Search

Background: Vitamin D was used to treat tuberculosis in the pre-antibiotic era, and its metabolites induce antimycobacterial immunity in vitro. Clinical trials investigating the effect of adjunctive vitamin D on sputum culture conversion are absent.Methods: We undertook a multicentre randomised controlled trial of adjunctive vitamin D in adults with sputum smear-positive pulmonary tuberculosis in London, UK. 146 patients were allocated to receive 2·5 mg vitamin D(3) or placebo at baseline and 14, 28, and 42 days after starting standard tuberculosis treatment. The primary endpoint was time from initiation of antimicrobial treatment to sputum culture conversion. Patients were genotyped for TaqI and FokI polymorphisms of the vitamin D receptor, and interaction analyses were done to assess the influence of the vitamin D receptor genotype on response to vitamin D(3). This trial is registered with ClinicalTrials.gov number NCT00419068.Findings: 126 patients were included in the primary efficacy analysis (62 assigned to intervention, 64 assigned to placebo). Median time to sputum culture conversion was 36·0 days in the intervention group and 43·5 days in the placebo group (adjusted hazard ratio 1·39, 95% CI 0·90-2·16; p=0.14). TaqI genotype modified the effect of vitamin D supplementation on time to sputum culture conversion (p(interaction)=0·03), with enhanced response seen only in patients with the tt genotype (8·09, 95% CI 1·36-48·01; p=0·02). FokI genotype did not modify the effect of vitamin D supplementation (p(interaction)=0·85). Mean serum 25-hydroxyvitamin D concentration at 56 days was 101·4 nmol/L in the intervention group and 22·8 nmol/L in the placebo group (95% CI for difference 68·6-88·2; p<0·0001).Interpretation: Administration of four doses of 2·5 mg vitamin D(3) increased serum 25-hydroxyvitamin D concentrations in patients receiving intensive-phase treatment for pulmonary tuberculosis. Vitamin D did not significantly affect time to sputum culture conversion in the whole study population, but it did significantly hasten sputum culture conversion in participants with the tt genotype of the TaqI vitamin D receptor polymorphism.Funding: British Lung Foundation. [ABSTRACT FROM AUTHOR]

WWOX is a bona fide tumour suppressor, with hypomorphic and knockout mouse models exhibiting increased tumour susceptibility. In ovarian cancer cells WWOX transfection abolishes tumourigenicity, suppresses tumour cell adhesion to extracellular matrix and induces apoptosis in non-adherent cells. One-third of ovarian tumours show loss of WWOX expression, and this loss significantly associates with clear cell and mucinous histology, advanced stage, low progesterone receptor expression and poor survival, suggesting that WWOX status affects ovarian cancer progression and prognosis. Genetic variation in other tumour suppressors (e.g. p53 and XPD) is reported to modify cancer progression/outcome, and single nucleotide polymorphisms (SNPs) within the WWOX gene are reported to associate with prostate cancer risk. We previously identified polymorphic variants within WWOX, some of which have potential to affect its expression. We therefore examined a cancer modifier role for these WWOX variants. Eight SNPs, based upon location, frequency and potential to affect WWOX expression, were genotyped in 554 ovarian cancer patients (CGP samples), and associations with pathological and survival data were examined. The CGP samples demonstrated significant associations after Bonferroni correction between Isnp1 and both tumour grade (p(corr)=0.033) and histology (p(corr)=0.046), Isnp8 and tumour grade (p(corr)=0.032) and T1497G and progression-free survival (p(corr)=0.037). None of these positive associations were confirmed in an independent ovarian cancer population (Scotroc1 samples, n=863). While these results may suggest that the associations are false positives, differences between the two populations cannot be excluded, and thus highlight the challenges in validation studies. [ABSTRACT FROM AUTHOR]

Background and Aims: Vagal parasympathetic dysfunction is strongly associated with impaired exercise tolerance, indicating that coordinated autonomic control is essential for optimizing exercise performance. This study tested the hypothesis that autonomic neuromodulation by non-invasive transcutaneous vagus nerve stimulation (tVNS) can improve exercise capacity in humans., Methods: This single-centre, randomized, double-blind, sham-controlled, crossover trial in 28 healthy volunteers evaluated the effect of bilateral transcutaneous stimulation of vagal auricular innervation, applied for 30 min daily for 7 days, on measures of cardiorespiratory fitness (peak oxygen consumption (VO2peak)) during progressive exercise to exhaustion. Secondary endpoints included peak work rate, cardiorespiratory measures, and the whole blood inflammatory response to lipopolysaccharide ex vivo., Results: tVNS applied for 30 min daily over 7 consecutive days increased VO2peak by 1.04 mL/kg/min (95% CI: .34-1.73; P = .005), compared with no change after sham stimulation (-0.54 mL/kg/min; 95% CI: -1.52 to .45). No carry-over effect was observed following the 2-week washout period. tVNS increased work rate (by 6 W; 95% CI: 2-10; P = .006), heart rate (by 4 bpm; 95% CI: 1-7; P = .011), and respiratory rate (by 4 breaths/min; 95% CI: 2-6; P < .001) at peak exercise. Analysis of the whole blood transcriptomic response to lipopolysaccharide in serial samples obtained from five participants showed that tVNS reduced the inflammatory response., Conclusions: Non-invasive vagal stimulation improves measures of cardiorespiratory fitness and attenuates inflammation, offering an inexpensive, safe, and scalable approach to improve exercise capacity., (© The Author(s) 2025. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Platelet function is driven by the expression of specialized surface markers. The concept of distinct circulating subpopulations of platelets has emerged in recent years, but their exact nature remains debatable., Objectives: To design a spectral flow cytometry-based phenotyping workflow to provide a more comprehensive characterization, at a global and individual level, of surface markers in resting and activated healthy platelets, and to apply this workflow to investigate how responses differ according to platelet age., Methods: A 14-marker flow cytometry panel was developed and applied to vehicle- or agonist-stimulated platelet-rich plasma and whole blood samples obtained from healthy volunteers, or to platelets sorted according to SYTO-13 (Thermo Fisher Scientific) staining intensity as an indicator of platelet age. Data were analyzed using both user-led and independent approaches incorporating novel machine learning-based algorithms., Results: The assay detected differences in marker expression in healthy platelets, at rest and on agonist activation, in both platelet-rich plasma and whole blood samples, that are consistent with the literature. Machine learning identified stimulated populations of platelets with high accuracy (>80%). Similarly, machine learning differentiation between young and old platelet populations achieved 76% accuracy, primarily weighted by forward scatter, cluster of differentiation (CD) 41, side scatter, glycoprotein VI, CD61, and CD42b expression patterns., Conclusion: Our approach provides a powerful phenotypic assay coupled with robust bioinformatic and machine learning workflows for deep analysis of platelet subpopulations. Cleavable receptors, glycoprotein VI and CD42b, contribute to defining shared and unique subpopulations. This adoptable, low-volume approach will be valuable in deep characterization of platelets in disease., (© 2024 The Author(s).)

Abstract: Visceral pain is a leading cause of morbidity in inflammatory bowel disease (IBD), contributing significantly to reduced quality of life. Currently available analgesics often lack efficacy or have intolerable side effects, driving the need for a more complete understanding of the mechanisms causing pain. Whole transcriptome gene expression analysis was performed by bulk RNA sequencing of colonic biopsies from patients with ulcerative colitis (UC) and Crohn's disease (CD) reporting abdominal pain and compared with noninflamed control biopsies. Potential pronociceptive mediators were identified based on gene upregulation in IBD biopsy tissue and cognate receptor expression in murine colonic sensory neurons. Pronociceptive activity of identified mediators was assessed in assays of sensory neuron and colonic afferent activity. RNA sequencing analysis highlighted a 7.6-fold increase in the expression of angiotensinogen transcripts, Agt , which encode the precursor to angiotensin II (Ang II), in samples from UC patients ( P = 3.2 × 10 -8 ). Consistent with the marked expression of the angiotensin AT 1 receptor in colonic sensory neurons, Ang II elicited an increase in intracellular Ca 2+ in capsaicin-sensitive, voltage-gated sodium channel subtype Na V 1.8-positive sensory neurons. Ang II also evoked action potential discharge in high-threshold colonic nociceptors. These effects were inhibited by the AT 1 receptor antagonist valsartan. Findings from our study identify AT 1 receptor-mediated colonic nociceptor activation as a novel pathway of visceral nociception in patients with UC. This work highlights the potential utility of angiotensin receptor blockers, such as valsartan, as treatments for pain in IBD., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

Background: Epigenetic changes can bring insight into gene regulatory mechanisms associated with disease pathogenicity, including chronicity and increased vulnerability. To date, we are yet to identify genes sensitive to epigenetic regulation that contribute to the maintenance of chronic pain and with an epigenetic landscape indicative of the susceptibility to persistent pain. Such genes would provide a novel opportunity for better pain management, as their epigenetic profile could be targeted for the treatment of chronic pain or used as an indication of vulnerability for prevention strategies. Here, we investigated the epigenetic profile of the gene Fkbp5 for this potential, using targeted bisulphite sequencing in rodent pre-clinical models of chronic and latent hypersensitive states., Results: The Fkbp5 promoter DNA methylation (DNAm) signature in the CNS was significantly different between models of persistent pain, and there was a significant correlation between CNS and peripheral blood Fkbp5 DNAm, indicating that further exploration of Fkbp5 promoter DNAm as an indicator of chronic pain pathogenic origin is warranted. We also found that maternal separation, which promotes the persistency of inflammatory pain in adulthood, was accompanied by long-lasting reduction in Fkbp5 DNAm, suggesting that Fkbp5 DNAm profile may indicate the increased vulnerability to chronic pain in individuals exposed to trauma in early life., Conclusions: Overall, our data demonstrate that the Fkbp5 promoter DNAm landscape brings novel insight into the differing pathogenic origins of chronic pain, may be able to stratify patients and predict the susceptibility to chronic pain., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production., (© 2023. The Author(s).)

Background: Hyperlinear palms are described as a feature of loss-of-function (LoF) variants in filaggrin (FLG)., Objectives: To explore the phenotype of participants (age < 31 years) with atopic eczema of Bangladeshi ancestry from East London and investigate which factors best associate with LoF FLG variants., Methods: A cross-sectional study with participants recruited between May 2018 and December 2020. Patterns of palmar linearity were categorized and modelled with the Eczema Area and Severity Index (EASI), transepidermal water loss (TEWL), skin hydration (SH) and LoF FLG variants., Results: There were 506 complete cases available. Five palm patterns were noted. The 'prominent diamond' pattern associated best with EASI [marginal effects (ME) 2.53, 95% confidence interval (CI) 1.74-3.67], SH (ME 0.85, 95% CI 0.78-0.96) and TEWL (ME 1.32, 95% CI 1.11-1.62). Using five palm patterns had some ability to discriminate LoF FLG variants [area under the receiver operator characteristic (AUROC) 76.32%, 95% CI 71.91-80.73], improving to 77.99% (73.70-82.28) with the addition of SH. In subgroup analysis with only fine perpendicular/prominent diamond patterns the AUROC was 89.11% (95% CI 84.02-94.19)., Conclusions: This was a single-centre study design with humans classifying clinical patterns. The stability of temperature and humidity was not guaranteed across TEWL and SH measurements despite using a climate-controlled room. Palm patterns associate with EASI and TEWL. The fine perpendicular/prominent diamond patterns are markers to detect the absence/presence of LoF FLG variants, respectively., Competing Interests: Conflicts of interest E.A.O’T. has received research funding paid to the university from Kamari Pharma and Unilever, consulting fees paid to the university from Azitra Inc., Kamari Pharma, Palvella Therapeutics and Timber Pharma unrelated to this work. All other authors have no conflicts of interest to declare., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Purpose: Genetic studies of multiple sclerosis (MS) susceptibility and severity have focused on populations of European ancestry. Studying MS genetics in other ancestral groups is necessary to determine the generalisability of these findings. The genetic Association study in individuals from Diverse Ancestral backgrounds with Multiple Sclerosis (ADAMS) project aims to gather genetic and phenotypic data on a large cohort of ancestrally-diverse individuals with MS living in the UK., Participants: Adults with self-reported MS from diverse ancestral backgrounds. Recruitment is via clinical sites, online (https://app.mantal.co.uk/adams) or the UK MS Register. We are collecting demographic and phenotypic data using a baseline questionnaire and subsequent healthcare record linkage. We are collecting DNA from participants using saliva kits (Oragene-600) and genotyping using the Illumina Global Screening Array V.3., Findings to Date: As of 3 January 2023, we have recruited 682 participants (n=446 online, n=55 via sites, n=181 via the UK MS Register). Of this initial cohort, 71.2% of participants are female, with a median age of 44.9 years at recruitment. Over 60% of the cohort are non-white British, with 23.5% identifying as Asian or Asian British, 16.2% as Black, African, Caribbean or Black British and 20.9% identifying as having mixed or other backgrounds. The median age at first symptom is 28 years, and median age at diagnosis is 32 years. 76.8% have relapsing-remitting MS, and 13.5% have secondary progressive MS., Future Plans: Recruitment will continue over the next 10 years. Genotyping and genetic data quality control are ongoing. Within the next 3 years, we aim to perform initial genetic analyses of susceptibility and severity with a view to replicating the findings from European-ancestry studies. In the long term, genetic data will be combined with other datasets to further cross-ancestry genetic discoveries., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Primary aldosteronism (PA) due to a unilateral aldosterone-producing adenoma is a common cause of hypertension. This can be cured, or greatly improved, by adrenal surgery. However, the invasive nature of the standard pre-surgical investigation contributes to fewer than 1% of patients with PA being offered the chance of a cure. The primary objective of our prospective study of 143 patients with PA ( NCT02945904 ) was to compare the accuracy of a non-invasive test, [ 11 C]metomidate positron emission tomography computed tomography (MTO) scanning, with adrenal vein sampling (AVS) in predicting the biochemical remission of PA and the resolution of hypertension after surgery. A total of 128 patients reached 6- to 9-month follow-up, with 78 (61%) treated surgically and 50 (39%) managed medically. Of the 78 patients receiving surgery, 77 achieved one or more PA surgical outcome criterion for success. The accuracies of MTO at predicting biochemical and clinical success following adrenalectomy were, respectively, 72.7 and 65.4%. For AVS, the accuracies were 63.6 and 61.5%. MTO was not significantly superior, but the differences of 9.1% (95% confidence interval = -6.5 to 24.1%) and 3.8% (95% confidence interval = -11.9 to 9.4) lay within the pre-specified -17% margin for non-inferiority (P = 0.00055 and P = 0.0077, respectively). Of 24 serious adverse events, none was considered related to either investigation and 22 were fully resolved. MTO enables non-invasive diagnosis of unilateral PA., (© 2022. The Author(s).)

The potential health consequences of glyphosate-induced gut microbiome alterations have become a matter of intense debate. As part of a multifaceted study investigating toxicity, carcinogenicity and multigenerational effects of glyphosate and its commercial herbicide formulations, we assessed changes in bacterial and fungal populations in the caecum microbiota of rats exposed prenatally until adulthood (13 weeks after weaning) to three doses of glyphosate (0.5, 5, 50 mg/kg body weight/day), or to the formulated herbicide products Roundup Bioflow and RangerPro at the same glyphosate-equivalent doses. Caecum bacterial microbiota were evaluated by 16S rRNA sequencing whilst the fungal population was determined by ITS2 amplicon sequencing. Results showed that both fungal and bacterial diversity were affected by the Roundup formulations in a dose-dependent manner, whilst glyphosate alone significantly altered only bacterial diversity. At taxa level, a reduction in Bacteroidota abundance, marked by alterations in the levels of Alloprevotella, Prevotella and Prevotellaceae UCG-003 , was concomitant to increased levels of Firmicutes (e.g., Romboutsia , Dubosiella , Eubacterium brachy group or Christensenellaceae ) and Actinobacteria (e.g., Enterorhabdus, Adlercreutzia , or Asaccharobacter ) . Treponema and Mycoplasma also had their levels reduced by the pesticide treatments. Analysis of fungal composition indicated that the abundance of the rat gut commensal Ascomycota Kazachstania was reduced while the abundance of Gibberella, Penicillium, Claviceps, Cornuvesica, Candida, Trichoderma and Sarocladium were increased by exposure to the Roundup formulations, but not to glyphosate. Altogether, our data suggest that glyphosate and its Roundup RangerPro and Bioflow caused profound changes in caecum microbiome composition by affecting the fitness of major commensals, which in turn reduced competition and allowed opportunistic fungi to grow in the gut, in particular in animals exposed to the herbicide formulations. This further indicates that changes in gut microbiome composition might influence the long-term toxicity, carcinogenicity and multigenerational effects of glyphosate-based herbicides., Competing Interests: RM has served as a consultant on glyphosate risk assessment issues as part of litigation in the US over glyphosate health effects. MP has provided expert consultation in legal proceedings related to occupational and environmental health issues including the health effects of pesticide exposures and exposure to COVID19. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mesnage, Panzacchi, Bourne, Mein, Perry, Hu, Chen, Mandrioli, Belpoggi and Antoniou.)

Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remain controversial. As GBHs are more cytotoxic than glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (United Kingdom), and MON 76207 (United States) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased miR-30, whereas miR-10 levels were increased. DNA methylation profiling of liver revealed 5727 and 4496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals, respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology.)

Small RNAs have emerged as a promising new type of biomarker to monitor health status and track the development of diseases. Here we report changes in the levels of small RNAs in the liver of rats exposed to a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole). Multivariate analysis with OPLS-DA methods showed that small RNA profiles can discriminate samples from pesticide treated rats from their concurrent controls. A total of 9 miRNAs were found to have their levels altered in the liver of the pesticide-treated rats in comparison to the controls, which included 7 that were downregulated (miR-22-5p, miR-193a-3p, miR-32-5p, miR-33-5p, miR-122-5p, miR-22-3p, miR-130a-3p) and 2 that were upregulated (miR-486-5p, miR-146a-5p). These miRNAs were predicted to regulate genes, which were found to have their expression altered by the pesticide mixture and have known health implications in the regulation of hepatic metabolism. This supports and extends our recent conclusions that high- throughput 'omics' analyses can reveal molecular perturbations, which can potentially act as sensitive and accurate markers of health risks arising from exposure to environmental pollutants such as pesticides., Competing Interests: Declaration of Competing Interest RM has served as a consultant on glyphosate risk assessment issues as part of litigation in the US over glyphosate health effects. The other authors declare no competing interests., (Copyright © 2021. Published by Elsevier B.V.)

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Betel-nut consumption is the fourth most common addictive habit globally and there is good evidence linking the habit to obesity, type 2 diabetes (T2D) and the metabolic syndrome. The aim of our pilot study was to identify gene expression relevant to obesity, T2D and the metabolic syndrome using a genome-wide transcriptomic approach in a human monocyte cell line incubated with arecoline and its nitrosated products., Results: The THP1 monocyte cell line was incubated separately with arecoline and 3-methylnitrosaminopropionaldehyde (MNPA) in triplicate for 24 h and pooled cDNA indexed paired-end libraries were sequenced (Illumina NextSeq 500). After incubation with arecoline and MNPA, 15 and 39 genes respectively had significant changes in their expression (q < 0.05, log fold change 1.5). Eighteen of those genes have reported associations with T2D and obesity in humans; of these genes there was most marked evidence for CLEC10A, MAPK8IP1, NEGR1, NQ01 and INHBE genes., Conclusions: Our preliminary studies have identified a large number of genes relevant to obesity, T2D and metabolic syndrome whose expression was changed significantly in human TPH1 cells following incubation with betel-nut derived arecoline or with MNPA. These findings require validation by further cell-based work and investigation amongst betel-chewing communities., (© 2021. The Author(s).)

The evaluation of bone diagenetic phenomena in archaeological timescales has a long history; however, little is known about the origins of the microbes driving bone diagenesis, nor about the extent of bone diagenesis in short timeframes-such as in forensic contexts. Previously, the analysis of non-collagenous proteins (NCPs) through bottom-up proteomics revealed the presence of potential biomarkers useful in estimating the post-mortem interval (PMI). However, there is still a great need for enhancing the understanding of the diagenetic processes taking place in forensic timeframes, and to clarify whether proteomic analyses can help to develop better models for estimating PMI reliably. To address these knowledge gaps, we designed an experiment based on whole rat carcasses, defleshed long rat bones, and excised but still-fleshed rat limbs, which were either buried in soil or exposed on a clean plastic surface, left to decompose for 28 weeks, and retrieved at different time intervals. This study aimed to assess differences in bone protein relative abundances for the various deposition modalities and intervals. We further evaluated the effects that extrinsic factors, autolysis, and gut and soil bacteria had on bone diagenesis via bottom-up proteomics. Results showed six proteins whose abundance was significantly different between samples subjected to either microbial decomposition (gut or soil bacteria) or to environmental factors. In particular, muscle- and calcium-binding proteins were found to be more prone to degradation by bacterial attack, whereas plasma and bone marrow proteins were more susceptible to exposure to extrinsic agents. Our results suggest that both gut and soil bacteria play key roles in bone diagenesis and protein decay in relatively short timescales, and that bone proteomics is a proficient resource with which to identify microbially-driven versus extrinsically-driven diagenesis.

The epigenome has been shown to deteriorate with age, potentially impacting on ageing-related disease. tRNA, while arising from only ˜46 kb (<0.002% genome), is the second most abundant cellular transcript. tRNAs also control metabolic processes known to affect ageing, through core translational and additional regulatory roles. Here, we interrogate the DNA methylation state of the genomic loci of human tRNA. We identify a genomic enrichment for age-related DNA hypermethylation at tRNA loci. Analysis in 4,350 MeDIP-seq peripheral-blood DNA methylomes (16-82 years), identifies 44 and 21 hypermethylating specific tRNAs at study-and genome-wide significance, respectively, contrasting with none hypomethylating. Validation and replication (450k array and independent targeted Bisuphite-sequencing) supported the hypermethylation of this functional unit. Tissue-specificity is a significant driver, although the strongest consistent signals, also independent of major cell-type change, occur in tRNA-iMet-CAT-1-4 and tRNA-Ser-AGA-2-6. This study presents a comprehensive evaluation of the genomic DNA methylation state of human tRNA genes and reveals a discreet hypermethylation with advancing age.

Health effects of pesticides are not always accurately detected using the current battery of regulatory toxicity tests. We compared standard histopathology and serum biochemistry measures and multi-omics analyses in a subchronic toxicity test of a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) in Sprague-Dawley rats. Analysis of water and feed consumption, body weight, histopathology and serum biochemistry showed little effect. Contrastingly, serum and caecum metabolomics revealed that nicotinamide and tryptophan metabolism were affected, which suggested activation of an oxidative stress response. This was not reflected by gut microbial community composition changes evaluated by shotgun metagenomics. Transcriptomics of the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways. Genome-wide DNA methylation analysis of the same liver samples showed that 4,255 CpG sites were differentially methylated. Overall, we demonstrated that in-depth molecular profiling in laboratory animals exposed to low concentrations of pesticides allows the detection of metabolic perturbations that would remain undetected by standard regulatory biochemical measures and which could thus improve the predictability of health risks from exposure to chemical pollutants.

Aspirin prevents thrombosis by inhibiting platelet cyclooxygenase (COX)-1 activity and the production of thromboxane (Tx)A 2 , a pro-thrombotic eicosanoid. However, the non-platelet actions of aspirin limit its antithrombotic effects. Here, we used platelet-COX-1-ko mice to define the platelet and non-platelet eicosanoids affected by aspirin. Mass-spectrometry analysis demonstrated blood from platelet-COX-1-ko and global-COX-1-ko mice produced similar eicosanoid profiles in vitro: for example, formation of TxA 2 , prostaglandin (PG) F 2α , 11-hydroxyeicosatraenoic acid (HETE), and 15-HETE was absent in both platelet- and global-COX-1-ko mice. Conversely, in vivo, platelet-COX-1-ko mice had a distinctly different profile from global-COX-1-ko or aspirin-treated control mice, notably significantly higher levels of PGI 2 metabolite. Ingenuity Pathway Analysis (IPA) predicted that platelet-COX-1-ko mice would be protected from thrombosis, forming less pro-thrombotic TxA 2 and PGE 2 . Conversely, aspirin or lack of systemic COX-1 activity decreased the synthesis of anti-aggregatory PGI 2 and PGD 2 at non-platelet sites leading to predicted thrombosis increase. In vitro and in vivo thrombosis studies proved these predictions. Overall, we have established the eicosanoid profiles linked to inhibition of COX-1 in platelets and in the remainder of the cardiovascular system and linked them to anti- and pro-thrombotic effects of aspirin. These results explain why increasing aspirin dosage or aspirin addition to other drugs may lessen antithrombotic protection., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Both vitamin D deficiency and single nucleotide polymorphisms (SNPs) in the gene encoding the vitamin D receptor (VDR) have been widely reported to associate with susceptibility to polycystic ovarian syndrome (PCOS). A case-control study was conducted to study the influence of vitamin D status and genotpye for 24 SNPs in four genes in the vitamin D pathway (VDR, DBP, CYP27B1, CYP24A1) on PCOS. Statistical analyses were conducted to identify phenotypic and genotypic factors associated with risk of PCOS and to test for interactions between genotype and vitamin D status. PCOS was independently associated with lower age, higher body mass index, lower waist-hip ratio, vitamin D deficiency (serum 25-hydroxyvitamin D concentration <10 ng/mL), lack of outdoor exercise, increased fasting glucose and a family history of PCOS in at least one first degree relative. No statistically significant association was observed between the genotype of any SNP investigated and risk of PCOS, either as a main effect or in interaction with vitamin D status. We report a strong and independent association between vitamin D deficiency and risk of PCOS in Pakistan, that was not modified by genetic variation in the vitamin D pathway.

Atrial fibrillation is a significant worldwide contributor to cardiovascular morbidity and mortality. Few studies have investigated the differences in gene expression between the left and right atrial appendages, leaving their characterization largely unexplored. In this study, differential gene expression was investigated in atrial fibrillation and sinus rhythm using left and right atrial appendages from the same patients. RNA sequencing was performed on the left and right atrial appendages from five sinus rhythm (SR) control patients and five permanent AF case patients. Differential gene expression in both the left and right atrial appendages was analyzed using the Bioconductor package edgeR. A selection of differentially expressed genes, with relevance to atrial fibrillation, were further validated using quantitative RT-PCR. The distribution of the samples assessed through principal component analysis showed distinct grouping between left and right atrial appendages and between SR controls and AF cases. Overall 157 differentially expressed genes were identified to be downregulated and 90 genes upregulated in AF. Pathway enrichment analysis indicated a greater involvement of left atrial genes in the Wnt signaling pathway whereas right atrial genes were involved in clathrin-coated vesicle and collagen formation. The differing expression of genes in both left and right atrial appendages indicate that there are different mechanisms for development, support and remodeling of AF within the left and right atria.

Exposure to endocrine disrupting chemicals is an established risk factor for obesity. The most commonly used pesticide active ingredients have never been tested in an adipogenesis assay. We tested for the first time the potential of glyphosate, 2, 4-dichlorophenoxyacetic acid, dicamba, mesotrione, isoxaflutole, and quizalofop-p-ethyl (QpE) to induce lipid accumulation in murine 3T3-L1 adipocytes. Only QpE caused a dose-dependent statistically significant triglyceride accumulation from a concentration of 5 up to 100 µM. The QpE commercial formulation Targa Super was 100 times more cytotoxic than QpE alone. Neither the estrogen receptor antagonist ICI 182, 780 nor the glucocorticoid receptor antagonist RU486 was able to block the QpE-induced lipid accumulation. RNAseq analysis of 3T3-L1 adipocytes exposed to QpE suggests that this compound exerts its lipid accumulation effects via a peroxisome proliferator-activated receptor gamma (PPARγ)-mediated pathway, a nuclear receptor whose modulation influences lipid metabolism. QpE was further shown to be active in a PPARγ reporter gene assay at 100 µM, reaching 4% of the maximal response produced by rosiglitazone, which acts as a positive control. This indicates that lipid accumulation induced by QpE is only in part caused by PPARγ activation. The lipid accumulation capability of QpE we observe suggest that this pesticide, whose use is likely to increase in coming years may have a hitherto unsuspected obesogenic property., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology.)

DNA methylation is an important epigenetic modification that can regulate gene expression following environmental encounters without changes to the genetic code. Using Infinium MethylationEPIC BeadChip Arrays (850,000 CpG sites) we analysed for the first time, DNA isolated from untrained human skeletal muscle biopsies (vastus lateralis) at baseline (rest) and immediately following an acute (single) bout of resistance exercise. In the same participants, we also analysed the methylome following a period of muscle growth (hypertrophy) evoked via chronic (repeated bouts-3 sessions/wk) resistance exercise (RE) (training) over 7-weeks, followed by complete exercise cessation for 7-weeks returning muscle back to baseline levels (detraining), and finally followed by a subsequent 7-week period of RE-induced hypertrophy (retraining). These valuable methylome data sets described in the present manuscript and deposited in an open-access repository can now be shared and re-used to enable the identification of epigenetically regulated genes/networks that are modified after acute anabolic stimuli and hypertrophy, and further investigate the phenomenon of epigenetic memory in skeletal muscle.

SNP in the vitamin D receptor (VDR) gene is associated with risk of lower respiratory infections. The influence of genetic variation in the vitamin D pathway resulting in susceptibility to upper respiratory infections (URI) has not been investigated. We evaluated the influence of thirty-three SNP in eleven vitamin D pathway genes (DBP, DHCR7, RXRA, CYP2R1, CYP27B1, CYP24A1, CYP3A4, CYP27A1, LRP2, CUBN and VDR) resulting in URI risk in 725 adults in London, UK, using an additive model with adjustment for potential confounders and correction for multiple comparisons. Significant associations in this cohort were investigated in a validation cohort of 737 children in Manchester, UK. In all, three SNP in VDR (rs4334089, rs11568820 and rs7970314) and one SNP in CYP3A4 (rs2740574) were associated with risk of URI in the discovery cohort after adjusting for potential confounders and correcting for multiple comparisons (adjusted incidence rate ratio per additional minor allele ≥1·15, P for trend ≤0·030). This association was replicated for rs4334089 in the validation cohort (P for trend=0·048) but not for rs11568820, rs7970314 or rs2740574. Carriage of the minor allele of the rs4334089 SNP in VDR was associated with increased susceptibility to URI in children and adult cohorts in the United Kingdom.

Rare heterozygous coding variants in the triggering receptor expressed in myeloid cells 2 (TREM2) gene, conferring increased risk of developing late-onset Alzheimer's disease, have been identified. We examined the transcriptional consequences of the loss of Trem2 in mouse brain to better understand its role in disease using differential expression and coexpression network analysis of Trem2 knockout and wild-type mice. We generated RNA-Seq data from cortex and hippocampus sampled at 4 and 8 months. Using brain cell-type markers and ontology enrichment, we found subnetworks with cell type and/or functional identity. We primarily discovered changes in an endothelial gene-enriched subnetwork at 4 months, including a shift toward a more central role for the amyloid precursor protein gene, coupled with widespread disruption of other cell-type subnetworks, including a subnetwork with neuronal identity. We reveal an unexpected potential role of Trem2 in the homeostasis of endothelial cells that goes beyond its known functions as a microglial receptor and signaling hub, suggesting an underlying link between immune response and vascular disease in dementia., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Use and thus exposure to quizalofop-p-ethyl, isoxaflutole, mesotrione and glyphosate, which are declared as active principles in commercial formulations of herbicides, is predicted to rapidly increase in coming years in an effort to overcome the wide-spread appearance of glyphosate-resistant weeds, especially in fields where glyphosate-tolerant genetically modified crops are cultivated in the USA. Thus, there is an urgent need for an evaluation of metabolic effects of new pesticide ingredients used to replace glyphosate. As the liver is a primary target of chemical pollutant toxicity, we have used the HepaRG human liver cell line as a model system to assess the toxicological insult from quizalofop-p-ethyl, isoxaflutole, mesotrione and glyphosate by determining alterations in the transcriptome caused by exposure to three concentrations of each of these compounds, including a low environmentally relevant dose. RNA-seq data were analysed with HISAT2, StringTie and Ballgown. Quizalofop-p-ethyl was found to be the most toxic of the pesticide ingredients tested, causing alterations in gene expression that are associated with pathways involved in fatty acid degradation and response to alcoholism. Isoxaflutole was less toxic, but caused detectable changes in retinol metabolism and in the PPAR signalling pathway at a concentration of 1 mM. ToxCast data analysis revealed that isoxaflutole activated PPAR gamma receptor and pregnane X responsive elements in reporter gene assays. Glyphosate and mesotrione caused subtle changes in transcriptome profiles, with too few genes altered in their function to allow a reliable pathway analysis. In order to explore the effects of glyphosate in greater depth and detail, we undertook a global metabolome profiling. This revealed a decrease in free long chain fatty acids and polyunsaturated fatty acid levels at the lowest concentration (0.06 μM) of glyphosate, although no effects were detected at the two higher concentrations tested, perhaps suggesting a non-linear dose response. This surprising result will need to be confirmed by additional studies. Overall, our findings contribute to filling the knowledge gap regarding metabolic toxicity that can potentially arise from exposure to these four herbicide active principles.

Chemical pollutant exposure is a risk factor contributing to the growing epidemic of non-alcoholic fatty liver disease (NAFLD) affecting human populations that consume a western diet. Although it is recognized that intoxication by chemical pollutants can lead to NAFLD, there is limited information available regarding the mechanism by which typical environmental levels of exposure can contribute to the onset of this disease. Here, we describe the alterations in gene expression profiles and metabolite levels in the human HepaRG liver cell line, a validated model for cellular steatosis, exposed to the polychlorinated biphenyl (PCB) 126, one of the most potent chemical pollutants that can induce NAFLD. Sparse partial least squares classification of the molecular profiles revealed that exposure to PCB 126 provoked a decrease in polyunsaturated fatty acids as well as an increase in sphingolipid levels, concomitant with a decrease in the activity of genes involved in lipid metabolism. This was associated with an increased oxidative stress reflected by marked disturbances in taurine metabolism. A gene ontology analysis showed hallmarks of an activation of the AhR receptor by dioxin-like compounds. These changes in metabolome and transcriptome profiles were observed even at the lowest concentration (100 pM) of PCB 126 tested. A decrease in docosatrienoate levels was the most sensitive biomarker. Overall, our integrated multi-omics analysis provides mechanistic insight into how this class of chemical pollutant can cause NAFLD. Our study lays the foundation for the development of molecular signatures of toxic effects of chemicals causing fatty liver diseases to move away from a chemical risk assessment based on in vivo animal experiments.

Secondary sexual trait expression can be influenced by fixed individual factors (such as genetic quality) as well as by dynamic factors (such as age and environmentally induced gene expression) that may be associated with variation in condition or quality. In particular, melanin-based traits are known to relate to condition and there is a well-characterized genetic pathway underpinning their expression. However, the mechanisms linking variable trait expression to genetic quality remain unclear. One plausible mechanism is that genetic quality could influence trait expression via differential methylation and differential gene expression. We therefore conducted a pilot study examining DNA methylation at a candidate gene (agouti-related neuropeptide: AgRP ) in the black grouse Lyrurus tetrix . We specifically tested whether CpG methylation covaries with age and multilocus heterozygosity (a proxy of genetic quality) and from there whether the expression of a melanin-based ornament (ultraviolet-blue chroma) correlates with DNA methylation. Consistent with expectations, we found clear evidence for age- and heterozygosity-specific patterns of DNA methylation, with two CpG sites showing the greatest DNA methylation in highly heterozygous males at their peak age of reproduction. Furthermore, DNA methylation at three CpG sites was significantly positively correlated with ultraviolet-blue chroma. Ours is the first study to our knowledge to document age- and quality-dependent variation in DNA methylation and to show that dynamic sexual trait expression across the lifespan of an organism is associated with patterns of DNA methylation. Although we cannot demonstrate causality, our work provides empirical support for a mechanism that could potentially link key individual factors to variation in sexual trait expression in a wild vertebrate.

We recently demonstrated that the major effector function of neonatal CD4 + T cells is to produce CXCL8, a prototypic cytokine of innate immune cells. In this article, we show that CXCL8 expression, prior to proliferation, is common in newly arising T cells (so-called "recent thymic emigrants") in adults, as well as in babies. This effector potential is acquired in the human thymus, prior to TCR signaling, but rather than describing end-stage differentiation, such cells, whether isolated from neonates or adults, can further differentiate into IFN-γ-producing CD4 + T cells. Thus, the temporal transition of host defense from innate to adaptive immunity is unexpectedly mirrored at the cellular level by the capacity of human innate-like CXCL8-producing CD4 + T cells to transition directly into Th1 cells., (Copyright © 2017 The Authors.)

High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P<0.05), and similarly significant increases in mean levels of methylation in high-grade tumors for the ATP5G2, VAX2, INSRR, PRDM14, VSX1, TFAP2b, PRRX1, and HIST1H4F genes (P<0.05). Although inappropriate promoter methylation was not invariantly associated with reduced transcript expression, a significant association was apparent for the ARHGEF4, PON3, STAT5a, and VAX2 gene transcripts (P<0.05). Herein, we present the first genome-wide DNA methylation analysis in a unique HG-NMIBC cohort, showing extensive and discrete methylation changes relative to normal bladder and low-intermediate-grade tumors. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination, with more conventional therapeutic options in the treatment of this clinically unpredictable disease.

Rationale: Asthma exacerbations are commonly precipitated by viral upper respiratory infections (URIs). Vitamin D insufficiency associates with susceptibility to URI in patients with asthma. Trials of vitamin D in adults with asthma with incidence of exacerbation and URI as primary outcome are lacking., Objective: To conduct a randomised controlled trial of vitamin D3 supplementation for the prevention of asthma exacerbation and URI (coprimary outcomes)., Measurements and Methods: 250 adults with asthma in London, UK were allocated to receive six 2-monthly oral doses of 3 mg vitamin D3 (n=125) or placebo (n=125) over 1 year. Secondary outcomes included asthma control test and St George's Respiratory Questionnaire scores, fractional exhaled nitric oxide and concentrations of inflammatory markers in induced sputum. Subgroup analyses were performed to determine whether effects of supplementation were modified by baseline vitamin D status or genotype for 34 single nucleotide polymorphisms in 11 vitamin D pathway genes., Main Results: 206/250 participants (82%) were vitamin D insufficient at baseline. Vitamin D3 did not influence time to first severe exacerbation (adjusted HR 1.02, 95% CI 0.69 to 1.53, p=0.91) or first URI (adjusted HR 0.87, 95% CI 0.64 to 1.16, p=0.34). No clinically important effect of vitamin D3 was seen on any of the secondary outcomes listed above. The influence of vitamin D3 on coprimary outcomes was not modified by baseline vitamin D status or genotype., Conclusions: Bolus-dose vitamin D3 supplementation did not influence time to exacerbation or URI in a population of adults with asthma with a high prevalence of baseline vitamin D insufficiency., Trial Registration Number: NCT00978315 (ClinicalTrials.gov)., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: The enzyme guanosine triphosphate-cyclohydrolase-1 (GCH-1) is a rate limiting step in the de novo synthesis of tetrahydrobiopterin (BH4) a co-factor in monoamine synthesis and nitric oxide production. GCH-1 is strongly implicated in chronic pain based on data generated using the selective GCH-1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP), and studies which have identified a pain protective GCH-1 haplotype associated with lower BH4 production and reduced pain., Methods: To investigate the role for GCH-1 in visceral pain we examined the effects of DAHP on pain behaviors elicited by colorectal injection of mustard oil in rats, and the pain protective GCH-1 haplotype in healthy volunteers characterized by esophageal pain sensitivity before and after acid injury, and assessed using depression and anxiety questionnaires., Key Results: In rodents pretreatment with DAHP produced a substantial dose related inhibition of pain behaviors from 10 to 180 mg/kg i.p. (p < 0.01 to 0.001). In healthy volunteers, no association was seen between the pain protective GCH-1 haplotype and the development of hypersensitivity following injury. However, a substantial increase in baseline pain thresholds was seen between first and second visits (26.6 ± 6.2 mA) in subjects who sensitized to esophageal injury and possessed the pain protective GCH-1 haplotype compared with all other groups (p < 0.05). Furthermore the same subjects who sensitized to acid and possessed the haplotype, also had significantly lower depression scores (p < 0.05)., Conclusions & Inferences: The data generated indicate that GCH-1 plays a role in visceral pain processing that requires more detailed investigation., (© 2015 John Wiley & Sons Ltd.)

Objective: To explore the hypothesis that blood transfusion contributes to an immunosuppressed phenotype in severely injured patients., Background: Despite trauma patients using disproportionately large quantities of blood and blood products, the immunomodulatory effects of blood transfusion in this group are inadequately described., Methods: A total of 112 ventilated polytrauma patients were recruited. Messenger RNA (mRNA) was extracted from PAXGene tubes collected within 2 hours of the trauma, at 24 hours, and at 72 hours. T-helper cell subtype specific cytokines and transcription factors were quantified using real-time polymerase chain reaction., Results: Median injury severity score was 29. Blood transfusion was administered to 27 (24%) patients before the 2-hour sampling point. Transfusion was associated with a greater immediate rise in IL-10 (P = 0.003) and IL-27 (P = 0.04) mRNA levels. Blood products were transfused in 72 (64%) patients within the first 24 hours. There was an association between transfusion at 24 hours and higher IL-10 (P < 0.0001), lower Foxp3 (P = 0.01), GATA3 (P = 0.006), and RORγt (P = 0.05) mRNA levels at 24 hours. There were greater reductions in T-bet (P = 0.03) mRNA levels and lesser increases in TNFα (P = 0.015) and IFNγ (P = 0.035) at 24 hours in those transfused. Multiple regression models confirmed that the transfusion of blood products was independently associated with altered patterns of gene expression. Blood stream infections occur in 15 (20.8%) of those transfused in the first 24 hours, compared with 1 patient (2.5%) not transfused (OR = 10.3 [1.3-81], P = 0.008)., Conclusions: The primarily immunosuppressive inflammatory response to polytrauma may be exacerbated by the transfusion of blood products. Furthermore, transfusion was associated with an increased susceptibility to nosocomial infections.

MIR137, transcribed as the microRNA miR-137, is one of the leading candidate schizophrenia susceptibility genes to arise from large genome-wide association studies (GWAS) of the disorder. Recent data suggest that miR-137 modulates the expression of other schizophrenia susceptibility genes. Although bioinformatic resources are available with which to predict genes regulated by individual microRNA, there has been a lack of empirical data on genome-wide gene expression changes following miR-137 manipulation. We have therefore performed a genome-wide assessment of transcriptional changes in a human neural progenitor cell line after miR-137 over-expression and inhibition in order to elucidate molecular pathways by which genetic perturbation of miR-137 could promote susceptibility to schizophrenia. Bioinformatically-predicted miR-137 targets showed a small but highly significant down-regulation following miR-137 over-expression. Genes that were significantly down-regulated in association with miR-137 over-expression were enriched for involvement in neuronal differentiation. Differentially expressed genes that were confirmed by qPCR included others at genome-wide significant risk loci for schizophrenia (MAD1L1 and DPYD) and BDNF. These data point to molecular pathways through which genetic variation at the MIR137 locus could confer risk for schizophrenia., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Affinity capture of DNA methylation combined with high-throughput sequencing strikes a good balance between the high cost of whole genome bisulfite sequencing and the low coverage of methylation arrays. We present BayMeth, an empirical Bayes approach that uses a fully methylated control sample to transform observed read counts into regional methylation levels. In our model, inefficient capture can readily be distinguished from low methylation levels. BayMeth improves on existing methods, allows explicit modeling of copy number variation, and offers computationally efficient analytical mean and variance estimators. BayMeth is available in the Repitools Bioconductor package.

The purpose of this study was the generation of central nervous system (CNS)-excluded cannabinoid receptor agonists to test the hypothesis that inhibition of spasticity, due to CNS autoimmunity, could be controlled by affecting neurotransmission within the periphery. Procedures included identification of chemicals and modeling to predict the mode of exclusion; induction and control of spasticity in the ABH mouse model of multiple sclerosis; conditional deletion of CB1 receptor in peripheral nerves; side-effect profiling to demonstrate the mechanism of CNS-exclusion via drug pumps; genome-wide association study in N2(129×ABH) backcross to map polymorphic cannabinoid drug pump; and sequencing and detection of cannabinoid drug-pump activity in human brain endothelial cell lines. Three drugs (CT3, SAB378 and SAD448) were identified that control spasticity via action on the peripheral nerve CB1 receptor. These were peripherally restricted via drug pumps that limit the CNS side effects (hypothermia) of cannabinoids to increase the therapeutic window. A cannabinoid drug pump is polymorphic and functionally lacking in many laboratory (C57BL/6, 129, CD-1) mice used for transgenesis, pharmacology, and toxicology studies. This phenotype was mapped and controlled by 1-3 genetic loci. ABCC1 within a cluster showing linkage is a cannabinoid CNS-drug pump. Global and conditional CB1 receptor-knockout mice were used as controls. In summary, CNS-excluded CB1 receptor agonists are a novel class of therapeutic agent for spasticity.

Tensin3 is a cytoskeletal regulatory protein that inhibits cell motility. Downregulation of the gene encoding Tensin3 (TNS3) in human renal cell carcinoma (RCC) may contribute to cancer cell metastatic behavior. We speculated that epigenetic mechanisms, e.g., gene promoter hypermethylation, might account for TNS3 downregulation. In this study, we identified and validated a TNS3 gene promoter containing a CpG island, and quantified the methylation level within this region in RCC. Using a luciferase reporter assay we demonstrated a functional minimal promoter activity for a 500-bp sequence within the TNS3 CpG island. Pyrosequencing enabled quantitative determination of DNA methylation of each CpG dinucleotide (a total of 43) in the TNS3 gene promoter. Across the entire analyzed CpG stretch, RCC DNA showed a higher methylation level than both non-tumor kidney DNA and normal control DNA. Out of all the CpGs analyzed, two CpG dinucleotides, specifically position 2 and 8, showed the most pronounced increases in methylation levels in tumor samples. Furthermore, CpG-specific higher methylation levels were correlated with lower TNS3 gene expression levels in RCC samples. In addition, pharmacological demethylation treatment of cultured kidney cells caused a 3-fold upregulation of Tensin3 expression. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter occurring in human RCC, suggesting an epigenetic mechanism for aberrant Tensin downregulation in human kidney cancer.

Genome-wide association studies (GWAS) have identified common variants of modest-effect size at hundreds of loci for common autoimmune diseases; however, a substantial fraction of heritability remains unexplained, to which rare variants may contribute. To discover rare variants and test them for association with a phenotype, most studies re-sequence a small initial sample size and then genotype the discovered variants in a larger sample set. This approach fails to analyse a large fraction of the rare variants present in the entire sample set. Here we perform simultaneous amplicon-sequencing-based variant discovery and genotyping for coding exons of 25 GWAS risk genes in 41,911 UK residents of white European origin, comprising 24,892 subjects with six autoimmune disease phenotypes and 17,019 controls, and show that rare coding-region variants at known loci have a negligible role in common autoimmune disease susceptibility. These results do not support the rare-variant synthetic genome-wide-association hypothesis (in which unobserved rare causal variants lead to association detected at common tag variants). Many known autoimmune disease risk loci contain multiple, independently associated, common and low-frequency variants, and so genes at these loci are a priori stronger candidates for harbouring rare coding-region variants than other genes. Our data indicate that the missing heritability for common autoimmune diseases may not be attributable to the rare coding-region variant portion of the allelic spectrum, but perhaps, as others have proposed, may be a result of many common-variant loci of weak effect.