protein selectively binds to necrotic cells via its N-terminus; in addition, it binds near necrotic glomerular sites of AAV patients and necrotic areas in atherosclerotic plaques. FHR1, but not factor H, FHR2 or FHR3 strongly induces inflammasome NLRP3 in blood-derived human monocytes, which subsequently secrete IL-1β, TNFα, IL-18 and IL-6. FHR1 triggers the phospholipase C-pathway via the G-protein coupled receptor EMR2 independent of complement. Moreover, FHR1 concentrations of AAV patients negatively correlate with glomerular filtration rates and associate with the levels of inflammation and progressive disease. These data highlight an unexpected role for FHR1 during sterile inflammation, may explain why FHR1-deficiency protects against certain diseases, and identifies potential targets for treatment of auto-inflammatory diseases.
    RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes
    Joel Johansson, Cell Stem Cell - 2019
    Abstract
    Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
    A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila
    Alessandro Scopelliti, Cell Metabolism - 2019
    Abstract
    The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. Here, we show that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon α, which signals via its neuronal receptor DLgr2. Bursicon α/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon α/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, our work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis.
    A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing
    Theophilus T. Tettey, Cell Reports - 2019
    Abstract
    FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
    Suppression of UV-B stress induced flavonoids by biotic stress: Is there reciprocal crosstalk?
    Dirk Schenke, Plant Physiology and Biochemistry - 2019
    Abstract
    Plants respond to abiotic UV-B stress with enhanced expression of genes for flavonoid production, especially the key-enzyme chalcone synthase (CHS). Some flavonoids are antioxidative, antimicrobial and/or UV-B protective secondary metabolites. However, when plants are challenged with concomitant biotic stress (simulated e.g. by the bacterial peptide flg22, which induces MAMP triggered immunity, MTI), the production of flavonoids is strongly suppressed in both Arabidopsis thaliana cell cultures and plants. On the other hand, flg22 induces the production of defense related compounds, such as the phytoalexin scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread within the host tissue. Since all these metabolites require the precursor phenylalanine for their production, suppression of the flavonoid production appears to allow the plant to focus its secondary metabolism on the production of pathogen defense related compounds during MTI. Interestingly, several flavonoids have been reported to display anti-microbial activities. For example, the plant flavonoid phloretin targets the Pseudomonas syringae virulence factors flagella and type 3 secretion system. That is, suppression of flavonoid synthesis during MTI might have also negative side-effects on the pathogen defense. To clarify this issue, we deployed an Arabidopsis flavonoid mutant and obtained genetic evidence that flavonoids indeed contribute to ward off the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Finally, we show that UV-B attenuates expression of the flg22 receptor FLS2, indicating that there is negative and reciprocal interaction between this abiotic stress and the plant-pathogen defense responses.
    Diet and diet‐associated bacteria shape early microbiome development in Yellowtail Kingfish (Seriola lalandi)
    Jackson Wilkes Walburn, Microbial Biotechnology - 2019
    Abstract
    The supply of quality juveniles via land‐based larviculture represents a major bottleneck to the growing finfish aquaculture industry. As the microbiome plays a key role in animal health, this study aimed to assess the microbial community associated with early larval development of commercially raised Yellowtail Kingfish (Seriola lalandi). We used qPCR and 16S rRNA gene amplicon sequencing to monitor changes in the microbiome associated with the development of S. lalandi from larvae to juveniles. We observed an increase in the bacterial load during larval development, which consisted of a small but abundant core microbiota including taxa belonging to the families Rhodobacteraceae, Lactobacillaceae and Vibrionaceae. The greatest change in the microbiome occurred as larvae moved from a diet of live feeds to formulated pellets, characterized by a transition from Proteobacteria to Firmicutes as the dominant phylum. A prediction of bacterial gene functions found lipid metabolism and secondary metabolite production were abundant in the early larval stages, with carbohydrate and thiamine metabolism functions increasing in abundance as the larvae age and are fed formulated diets. Together, these results suggest that diet is a major contributor to the early microbiome development of commercially raised S. lalandi.
    Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model
    Benedikt Linder, Cancers - 2019
    Abstract
    Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12–15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability
    On resolving ambiguities in microbial community analysis of partial nitritation anammox reactors
    Laura Orschler, Scientific Reports - 2019
    Abstract
    PCR-based methods have caused a surge for integration of eco-physiological approaches into research on partial nitritation anammox (PNA). However, a lack of rigorous standards for molecular analyses resulted in widespread data misinterpretation and consequently lack of consensus. Data consistency and accuracy strongly depend on the primer selection and data interpretation. An in-silico evaluation of 16S rRNA gene eubacterial primers used in PNA studies from the last ten years unraveled the difficulty of comparing ecological data from different studies due to a variation in the coverage of these primers. Our 16S amplicon sequencing approach, which includes parallel sequencing of six 16S rRNA hypervariable regions, showed that there is no perfect hypervariable region for PNA microbial communities. Using qPCR analysis, we emphasize the significance of primer choice for quantification and caution with data interpretation. We also provide a framework for PCR based analyses that will improve and assist to objectively interpret and compare such results.
    Infection with Toxoplasma gondii (Eucoccidiorida: Sarcocystidae) in bats of Campeche and Yucatán, Mexico
    Marco Torres-Castro, International Journal of Tropical Biology and Conservation - 2019
    Abstract
    Toxoplasma gondii is a protozoan parasite recognized as the causative agent of toxoplasmosis, a zoonotic disease that affects humans and domestic or wild animals. In Mexico, it represents a public and animal health problem, especially in regions with tropical and subtropical climates. Bats have been reported as accidental hosts in the transmission cycle; however, there is no preceding information in Mexico. Therefore, the aim of the present study is to report the T. gondii infection in bats captured in sites of Campeche and Yucatan states, Mexico. Bats were captured in two sites in Yucatan (X’matkuil and Panaba) and one in Campeche (Hampolol), located in the Yucatan Peninsula. Kidneys, spleen, and liver were collected and used in the total DNA extraction. Toxoplasma gondii infection was detected through the amplification of a B1 gene fragment, using nested PCR. The positive PCR products were purified and sent to sequencing for a posterior sequence identity analysis. Additionally, a phylogenetic tree was made. A total of 69 bats belonging to eight different species were processed: 41 (59.4 %, 41/69) Artibeus jamaicensis; six (8.7 %, 6/69) Pteronotus parnellii; six (8.7 %, 6/69) Noctilio leporinus; six (8.7 %, 6/69) Chiroderma villosum; four (5.8 %, 4/69) Glossophaga soricina; two (2.9 %, 2/69) Carollia sowelli; two (2.89 %, 2/69) Artibeus lituratus; and two (2.9 %, 2/69) Rhogeessa aeneus. The nested PCR identified eight (11.6 %, 8/69) infected bats: six (75 %, 6/8) A. jamaicensis, captured in X'matkuil and Panaba, one (12.5 %, 1/8) G. soricina, and one (12.5 %, 1/8) C. villosum, both captured in Panaba. The alignment analysis yielded 99-100 % for cover and 97-99 % for identity to T. gondii sequences. Our results contribute to the understanding of the T. gondii transmission cycle in the region; however, future research is needed to determine circulating genotypes, as previous studies have demonstrated that these animals might be infected with identified genotypes in other domestic or wild animals and even in humans.
    Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST
    Yoko Nakano, Cell Reports - 2019
    Abstract
    A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST.
    Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts
    Zoe E. Gillespie, Scientific Reports - 2019
    Abstract
    Metformin is a widely-used treatment for type 2 diabetes and is reported to extend health and lifespan as a caloric restriction (CR) mimetic. Although the benefits of metformin are well documented, the impact of this compound on the function and organization of the genome in normal tissues is unclear. To explore this impact, primary human fibroblasts were treated in culture with metformin resulting in a significant decrease in cell proliferation without evidence of cell death. Furthermore, metformin induced repositioning of chromosomes 10 and 18 within the nuclear volume indicating altered genome organization. Transcriptome analyses from RNA sequencing datasets revealed that alteration in growth profiles and chromosome positioning occurred concomitantly with changes in gene expression profiles. We further identified that different concentrations of metformin induced different transcript profiles; however, significant enrichment in the activator protein 1 (AP-1) transcription factor network was common between the different treatments. Comparative analyses revealed that metformin induced divergent changes in the transcriptome than that of rapamycin, another proposed mimetic of CR. Promoter analysis and chromatin immunoprecipitation assays of genes that changed expression in response to metformin revealed enrichment of the transcriptional regulator forkhead box O3a (FOXO3a) in normal human fibroblasts, but not of the predicted serum response factor (SRF). Therefore, we have demonstrated that metformin has significant impacts on genome organization and function in normal human fibroblasts, different from those of rapamycin, with FOXO3a likely playing a role in this response.
    The chemokine receptor CXCR2 contributes to murine adipocyte development
    Douglas P. Dyer, Journal of Leukocyte Biology - 2019
    Abstract
    Chemokines are members of a large family of chemotactic cytokines that signal through their receptors to mediate leukocyte recruitment during inflammation and homeostasis. The chemokine receptor CXCR2 has largely been associated with neutrophil recruitment. However, there is emerging evidence of roles for chemokines and their receptors in processes other than leukocyte migration. We have previously demonstrated that CXCR2 knockout (KO) mice have thinner skin compared to wild‐type mice. Herein we demonstrate that this is due to a thinner subcutaneous adipose layer, as a result of fewer and smaller individual adipocytes. We observe a similar phenotype in other fat depots and present data that suggests this may be due to reduced expression of adipogenesis related genes associated with adipocyte specific CXCR2 signaling. Interestingly, this phenotype is evident in female, but not male, CXCR2 KO mice. These findings expand our understanding of nonleukocyte related chemokine receptor functions and help to explain some previously observed adipose‐related phenotypes in CXCR2 KO mice.
    Cytoprotective effects of Avenathramide C against oxidative and inflammatory stress in normal human dermal fibroblasts
    Chenxuan Wang, Scientific Reports - 2019
    Abstract
    Natural polyphenols are promising anti-aging compounds not only for their antioxidant activity, but also their ability to activate specific cellular pathways mediating the aging process. Avenanthramide C (Avn C), found exclusively in oats, is a natural antioxidant associated with free radical scavenging; however, it is how this compound elicits other protective effects. We investigated the intracellular antioxidant activity of Avn C and other cytoprotective potential in normal human skin fibroblasts exposed to extracellular stress. Avn C reduced H2O2-induced oxidative stress by reducing intracellular free radical levels and antioxidant gene transcripts. Avn C also resulted in decreased levels of gene transcripts encoding pro-inflammatory cytokines in response to H2O2 or tumor necrosis factor-α (TNF-α). This reduction in cytokine gene transcription occurred concomitantly with reduced phosphorylated nuclear factor-κB (NF-κB) p65, and decreased NF-κB DNA binding. Avn C further induced heme oxygense-1 (HO-1) expression through increased Nrf2 DNA binding activity, demonstrating a second mechanism by which Avn C attenuates cellular stress. Collectively, our findings indicate that Avn C protects normal human skin fibroblasts against oxidative stress and inflammatory response through NF-κB inhibition and Nrf2/HO-1 activation
    Effects of 50 Hz magnetic fields on circadian rhythm control in mice
    Louise Lundberg, Bio Electro Magnetics - 2019
    Abstract
    Artificial light and power frequency magnetic fields are ubiquitous in the built environment. Light is a potent zeitgeber but it is unclear whether power frequency magnetic fields can influence circadian rhythm control. To study this possibility, 8–12‐week‐old male C57BL/6J mice were exposed for 30 min starting at zeitgeber time 14 (ZT14, 2 h into the dark period of the day) to 50 Hz magnetic fields at 580 μT using a pair of Helmholtz coils and/or a blue LED light at 700 lux or neither. Our experiments revealed an acute adrenal response to blue light, in terms of increased adrenal per1 gene expression, increased serum corticosterone levels, increased time spent sleeping, and decreased locomotor activity (in all cases, P < 0.0001) compared to an unexposed control group. There appeared to be no modulating effect of the magnetic fields on the response to light, and there was also no effect of the magnetic fields alone (in both cases, P > 0.05) except for a decrease in locomotor activity (P < 0.03). Gene expression of the cryptochromes cry1 and cry2 in the adrenals, liver, and hippocampus was also not affected by exposures (in all cases, P > 0.05). In conclusion, these results suggest that 50 Hz magnetic fields do not significantly affect the acute light response to a degree that can be detected in the adrenal response. Bioelectromagnetics. 2019;9999:XX–XX. © 2019 Bioelectromagnetics Society.
    Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface
    Maria Salvany-Celades, Cell Reports - 2019
    Abstract
    During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.
    Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine
    May Levin, Cell Death and Disease - 2019
    Abstract
    Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.
    Transcriptome Response of Female Culicoides sonorensis Biting Midges (Diptera: Ceratopogonidae) to Early Infection with Epizootic Hemorrhagic Disease Virus (EHDV-2)
    Dana Nayduch, Viruses - 2019
    Abstract
    Female Culicoides sonorensis biting midges are vectors of epizootic hemorrhagic disease virus (EHDV), which causes morbidity and mortality in wild and domesticated ruminants. The aims in this study were to identify key changes in female midge transcriptome profiles occurring during early infection with EHDV-2. Midges were fed either negative control bloodmeals or bloodmeals containing EHDV-2 and transcriptomes were acquired at 36 h through deep sequencing. Reads were de novo assembled into a transcriptome comprised of 18,754 unigenes. Overall, there were 2401 differentially expressed unigenes and ~60% were downregulated in response to the virus (953 up; 1448 down). Downstream Gene Ontology enrichment, KEGG pathway mapping, and manual analyses were used to identify the effect of virus ingestion at both the gene and pathway levels. Downregulated unigenes were predominantly assigned to pathways related to cell/tissue structure and integrity (actin cytoskeleton, adherens junction, focal adhesion, hippo signaling), calcium signaling, eye morphogenesis and axon guidance. Unigenes attributed to sensory functions (especially vision), behavior, learning and memory were largely downregulated. Upregulated unigenes included those coding for innate immune processes, olfaction and photoreceptor pigments. Our results suggest that midges respond to virus infection as soon as 36 h post-ingestion, and that EHDV-2 may have a significant phenotypic effect on sensory and neural tissues.
    CYLD Regulates Centriolar Satellites Proteostasis by Counteracting the E3 Ligase MIB1
    Tiphaine Douanne, Cell Reports - 2019
    Abstract
    The tumor suppressor CYLD is a deubiquitinatingenzyme that removes non-degradative ubiquitin link-ages bound to a variety of signal transduction adap-tors. CYLD participates in the formation of primarycilia, a microtubule-based structure that protrudesfrom the cell body to act as a ‘‘sensing antenna.’’Yet, how exactly CYLD regulates ciliogenesis is notfully understood. Here, we conducted an unbiasedproteomic screen of CYLD binding partners andidentified components of the centriolar satellites.These small granular structures, tethered to the scaf-fold protein pericentriolar matrix protein 1 (PCM1),gravitate toward the centrosome and orchestrateciliogenesis. CYLD knockdown promotes PCM1degradation and the subsequent dismantling of thecentriolar satellites. We found that CYLD marshalsthe centriolar satellites by deubiquitinating andpreventing the E3 ligase Mindbomb 1 (MIB1) frommarking PCM1 for proteasomal degradation. Theseresults link CYLD to the regulation of centriolar satel-lites proteostasis and provide insight into howreversible ubiquitination finely tunes ciliogenesis.
    Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune Uveitis
    Cathleen Janowitz, Investigative Ophthalmology & Visual Science - 2019
    Abstract
    Purpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis. Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation. Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis. Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.
    Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing Mice
    Nicholas Moon, Nutrients - 2019
    Abstract
    Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1β and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.
    Insulin-like Growth Factor (IGF)-1 treatment stabilizes the microvascularcytoskeleton under ischemic conditions
    Shameena Bake, Experimental Neurology - 2019
    Abstract
    Our previous studies showed that Insulin-like Growth Factor (IGF)-1 reduced blood brain barrier permeabilityand decreased infarct volume caused by middle cerebral artery occlusion (MCAo) in middle aged female rats.Similarly, cultures of primary brain microvessel endothelial cells from middle-aged female rats and exposed tostroke-like conditions (oxygen glucose deprivation; OGD) confirmed that IGF-1 reduced dye transfer across thiscell monolayer. Surprisingly, IGF-1 did not attenuate endothelial cell death caused by OGD. To reconcile thesefindings, the present study tested the hypothesis that, at the earliest phase of ischemia, IGF-1 promotes barrierfunction by increasing anchorage and stabilizing cell geometry of surviving endothelial cells. Cultures of humanbrain microvessel endothelial cells were subject to oxygen-glucose deprivation (OGD) in the presence of IGF-1,IGF-1 + JB-1 (IGFR inhibitor) or vehicle. OGD disrupted the cell monolayer and reduced cell-cell interactions,which was preserved in IGF-1-treated cultures and reversed by concurrent treatment with JB-1. IGF-1-mediatedpreservation of the endothelial monolayer was reversed with LY294002 treatment, but not by Rapamycin, in-dicating that IGF-1 s actions on cell-cell contacts are likely mediated via the PI3K pathway. In vivo, microvesselmorphology was evaluated in middle-aged female rats that were subjected to ischemia by MCAo, and treated ICVwith IGFeI, IGF-1 + JB-1, or artificial CSF (aCSF; vehicle) after reperfusion. Compared to vehicle controls, IGF-1treated animals displayed larger microvessel diameters in the peri-infarct area and increased staining density forvinculin, an anchorage protein. Both these measures were reversed by concurrent IGF-1 + JB-1 treatment.Moreover these effects were restricted to 24 h after ischemia-reperfusion and no treatment effects were seen at5d post stroke. Collectively, these data suggest that in the earliest hours during ischemia, IGF-1 promotes re-ceptor-mediated anchorage of endothelial cells, and its actions may be accurately characterized as vasculo-protective.
    High diagnostic yield and novel variants in very late-onset spasticity
    Momen Almomen, Journal of Neurogenetics - 2019
    Abstract
    Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to support the pathogenicity of these variants, and provide observations regarding the poor genotypephenotype correlation in these conditions that should be the subject of future study
    Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bonelike Matrix Deposition in Vitro
    Rayan Fairag, ACS Applied Materials & Interfaces - 2019
    Abstract
    Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 μm), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 μm pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 μm pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 μm pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.
    Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening
    Konrad Schwefel, Journal of Cellular and Molecular Medicine - 2019
    Abstract
    CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.
    S-adenosyl methionine prevents ASD like behaviors triggered by earlypostnatal valproic acid exposure in very young mice
    asher Ornoy, Neurotoxicology and Terattology - 2019
    Abstract
    Introduction:A common animal model of ASD is the one induced by valproic acid (VPA), inducing epigeneticchanges and oxidative stress. We studied the possible preventive effect of the methyl donor for epigenetic en-zymatic reactions, S-adenosine methionine (SAM), on ASD like behavioral changes and on redox potential in thebrain and liver in this model.Methods:ICR albino mice were injected on postnatal day 4 with one dose of 300 mg/kg of VPA, with normalsaline (controls) or with VPA and SAM that was given orally for 3 days at the dose of 30 mg/kg body weight.From day 50, we carried out neurobehavioral tests and assessment of the antioxidant status of the prefrontalcerebral cortex, liver assessing SOD and CAT activity, lipid peroxidation and the expression of antioxidant genes.Results:Mice injected with VPA exhibited neurobehavioral deficits typical of ASD that were more prominent inmales. Changes in the activity of SOD and CAT increased lipid peroxidation and changes in the expression ofantioxidant genes were observed in the prefrontal cortex of VPA treated mice, more prominent in females, whileASD like behavior was more prominent in males. There were no changes in the redox potential of the liver. Theco-administration of VPA and SAM alleviated most ASD like neurobehavioral symptoms and normalized theredox potential in the prefrontal cortex.Conclusions:Early postnatal VPA administration induces ASD like behavior that is more severe in males, whilethe redox status changes are more severe in females; SAM corrects both. VPA-induced ASD seems to result fromepigenetic changes, while the redox status changes may be secondary.
    Human-based fibrillar nanocomposite hydrogels as bioinstructive matrices to tune stem cell behavior
    Bárbara B. Mendes, Nanoscale - 2018
    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.
    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.
    Probe-based Detection
    Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application
    Dongmei Li, Journal of Applied Entomology - 2019
    Abstract
    Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.
    Digital Loop-Mediated Isothermal Amplification on a Commercial Membrane
    Xingyu Lin, ACS Sensors - 2019
    Abstract
    In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a commercial membrane, without the need for complex chip fabrication or use of specialized equipment. Due to the pore size distribution, the theoretical error for digital LAMP on these membranes was analyzed, using a combination of Random Distribution Model and Multivolume Theory. A facile peel-off process was developed for effective droplet formation on the commercial track-etched polycarbonate (PCTE) membrane. Each pore functions as an individual nanoreactor for single DNA amplification. Absolute quantification of bacteria genomic DNA was realized with a dynamic range from 11 to 1.1 × 105 copies/μL. One-step digital RT-LAMP was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence intensities. Finally, the cost of a disposable membrane is less than $0.10/piece, which, to the best of our knowledge, is the most inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible, and simplified way.
    Investigation of an outbreak caused by antibiotic‐susceptible Klebsiella oxytoca in a neonatal intensive care unit in Norway
    Torunn Gresdal Ronning, Acta Paediatrica - 2019
    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.
    A Brief Review of Non-Avian Reptile Environmental DNA (eDNA), with a Case Study of Painted Turtle (Chrysemys picta) eDNA Under Field Conditions
    Clare I. M. Adams, Diversity - 2019
    Abstract
    Environmental DNA (eDNA) is an increasingly used non-invasive molecular tool for detecting species presence and monitoring populations. In this article, we review the current state of non-avian reptile eDNA work in aquatic systems, and present a field experiment on detecting the presence of painted turtle (Chrysemys picta) eDNA. Thus far, turtle and snake eDNA studies have shown mixed results in detecting the presence of these animals under field conditions. However, some instances of low detection rates and non-detection occur for these non-avian reptiles, especially for squamates. We explored non-avian reptile eDNA quantification by sampling four lentic ponds with different densities (0 kg/ha, 6 kg/ha, 9 kg/ha, and 13 kg/ha) of painted turtles over three months to detect differences in eDNA using a qPCR assay amplifying the COI gene of the mtDNA genome. Only one sample of the highest-density pond amplified eDNA for a positive detection. Yet, estimates of eDNA concentration from pond eDNA were rank-order correlated with turtle density. We present the “shedding hypothesis”—the possibility that animals with hard, keratinized integument do not shed as much DNA as mucus-covered organisms—as a potential challenge for eDNA studies. Despite challenges with eDNA inhibition and availability in water samples, we remain hopeful that eDNA can be used to detect freshwater turtles in the field. We provide key recommendations for biologists wishing to use eDNA methods for detecting non-avian reptiles.
    RAPID GENE EXPRESSION BASED DOSE ESTIMATION FOR RADIOLOGICAL EMERGENCIES
    Stanislav Polozov, Radiation Protection Dosimetry - 2019
    Abstract
    Gene expression (GE) assays have shown great potential for rapid individual radiation dose exposure assessment. The aim of the present study was to optimise GE-based biological dosimetry protocols for radiological emergencies. Experiments were carried out to validate a newly developed protocol (P2) where several steps were optimised and to compare it with the current validated protocol in place in our laboratory (P1). Several donor blood samples from were exposed ex vivo to of the following doses: 0, 0.5, 1, 2 Gy X-rays. Concomitant measurement of transcription level of genes FDXR, P21, PHPT1, CCNG1 and SESN1 plus HPRT (control) was performed. To summarise, both protocols provided similar dose estimates, P1 being completed in 7 hours while P2 in merely 4 hours. Thus, a significant time shortening was achieved leading to a potential increase of throughput capacity. Hence, this new protocol can be recommended for mass radiation casualties triage purposes.
    Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR
    Bingqian Qu, Bio-Protocol - 2019
    Abstract
    Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate “cccDNA copies per (infected) cell”.
    Linking the resistome and plasmidome to the microbiome
    Thibault Stalder, The ISME Journal - 2019
    Abstract
    The rapid spread of antibiotic resistance among bacterial pathogens is a serious human health threat. While a range of environments have been identified as reservoirs of antibiotic resistance genes (ARGs), we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the natural bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrate that the in vivo proximity-ligation method Hi-C can reconstruct a known plasmid-host association from a wastewater community, and identify the in situ host range of ARGs, plasmids, and integrons by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile genetic elements and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the broadest host range in this wastewater, and identified bacteria belonging to Moraxellaceae, Bacteroides, and Prevotella, and especially Aeromonadaceae as the most likely reservoirs of ARGs in this community. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
    Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
    Patrick Guertler, Biomolecular Detection andQuantification - 2019
    Abstract
    Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, eventspecific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.
    A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis
    Henk J. Wisselink, BMC Veterinary Research - 2019
    Abstract
    Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
    Effect of Thermotherapy on the Acquisition of Candidatus Liberibacter Asiaticus by the Asian Citrus Psyllid (Hemiptera: Liviidae)
    Alicia J. Kelley, Florida Entomologist - 2019
    Abstract
    The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the most detrimental insect pest of citrus crops due to its role as a vector of Candidatus Liberibacter asiaticus (Las), the bacterial causal agent of huanglongbing, also known as citrus greening disease. Trees infected with Las decline rapidly and fruit production decreases until eventual tree death. Few treatment options for infected trees are available for disease management. A technique called “thermotherapy” is under development to reduce bacterial titers in infected trees; however, the effect of these treatments on the transmission cycle of Las is not known. Field and laboratory assays were conducted to determine whether thermotherapy treatment reduced Las acquisition by D. citri. Trees in the field were treated with a mobile heat treatment system. Potted trees in the laboratory were treated in a steam chamber. We monitored acquisition rates in D. citri following thermal treatment of Las-positive Citrus sinensis (L.) (Rutaceae). Psyllid acquisition and Las titer in thermotherapy-treated trees were compared with untreated Las-positive and untreated Las-negative trees. Our results confirmed the efficacy of whole-tree thermotherapy on Las in potted citrus trees. In contrast, thermotherapy did not significantly reduce plant Las titers or acquisition of Las by D. citri under field conditions. These results suggest that further development of field application methods is needed to determine the utility of thermotherapy as a tool for huanglongbing management.
    Using discrete and online ATP measurements to evaluate regrowthpotential following ozonation and (non)biological drinking watertreatment
    Glen Andrew de Vera, Water Research - 2019
    Abstract
    Water utilities must control microbial regrowth in the distribution system to protect public health. In thisstudy, an adenosine triphosphate (ATP)-based biomass production potential test using indigenous bac-terial communities were used to evaluate regrowth potential following ozonation with either bio-filtration (BF) or sustained chlorination (SCl2). Two full-scale water treatment plants with differentupstream processes (i.e., WTP-BF: ozonation, coagulation/flocculation, biofiltration, UV irradiation,chlorination; and WTP-SCl2: ozonation, chlorination, coagulation/flocculation,filtration, chlorination)were compared. Characterization of indigenous bacteria using 16S rRNA gene sequencing, qPCR, andcellular ATP (cATP) showed microbial diversity changes across treatment, biomass sloughing from bio-filters (effluent cATP¼30±1 ng/L), and disinfection by chlorine (cATP<1 ng/L). For both WTPs, 14-daycumulative biomass production (CBPt¼Ptt¼0ATPðtÞDt) was highest for ozonated water samples(CBP14¼1.2103e3.0103d ngATP/L). CBP further increased with increasing ozone dose due to pro-duction of more biodegradable carbon. Growth promotion by carbon was confirmed from the con-sumption of ozonation byproducts (carboxylic acids, aldehydes) and the increase in CBP (9.5102e2.9103d ngATP/L) after addition of 50e300mgC/L acetate. Ozone followed by sustained chlorination(WTP-SCl2) effectively controlled biomass growth across the treatment process (CBP14<10 d ngATP/L). Incontrast, ozone followed by biofiltration (WTP-BF) reduced regrowth potential by 30% (biofilter influentCBP14¼1.3103d ngATP/L; biofilter effluent CBP14¼9.3102d ngATP/L). After adding chlorine to thebiofilter effluent, CBP14was reduced to<10 d ngATP/L. Lastly, online ATP measurements confirmed thediscrete measurements and improved identification of the cATP peak and growth phases of indigenousbacteria.
    Using environmental DNA to extend the window of early detection for dreissenid mussels
    Adam J. Sepulveda, Management of Biological Invasions - 2019
    Abstract
    Tools that bolster early detection of invasive dreissenid mussels are needed to prevent their spread across western North America. In this study, we assessed if environmental DNA (eDNA) can extend the seasonal window for dreissenid mussel early detection beyond that of plankton tows, which are limited to warmer seasons when mussel larvae are present. We focused eDNA sampling efforts at multiple sites in Tiber Reservoir (Montana) where dreissenid mussel abundance is hypothesized to be low. Samples were collected in June and October 2017, when water temperatures were cooler than thermal optima for dreissenid reproduction, and in July 2017 when water temperatures were warmer and conducive for reproduction. We detected dreissenid mussel DNA in June, July and October even though no dreissenid mussels were observed using non-molecular tools in 2017. A subset of positive and negative eDNA samples was analyzed by an independent lab and results were corroborated. We then estimated the effort needed for 95% probability detection of dreissenid DNA at each site within Tiber Reservoir and found that as many as 27, 14, and 34 samples needed to be collected in June, July and October, respectively. To further validate the utility of eDNA, we also present ancillary eDNA results from other waters in the Flathead Reservation (Montana) where dreissenid mussels have never been detected and from waters with established zebra mussel populations in the upper Mississippi River, which were sampled in the spring when water temperatures were cooler than thermal optima for dreissenid reproduction. All Flathead Reservation samples were negative for dreissenid mussel DNA, while all upper Mississippi River samples were positive. This study adds to a growing body of research that demonstrates eDNA is a highly sensitive tool for dreissenid mussel surveillance in newly invaded waters, including colder seasons when non-molecular tools are likely to be less effective or more challenging to employ
    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.
    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.
    Conventional PCR
    British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy Bacilli
    Anna-Katrina Schilling, Frontiers in Veterinary Science - 2019
    Abstract
    Eurasian red squirrels (Sciurus vulgaris) in the British Isles are the most recently discovered animal reservoir for the leprosy bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others (Siberian chipmunk [Tamias sibiricus], and Thirteen-lined ground squirrel [Ictidomys tridecemlineatus]) having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species [96 Eurasian red squirrels, 67 Eastern gray squirrels (Sciurus carolinensis), 35 Siberian chipmunks, and 103 Pallas's squirrels (Callosciurus erythraeus)] from Europe and 72 Mexican white-throated woodrats (Neotoma albigula) for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.
    Epigenome-wide analysis of sperm cells identifies IL22 as a possible germ line risk locus for psoriatic arthritis
    Remy A. Pollock, PLOS ONE - 2019
    Abstract
    Psoriasis and its associated inflammatory arthritis, psoriatic arthritis (PsA), have a clear heritable component, but a large proportion of the heritable risk remains unexplained by gene sequence variation. This study aimed to determine if epigenetic factors contribute to the missing heritability in psoriatic disease. DNA methylation profiling was performed on sperm cells from 23 probands with psoriasis without PsA (PsC), 13 PsA probands, and 18 unaffected controls. Differentially methylated CpGs and regions (DMRs) were identified and validated by pyrosequencing. Underlying AluY and copy number variation (CNV) in the HCG26 and IL22 genes, respectively, were assessed by genotyping. Array, subject’s age, age of psoriasis onset, psoriasis severity, and medication usage were found to influence methylation at many genes and were included as covariates in the analysis. Between PsC probands vs. controls, 169 DMRs were found; 754 DMRs were found between PsA probands vs. controls, and 86 between PsA and PsC probands (adjusted p<0.05). Differences in methylation across DMRs were generally subtle (<10%) but correlated well with pyrosequencing. Biological inference prioritized notable DMRs associated with skin disease (SIGLEC14, JAM3, PCOLCE, RXRB), skin and/or joint disease (MBP, OSBPL5, SNORD115, HCG26), and joint disease (IL22, ELF5, PPP2R2D, PTPRN2, HCG26). Hypermethylation of the DMR within the first exon of arthritis-associated IL22 showed significant correlation (rho = 0.34, 95% CI 0.06–0.57, p = 0.01) between paired sperm and blood samples, independent of a CNV within the same region. Further studies are needed to rule out underlying genetic causes and determine if these represent heritable, constitutional epimutations, or are the result of exposure of germ cells to endogenous or exogenous environmental factors.
    Investigation of the relationship between virulence factors and antibiotic resistance of Enterococci isolates
    Umut Safiye Say Coskun, Cellular and Molecular Biology - 2019
    Abstract
    The aim of this study was to determine the relationship between aggregation factor (asa1), enterococcal surface protein (esp), cytolysin (cyl), gelatinase (gelE), hyaluronidase (hyl) virulence factors and antibiotic resistance in Enterococci. VITEK 2 ID system was used to identify the isolates and determine their antibiotic susceptibility. Virulence genes were investigated by polymerase chain reaction. Of the 93 isolates, 62 (66 %) were Enterococcus faecium, 31 (44 %) were Enterococcus faecalis (E. faecialis ). E. faecium isolates were more resistant to ampicillin, ciprofloxacin, linezolid, teicoplanin and vancomycin than E. faecalis. High-level gentamycin rate were higher in E. faecium than E. faecalis (p <0.05). The most prevalant virulence genes were esp (60.9 %) and asa1 (25 %) followed by gelE (22.8 %), cyl (16.3 %) and hyl (8.7 %). Asa1, cyl, gelE genes positivity were higer in E. faecalis than E. faecium. Hyl positivity was higher in E. faecalis than E. faecium isolates. Ampicillin resistance was higher in gelE positive E. faecalis than gelE negative E. faecalis (p <0.05). Ciprofloxacin resistance was higher in gelE negative E. faecalis than gelE positive E. faecalis (p <0.05). Asa, cyl, hyl, gelE positive E. faecium isolates were more susceptible to teicoplanin than the isolates that did not have these genes (p <0.05). Cyl, asa, gelE positive E. faecalis isolates were more susceptible to vancomycin than cyl, asa, gelE negative E. faecalis isoates (p <0.05). Hyl positive E. faecium isolates were more susceptible to vancomycin than hyl negative E. faecium isolates (p <0.05). E. faecalis isolates that have virulence genes were more susceptible to vancomycin (p <0.05). The resistance to antibiotics in E. faecalis should be a concern for the treatment of infectious disease.
    Diversity and distribution of Laonice species (Annelida: Spionidae) in the tropical North Atlantic and Puerto Rico Trench
    Theresa Guggolz, Scientific Reports - 2019
    Abstract
    Laonice Malmgren, 1867 (Annelida: Spionidae) is a common polychaete genus in the deep-sea. Although most species are quite well studied morphologically, fragmentation and other damage that occurs during sampling often hampers morphological species identification of deep-sea specimens. In this study, we employ three molecular markers (16S, COI and 18S) to study the biodiversity and the distribution patterns of Laonice from the tropical North Atlantic and the Puerto Rico Trench. Based upon different molecular analyses (Automated Barcode Gap Discovery, pairwise genetic distances, phylogenetics, haplotype networks) we were able to identify and differentiate eight Laonice species. Up to four of these species co-occurred sympatrically at the same station. The majority of species were found at multiple stations and two species in the eastern as well as western Atlantic had ranges of up to 4,000 km. Genetic differentiation across these extensive geographic distances was very low. Surprisingly, one 16S haplotype was shared between individuals 2,776 km apart and individuals from the Caribbean and the abyssal plain in the eastern Atlantic (>3,389 km) differed in only a single mutation in 16S. Our results suggest that members of this genus successfully disperse across large geographic distances and are largely unaffected by topographic barriers.
    The genus Syrrhoe (Crustacea, Amphipoda, Synopiidae) from the North Atlantic
    Luisa Fuchs, Evolutionary Systematics - 2019
    Abstract
    Three species of the amphipod genus Syrrhoe are described from the North Atlantic. The differences between these species are primarily the patterns of serration of the posterior margins of pleonite 3 and urosomite 1 and 2: Syrrhoe affinis has a wide convex space on the posterior margin between the epimeron 3 and the dorsal serration. In Syrrhoe crenulata and Syrrhoe anneheleneae sp. nov. there is only a small notch on the posterior margin of pleonite 3. Syrrhoe anneheleneae sp. nov., otherwise similar to S. crenulata, has an additional serration on the posterior margin of urosomite 1. The inter- and intraspecific distances analyzed from COI confirm the morphological species concept of North Atlantic Syrrhoe.
    Genomic and metagenomic insights into the microbial community of a thermal spring
    Renato Pedron, Microbiome - 2019
    Abstract
    Background: Water springs provide important ecosystem services including drinking water supply, recreation, and balneotherapy, but their microbial communities remain largely unknown. In this study, we characterized the spring water microbiome of Comano Terme (Italy) at four sampling points of the thermal spa, including natural (spring and well) and human-built (storage tank, bathtubs) environments. We integrated large-scale culturing and metagenomic approaches, with the aim of comprehensively determining the spring water taxonomic composition and functional potential. Results: The groundwater feeding the spring hosted the most atypical microbiome, including many taxa known to be recalcitrant to cultivation. The core microbiome included the orders Sphingomonadales, Rhizobiales, and Caulobacterales, and the families Bradyrhizobiaceae and Moraxellaceae. A comparative genomic analysis of 72 isolates and 30 metagenome-assembled genomes (MAGs) revealed that most isolates and MAGs belonged to new species or higher taxonomic ranks widely distributed in the microbial tree of life. Average nucleotide identity (ANI) values calculated for each isolated or assembled genome showed that 10 genomes belonged to known bacterial species (> 95% ANI), 36 genomes (including 1 MAG) had ANI values ranging 85–92.5% and could be assigned as undescribed species belonging to known genera, while the remaining 55 genomes had lower ANI values (< 85%). A number of functional features were significantly over- or underrepresented in genomes derived from the four sampling sites. Functional specialization was found between sites, with for example methanogenesis being unique to groundwater whereas methanotrophy was found in all samples. Conclusions: Current knowledge on aquatic microbiomes is essentially based on surface or human-associated environments. We started uncovering the spring water microbiome, highlighting an unexpected diversity that should be further investigated. This study confirms that groundwater environments host highly adapted, stable microbial communities composed of many unknown taxa, even among the culturable fraction.
    Competition and predation as possible causes of bacterial rarity
    Viola Kurm, Environmental Biology - 2019
    Abstract
    We assembled communities of bacteria and exposed them to different nutrient concentrations with or without predation by protists. Taxa that were rare in the field were less abundant at low nutrient concentrations than common taxa, independent of predation. However, some taxa that were rare in the field became highly abundant in the assembled communities, especially under ample nutrient availability. This high abundance points at a possible competitive advantage of some rare bacterial taxa under nutrient‐rich conditions. In contrast, the abundance of most rare bacterial taxa decreased at low resource availability. Since low resource availability will be the prevailing situation in most soils, our data suggests that under those conditions poor competitiveness for limiting resources may contribute to bacterial rarity. Interestingly, taxa that were rare in the field and most successful under predator‐free conditions in the lab also tended to be more reduced by predation than common taxa. This suggests that predation contributes to rarity of bacterial taxa in the field. We further discuss whether there may be a trade‐off between competitiveness and predation resistance. The substantial variability among taxa in their responses to competition and predation suggests that other factors, for example abiotic conditions and dispersal ability, also influence the local abundance of soil bacteria.
    Bacterial release from pipe biofilm in a full-scale drinking water distribution system
    Sandy Chan, npj Biofilms and Microbiomes - 2019
    Abstract
    Safe drinking water is delivered to the consumer through kilometres of pipes. These pipes are lined with biofilm, which is thought to affect water quality by releasing bacteria into the drinking water. This study describes the number of cells released from this biofilm, their cellular characteristics, and their identity as they shaped a drinking water microbiome. Installation of ultrafiltration (UF) at full scale in Varberg, Sweden reduced the total cell count to 1.5 × 103 ± 0.5 × 103 cells mL−1 in water leaving the treatment plant. This removed a limitation of both flow cytometry and 16S rRNA amplicon sequencing, which have difficulties in resolving small changes against a high background cell count. Following installation, 58% of the bacteria in the distributed water originated from the pipe biofilm, in contrast to before, when 99.5% of the cells originated from the treatment plant, showing that UF shifts the origin of the drinking water microbiome. The number of bacteria released from the biofilm into the distributed water was 2.1 × 103 ± 1.3 × 103 cells mL−1 and the percentage of HNA (high nucleic acid) content bacteria and intact cells increased as it moved through the distribution system. DESeq2 analysis of 16S rRNA amplicon reads showed increases in 29 operational taxonomic units (OTUs), including genera identified as Sphingomonas, Nitrospira, Mycobacterium, and Hyphomicrobium. This study demonstrated that, due to the installation of UF, the bacteria entering a drinking water microbiome from a pipe biofilm could be both quantitated and described.
    Mine reclamation using biofuel crops: Insights into the microbial ecology of the switchgrass (Panicum virgatum) microbiome
    Brianna L. Mayfield, Graduate Theses, Dissertations, and Problem reports - 2019
    Abstract
    Bioenergy crop production has steadily increased due to growing political support for renewable energy, thus initiating a demand to find alternative agricultural land. An innovative option is the use of marginal soils, such as reclaimed mine lands, to produce bioenergy crops. Switchgrass (Panicum virgatum) is a promising bioenergy crop that can be grown on marginal lands due to its robust growth in various soil types and climates. However, little is known regarding plant-microbe interactions among switchgrass systems within reclaimed mine lands. A study conducted in 2008 grew switchgrass on high- and low- quality reclaimed mine sites (Hampshire and Hobet, respectively) in West Virginia to examine the resilience of switchgrass as a reclamation-friendly bioenergy crop. Switchgrass yields at Hampshire were nearly an order of magnitude higher than Hobet (8.4 Mg ha−1 vs 1.0 Mg ha−1). Within Hampshire, the Cave-in-Rock cultivar yield was approximately 2-fold greater than that of Shawnee (12.9 Mg ha-1 vs. 7.6 Mg ha-1). Here, I sought to illuminate plant-microbial interactions that may account for this drastic shift in cultivar yield by assessing the soil microbial community’s function and composition. I tested two hypotheses: i) that the microbial community’s ability to acquire C, N, and P will be greatest in Hampshire soils compared to that of Hobet and ii) that there will be a cultivar-specific root-associated microbiome that may drive previously observed greater, but differential yields across switchgrass cultivars at Hampshire. I found that reclamation strategy substantially impacts the switchgrass microbiome’s composition as well as its ability to acquire critical nutrients like carbon, nitrogen, and phosphorus. I also found that a functionally, but not necessarily compositionally, unique microbiome exists in the root-associated soils compared to that of the bulk soil. Additionally, there were indicators that organic amendments to the topsoil may induce cultivar-specific soil microbiomes that mediate or facilitate differential yields within Hampshire. Taken together, I suggest that organic amendments to the topsoil during reclamation selects for a cultivar-specific microbiome more adept to acquiring critical nutrients and thus, increases aboveground productivity.
    Temporal escalation of Pyrethroid Resistance in the major malaria vector Anopheles coluzzii from Sahelo-Sudanian Region of northern Nigeria
    Sulaiman S. Ibrahim, Scientific Reports - 2019
    Abstract
    Despite the highest global burden of malaria, information on bionomics and insecticide resistance status of malaria vectors is grossly lacking in the densely populated Sahelo-Sudanian region of Nigeria. To support evidence-based vector control we characterised transmission and resistance profiles of Anopheles coluzzii populations from three sites in northern Nigeria. High sporozoite infection (~19.51%) was found in the An. coluzzii populations. A high pyrethroid resistance was observed with only 1% mortality against deltamethrin, a high LD50 (96.57 µg/ml), and a high LT50 (170.27 min, resistance ratio of ~51 compared with the fully susceptible Ngoussou colony). Moderate carbamate resistance was observed. Synergist bioassays significantly recovered deltamethrin susceptibility implicating CYP450s (mortality = 85%, χ2 = 134.04, p < 0.0001) and esterases (mortality = 56%, χ2 = 47.31, p < 0.0001). Reduced bed net efficacy was also observed, with mortalities on exposure to the roof of PermaNet3.0 (PBO + deltamethrin) more than 22 times compared to the side panel (deltamethrin). TaqMan genotyping revealed a high frequency of 1014F kdr mutation (82%) with significant difference in genotype distribution associated with permethrin resistance [OR = 4.69 (CI:1.53–14.35, χ2 = 8.22 p = 0.004]. Sequencing of exons 18–21 of the VGSC led to detection of two additional nonsynonymous mutations, Ile10148Asn and Ser1156Gly. These findings highlight the threats posed by the highly resistant An. coluzzii to malaria control in Nigeria.
    Gamma Irradiation Influences the Survival and Regrowth of Antibiotic-Resistant Bacteria and Antibiotic-Resistance Genes on Romaine Lettuce
    Vaishali Dharmarha, Frontiers in Microbiology - 2019
    Abstract
    Contamination of romaine lettuce with human pathogens, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs) occurs during production. Post-harvest interventions are emplaced to mitigate pathogens, but could also mitigate ARB and ARGs on vegetables. The objective of this research was to determine changes to lettuce phyllosphere microbiota, inoculated ARB, and the resistome (profile of ARGs) following washing with a sanitizer, gamma irradiation, and cold storage. To simulate potential sources of pre-harvest contamination, romaine lettuce leaves were inoculated with compost slurry containing antibiotic-resistant strains of pathogenic (Escherichia coli O157:H7) and representative of spoilage bacteria (Pseudomonas aeruginosa). Various combinations of washing with sodium hypochlorite (50 ppm free chlorine), packaging under modified atmosphere (98% nitrogen), irradiating (1.0 kGy) and storing at 4°C for 1 day versus 14 days were compared. Effects of post-harvest treatments on the resistome were profiled by shotgun metagenomic sequencing. Bacterial 16S rRNA gene amplicon sequencing was performed to determine changes to the phyllosphere microbiota. Survival and regrowth of inoculated ARB were evaluated by enumeration on selective media. Washing lettuce in water containing sanitizer was associated with reduced abundance of ARG classes that confer resistance to glycopeptides, β-lactams, phenicols, and sulfonamides (Wilcoxon, p < 0.05). Washing followed by irradiation resulted in a different resistome chiefly due to reductions in multidrug, triclosan, polymyxin, β-lactam, and quinolone ARG classes (Wilcoxon, p < 0.05). Irradiation followed by storage at 4°C for 14 days led to distinct changes to the β-diversity of the host bacteria of ARGs compared to 1 day after treatment (ANOSIM, R = 0.331; p = 0.003). Storage of washed and irradiated lettuce at 4°C for 14 days increased the relative abundance of Pseudomonadaceae and Carnobacteriaceae (Wilcoxon, p < 0.05), two groups whose presence correlated with detection of 10 ARG classes on the lettuce phyllosphere (p < 0.05). Irradiation resulted in a significant reduction (∼3.5 log CFU/g) of inoculated strains of E. coli O157:H7 and P. aeruginosa (ANOVA, p < 0.05). Results indicate that washing, irradiation and storage of modified atmosphere packaged lettuce at 4°C are effective strategies to reduce antibiotic-resistant E. coli O157:H7 and P. aeruginosa and relative abundance of various ARG classes.
    Identification and removal of contaminating microbial DNA from PCR reagents: impact on low‐biomass microbiome analyses
    L.F. Stinson, Letters in Applied Microbiology - 2019
    Abstract
    Reagent‐derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the ‘kitome’ (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents. In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing. Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads. We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low‐biomass microbiome studies and producing more robust and reliable data.
    Pyrethroid exposure alters internal and cuticle surface bacterial communities in Anopheles albimanus
    Nsa Dada, The ISME Journal - 2019
    Abstract
    A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Following previously identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, the effects of pyrethroid exposure on the microbiota of F1 progeny of field-collected Anopheles albimanus. Larval and adult mosquitoes were exposed to the pyrethroids alphacypermethrin (only adults), permethrin, and deltamethrin. While there were no significant differences in bacterial composition between insecticide-resistant and insecticide-susceptible mosquitoes, bacterial composition between insecticide-exposed and non-exposed mosquitoes was significantly different for alphacypermethrin and permethrin exposure. Along with other bacterial taxa not identified to species, Pantoea agglomerans (a known insecticide-degrading bacterial species) and Pseudomonas fragi were more abundant in insecticide-exposed compared to non-exposed adults, demonstrating that insecticide exposure can alter mosquito bacterial communities. We also show for the first time that the cuticle surfaces of both larval and adult An. albimanus harbor more diverse bacterial communities than their internal microbial niches. Together, these findings demonstrate how insecticide pressure could be selecting for certain bacteria within mosquitoes, especially insecticide-metabolizing bacteria, thus potentially contributing to insecticide resistance.
    High Plasmodium infection and multiple insecticide resistance in a major malaria vector Anopheles coluzzii from Sahel of Niger Republic
    Sulaiman S. Ibrahim, BMC - 2019
    Abstract
    Background: Information on insecticide resistance and the mechanisms driving it in the major malaria vectors is grossly lacking in Niger Republic, thus hindering control eforts. To facilitate evidence-based malaria control, the role of Anopheles coluzzii population from southern Niger, in malaria transmission, its insecticides resistance profle and the molecular mechanisms driving the resistance were characterized. Methods: Blood fed female Anopheles gambiae sensu lato resting indoor were collected at Tessaoua, Niger. Source of blood was established using PCR and infection with Plasmodium determined using TaqMan assay. Resistance profle was established with the major public health insecticides, and resistance intensity determined with deltamethrin. Synergist assays were conducted with piperonyl butoxide and diethyl maleate. Presence of L1014F and L1014S knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) was investigated using TaqMan genotyping, and strength of selection pressure acting on the Anopheles populations determined by assessing the genetic diversity of a fragment spanning exon-20 of the VGSC from alive and dead females. Results: High human blood index (96%) and high Plasmodium falciparum infection (~13%) was observed in the An. coluzzii population. Also, a single mosquito was found infected with Plasmodium vivax. High pyrethroid and organochloride resistance was observed with mortalities of less than 20% for deltamethrin, permethrin, α-cypermethrin, and DDT. A high LD50 (156.65 min) was obtained for deltamethrin, with a resistance ratio of~47.18 compared to the susceptible Ngoussou colony. Moderate carbamate resistance was observed, and a full susceptibility to organophosphates recorded. Synergist bioassays with piperonyl butoxide and diethyl maleate signifcantly recovered deltamethrin and DDT susceptibility, respectively implicating CYP450 s (mortality=82%, χ2=84.51, p<0.0001) and glutathione S-transferases (mortality=58%, χ2=33.96, p<0.001) in resistance. A high frequency of 1014F kdr mutation (82%) was established, with signifcant diference in genotype distribution associated with permethrin resistance [odds ratio=7.71 (95% CI 2.43–14.53, χ2=13.67, p=0.001]. Sequencing of intron-1 of the voltage-gated sodium channel (VGSC) revealed a low genetic diversity. Conclusion: High pyrethroid resistance highlight the challenges to the efectiveness of the pyrethroids-based ITNs and indoor residual spraying (IRS) against An. coluzzii in Niger. The pyrethroids-synergists LLINs and organophosphatebased IRS maybe the alternatives for malaria control in southern Niger
    Community ecology across bacteria, archaea and microbial eukaryotes in the sediment and seawater of coastal Puerto Nuevo, Baja California
    Sabah UI-Hasan, PLOS ONE - 2019
    Abstract
    Microbial communities control numerous biogeochemical processes critical for ecosystem function and health. Most analyses of coastal microbial communities focus on the characterization of bacteria present in either sediment or seawater, with fewer studies characterizing both sediment and seawater together at a given site, and even fewer studies including information about non-bacterial microbial communities. As a result, knowledge about the ecological patterns of microbial biodiversity across domains and habitats in coastal communities is limited–despite the fact that archaea, bacteria, and microbial eukaryotes are present and known to interact in coastal habitats. To better understand microbial biodiversity patterns in coastal ecosystems, we characterized sediment and seawater microbial communities for three sites along the coastline of Puerto Nuevo, Baja California, Mexico using both 16S and 18S rRNA gene amplicon sequencing. We found that sediment hosted approximately 500-fold more operational taxonomic units (OTUs) for bacteria, archaea, and microbial eukaryotes than seawater (p < 0.001). Distinct phyla were found in sediment versus seawater samples. Of the top ten most abundant classes, Cytophagia (bacterial) and Chromadorea (eukaryal) were specific to the sediment environment, whereas Cyanobacteria and Bacteroidia (bacterial) and Chlorophyceae (eukaryal) were specific to the seawater environment. A total of 47 unique genera were observed to comprise the core taxa community across environment types and sites. No archaeal taxa were observed as part of either the abundant or core taxa. No significant differences were observed for sediment community composition across domains or between sites. For seawater, the bacterial and archaeal community composition was statistically different for the Major Outlet site (p < 0.05), the site closest to a residential area, and the eukaryal community composition was statistically different between all sites (p < 0.05). Our findings highlight the distinct patterns and spatial heterogeneity in microbial communities of a coastal region in Baja California, Mexico.
    Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring
    Vasco Elbrecht, Freshwater Biology - 2019
    Abstract
    The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the recent years. The method has matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high‐throughput sequencing capacity. However, workflows and sample tagging need to be optimised to accommodate for hundreds of samples within a single sequencing run. Here, we conceptualise a streamlined metabarcoding workflow, in which samples are processed in 96‐well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2 + BR2 primer pair up to three 96‐well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large‐scale biodiversity assessments involving freshwater invertebrates. However, as this is just one possible metabarcoding approach, we hope this article will stimulate discussion and publication of alternatives and extensions to this method.
    Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct
    Holly Packard, PLOS ONE - 2019
    Abstract
    A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion
    Scalp bacterial shift in Alopecia areata
    Daniela Pinto, PLOS ONE - 2019
    Abstract
    The role of microbial dysbiosis in scalp disease has been recently hypothesized. However, little information is available with regards to the association between microbial population on the scalp and hair diseases related to hair growth. Here we investigated bacterial communities in healthy and Alopecia areata (AA) subjects. The analysis of bacterial distribution at the genus level highlighted an increase of Propionibacterium in AA subjects alongside a general decrease of Staphylococcus. Analysis of log Relative abundance of main bacterial species inhabiting the scalp showed a significant increase of Propionibacterium acnes in AA subjects compared to control ones. AA scalp condition is also associated with a significant decrease of Staphylococcus epidermidis relative abundance. No significant changes were found for Staphylococcus aureus. Therefore, data from sequencing profiling of the bacterial population strongly support a different microbial composition of the different area surrounded hair follicle from the epidermis to hypodermis, highlighting differences between normal and AA affected the scalp. Our results highlight, for the first time, the presence of a microbial shift on the scalp of patients suffering from AA and gives the basis for a larger and more complete study of microbial population involvement in hair disorders.
    Downregulation of female doublesex expression by oral-mediated RNA interference reduces number and fitness of Anopheles gambiae adult females
    Mabel L. Taracena, Parasites and Vectors - 2019
    Abstract
    Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies. Alternative control technologies are in development, including genetic control such as the sterile insect technique (SIT). The SIT is a pivotal tool in integrated agricultural pest management and could be used to improve malaria vector control. To apply the SIT and most other newer technologies against disease transmitting mosquitoes, it is essential that releases are composed of males with minimal female contamination. The removal of females is an essential requirement because released females can themselves contribute towards nuisance biting and disease transmission. Thus, females need to be eliminated from the cohorts prior to release. Manual separation of Anopheles gambiae pupae or adult mosquitoes based on morphology is time consuming, is not feasible on a large scale and has limited the implementation of the SIT technique. The doublesex (dsx) gene is one of the effector switches of sex determination in the process of sex differentiation in insects. Both males and females have specific splicing variants that are expressed across the different life stages. Using RNA interference (RNAi) to reduce expression of the female specific (dsxF) variant of this gene has proven to have detrimental effects to the females in other mosquito species, such as Aedes aegypti. We tested oral RNAi on dsx (AgdsxF) in An. gambiae. Methods We studied the expression pattern of the dsx gene in the An. gambiae G3 strain. We knocked down AgdsxF expression in larvae through oral delivery of double stranded RNA (dsRNA) produced by bacteria and observed its effects in adults. Results Our results show that feeding of AgdsxF dsRNA can effectively reduce (> 66%) the mRNA of female dsx transcript and that there is a concomitant reduction in the number of female larvae that achieve adulthood. Control groups produced 52% (± 3.9% SE) of adult males and 48% (± 4.0% SE) females, while AgdsxF dsRNA treated groups had 72.1% (± 4.0% SE) males vs 27.8% females (± 3.3% SE). In addition, the female adults produce fewer progeny, 37.1% (± 8.2% SE) less than the controls. The knockdown was sex-specific and had no impact on total numbers of viable male adults, in the male dsx transcripts or male fitness parameters such as longevity or body size. Conclusions These findings indicate that RNAi could be used to improve novel mosquito control strategies that require efficient sex separation and male-only release of An. gambiae by targeting sex determination genes such as AgdsxF. The advantages of using RNAi in a controlled setting for mosquito rearing are numerous, as the dose and time of exposure are controlled, and the possibility of off-target effects and the waste of female production would be significantly reduced.
    A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay
    Nikol Reslova, Scientific Reports - 2019
    Abstract
    Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.
    The Microbial Communities of Leaves and Roots Associated with Turtle Grass (Thalassia testudinum) and Manatee Grass (Syringodium filliforme) are Distinct from Seawater and Sediment Communities, but Are Similar between Species and Sampling Sites
    Kelly Ugarelli, Microorganisms - 2019
    Abstract
    Seagrasses are vital members of coastal systems, which provide several important ecosystem services such as improvement of water quality, shoreline protection, and serving as shelter, food, and nursery to many species, including economically important fish. They also act as a major carbon sink and supply copious amounts of oxygen to the ocean. A decline in seagrasses has been observed worldwide, partly due to climate change, direct and indirect human activities, diseases, and increased sulfide concentrations in the coastal porewaters. Several studies have shown a symbiotic relationship between seagrasses and their microbiome. For instance, the sulfur, nitrogen, and carbon cycles are important biochemical pathways that seem to be linked between the plant and its microbiome. The microbiome presumably also plays a key role in the health of the plant, for example in oxidizing phyto-toxic sulfide into non-toxic sulfate, or by providing protection for seagrasses from pathogens. Two of the most abundant seagrasses in Florida include Thalassia testudinum (turtle grass) and Syringodium filliforme (manatee grass), yet there is little data on the composition of the microbiome of these two genera. In this study, the microbial composition of the phyllosphere and rhizosphere of Thalassia testudinum and Syringodium filiforme were compared to water and sediment controls using amplicon sequencing of the V4 region of the 16S rRNA gene. The microbial composition of the leaves, roots, seawater, and sediment differ from one another, but are similar between the two species of seagrasses.
    A Method for Targeted 16S Sequencing of Human Milk Samples
    Tobin, N. H., J. Vis. Exp. - 2018
    Abstract
    Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.
    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.
    Cloning
    Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
    Ryan C. Heller, Nucleic Acids Research - 2019
    Abstract
    Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5′→3′ nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.
  • 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