Characterization of the bovine pregnancy-associated glycoprotein gene family – analysis of gene sequences, regulatory regions within the promoter and expression of selected genes

<p>Abstract</p> <p>Background</p> <p>The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the <it>Artiodactyla </it>order. In cattle, the <it>PAG </it>gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an <it>in silico </it>analysis of the bovine genome to further characterize the <it>PAG </it>gene family, 2) we scrutinized proximal promoter sequences of the <it>PAG </it>genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected <it>PAGs </it>during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, <it>bovine </it>(<it>bo</it>) <it>PAG-2</it>.</p> <p>Results</p> <p>From our analysis of the bovine genome, we identified 18 distinct <it>PAG </it>genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all <it>boPAGs</it>. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern <it>boPAG </it>grouping, but not the ancient <it>boPAGs</it>. We gathered evidence by means of Q-PCR and screening of EST databases to show that <it>boPAG-2 </it>is the most abundant of all <it>boPAG </it>transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the <it>boPAG-2 </it>gene.</p> <p>Conclusion</p> <p><it>PAGs </it>represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that <it>boPAG-2 </it>is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the <it>boPAG </it>gene family.</p

Ethnic-Specific Differences in Vitamin D Status Is Associated with Adiposity

BackgroundLow circulating 25 hydroxyvitamin D [25(OH)D] concentrations are common in obesity (BMI ≥30 kg/m2) and a negative relationship with body fat distribution has recently been reported. Ethnic-specific differences in body fat distribution have been described with South Asians are reported to have greater visceral adipose tissue (VAT), which could influence circulating 25(OH)D concentrations. The objective of this study is to investigate the relationship between plasma 25(OH)D, adiposity, and body fat distribution in Europeans and South Asians.Methods/Principal Findings187 Europeans and 192 South Asians were assessed for demographics, anthropometrics, and plasma 25(OH)D concentrations. Subcutaneous adipose tissue (SAT) and VAT were quantified by CT scan, and percent body fat by DEXA. Data were assessed by general linear models. South Asians had lower (P&lt;0.001) plasma 25(OH)D concentrations and higher VAT (P = 0.04) than Europeans. Plasma 25(OH)D concentrations were negatively (P&lt;0.05) associated with BMI, waist circumference, percent body fat, total adipose tissue, VAT, and SAT in unadjusted models and negatively (P&lt;0.05) associated with VAT, SAT, and percent body fat after adjusting for BMI, ethnicity, age, and season of blood collection in males and females. When percent body fat, VAT, and SAT were included in the same model, only VAT remained negatively (P&lt;0.05) associated with plasma 25(OH)D concentrations. Ethnicity remained significant in all models (P&lt;0.001).ConclusionCompared to other adipose tissue compartments, VAT may have a distinct role in determining plasma 25(OH)D concentrations, which may account for the lower levels in South Asians

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxinand model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses

Sex differences in the ability of corticostriatal oscillations to predict rodent alcohol consumption

Background: Although male and female rats differ in their patterns of alcohol use, little is known regarding the neural circuit activity that underlies these differences in behavior. The current study used a machine learning approach to characterize sex differences in local field potential (LFP) oscillations that may relate to sex differences in alcohol-drinking behavior. Methods: LFP oscillations were recorded from the nucleus accumbens shell and the rodent medial prefrontal cortex of adult male and female Sprague-Dawley rats. Recordings occurred before rats were exposed to alcohol (n = 10/sex × 2 recordings/rat) and during sessions of limited access to alcohol (n = 5/sex × 5 recordings/rat). Oscillations were also recorded from each female rat in each phase of estrous prior to alcohol exposure. Using machine learning, we built predictive models with oscillation data to classify rats based on: (1) biological sex, (2) phase of estrous, and (3) alcohol intake levels. We evaluated model performance from real data by comparing it to the performance of models built and tested on permutations of the data. Results: Our data demonstrate that corticostriatal oscillations were able to predict alcohol intake levels in males (p \u3c 0.01), but not in females (p = 0.45). The accuracies of models predicting biological sex and phase of estrous were related to fluctuations observed in alcohol drinking levels; females in diestrus drank more alcohol than males (p = 0.052), and the male vs. diestrus female model had the highest accuracy (71.01%) compared to chance estimates. Conversely, females in estrus drank very similar amounts of alcohol to males (p = 0.702), and the male vs. estrus female model had the lowest accuracy (56.14%) compared to chance estimates. Conclusions: The current data demonstrate that oscillations recorded from corticostriatal circuits contain significant information regarding alcohol drinking in males, but not alcohol drinking in females. Future work will focus on identifying where to record LFP oscillations in order to predict alcohol drinking in females, which may help elucidate sex-specific neural targets for future therapeutic development

Examining the Polymorphisms in the Hypoxia Pathway Genes in Relation to Outcome in Colorectal Cancer

Introduction Colorectal cancer is a common malignancy. Identification of genetic prognostic markers may help prognostic estimations in colorectal cancer. Genes that regulate response to hypoxia and other genes that are regulated under the hypoxic conditions have been shown to play roles in cancer progression. In this study, we hypothesized that genetic variations in the hypoxia pathway genes were associated with the risk of outcome in colorectal cancer patients. Methods This study was performed in two phases. In the first phase, 49 SNPs from six hypoxia pathway genes (HIF1A, HIF1B, HIF2A, LOX, MIF and CXCL12) in 272 colorectal cancer patients were analyzed. In the second phase, 77 SNPs from seven hypoxia pathway genes (HIF1A, HIF1B, HIF2A, HIF2B, HIF3A, LOX and CXCL12) were analyzed in an additional cohort of 535 patients. Kaplan Meier, Cox univariate and multivariable regression analyses were performed to analyze the relationship between the SNPs and overall survival (OS), disease free survival (DFS) or disease specific survival (DSS). Since this was a hypothesis-generating study, no correction for multiple testing was applied. Results In phase I, one SNP (HIF2A rs11125070) was found to be associated with DFS in multivariable analysis; yet association of a proxy polymorphism (HIF2A rs4953342) was not detected in the phase II patient cohort. In phase II, associations of two SNPs (HIF2A rs4953352 and HIF2B rs12593988) were significant in both OS and DFS multivariable analyses. However, association of HIF2A rs4953352 was not replicated in the phase I cohort using a proxy SNP (HIF2A rs6706003). Conclusion Overall, our study did not find a convincing evidence of association of the investigated polymorphisms with the disease outcomes in colorectal cance

L-5-methyltetrahydrofolate supplementation increases blood folate concentrations to a greater extent than folic acid supplementation in Malaysian women

Background: Folic acid fortification of grains is mandated in many countries to prevent neural tube defects. Concerns regarding excessive intakes of folic acid have been raised. A synthetic analog of the circulating form of folate, l-5-methyltetrahydrofolate (l-5-MTHF), may be a potential alternative. Objective: The objective of this study was to determine the effects of folic acid or l-5-MTHF supplementation on blood folate concentrations, methyl nutrient metabolites, and DNA methylation in women living in Malaysia, where there is no mandatory fortification policy. Methods: In a 12-wk, randomized, placebo-controlled intervention trial, healthy Malaysian women (n = 142, aged 20–45 y) were randomly assigned to receive 1 of the following supplements daily: 1 mg (2.27 μmol) folic acid, 1.13 mg (2.27 μmol) l-5-MTHF, or a placebo. The primary outcomes were plasma and RBC folate and vitamin B-12 concentrations. Secondary outcomes included plasma total homocysteine, total cysteine, methionine, betaine, and choline concentrations and monocyte long interspersed nuclear element-1 (LINE-1) methylation. Results: The folic acid and l-5-MTHF groups had higher (P < 0.001) RBC folate (mean ± SD: 1498 ± 580 and 1951 ± 496 nmol/L, respectively) and plasma folate [median (25th, 75th percentiles): 40.1 nmol/L (24.9, 52.7 nmol/L) and 52.0 nmol/L (42.7, 73.1 nmol/L), respectively] concentrations compared with RBC folate (958 ± 345 nmol/L) and plasma folate [12.6 nmol/L (8.80, 17.0 nmol/L)] concentrations in the placebo group at 12 wk. The l-5-MTHF group had higher RBC folate (1951 ± 496 nmol/L; P = 0.003) and plasma folate [52.0 nmol/L (42.7, 73.1 nmol/L); P = 0.023] at 12 wk than did the folic acid group [RBC folate, 1498 ± 580 nmol/L; plasma folate, 40.1 nmol/L (24.9, 52.7 nmol/L)]. The folic acid and l-5-MTHF groups had 17% and 15%, respectively, lower (P < 0.001) plasma total homocysteine concentrations than did the placebo group at 12 wk; there were no differences between the folic acid and l-5-MTHF groups. No differences in plasma vitamin B-12, total cysteine, methionine, betaine, and choline and monocyte LINE-1 methylation were observed. Conclusion: These findings suggest differential effects of l-5-MTHF compared with folic acid supplementation on blood folate concentrations but no differences on plasma total homocysteine lowering in Malaysian women. This trial was registered at clinicaltrials.gov as NCT01584050

Antagonism of STAT3 signalling by Ebola virus

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer