The role of GPR65 and GALC in Multiple Sclerosis: Establishing the functional consequences of an MS associated variant on chromosome 14q31.3

Genome-wide association studies have identified an extensive catalogue of unequivocally associated genetic variants, each of which provides a clue to the aetiology of multiple sclerosis (MS). Unfortunately, for the vast majority of these quite how the genetic change contributes to the development of the disease is unknown. However, preliminary work undertaken by one of my predecessors suggested that the functionally relevant variant underlying the association identified on chromosome 14q31.3 might exert its effect by influencing T cell activation in a pH dependent manner. From a genetics perspective the ImmunoChip study showed that the lead most strongly associated single nucleotide polymorphism (SNP) tagging this association was rs74796499, which lies in a region of genome containing two protein coding genes; the lysosomal enzyme galactosylceramidase (GALC) and acid sensing surface receptor G-protein coupled receptor 65 (GPR65). In my thesis I attempted to validate and replicate this finding, and also to explore the possibility of other pH dependent effects of this SNP. In order to undertake this work, I first developed a system capable of maintain cultured cells at a stable pH for prolonged periods. Using this system I was able to show that the activation of human lymphocytes to stimulation with anti-CD3/CD28 antibodies is maximal at neutral pH and reduced in both acidic and alkaline conditions. Unfortunately, I was unable to replicate the provisional finding that carrying the protective allele at rs74796499 resulted in greater inhibition of CD25 expression in acidic conditions. However, I did observe nominally significant evidence that the expression of the early activation marker CD69 was induced by culturing cells in unstimulated but acidic conditions, and that this expression was highest in individuals carrying the protective allele at rs74796499. In my first attempt to validate and replicate this new finding I not only found further evidence to support this effect on CD69 in unstimulated cells but also found nominally significant evidence that the expression of lactosylceramide (LacCer, CD17) was lower in ex-vivo cells from individuals carrying the protective allele at rs74796499. Unfortunately, in my final confirmatory experiments I was unable to replicate either of these effects. In summary despite carefully studying over 160 healthy subjects, I was unable to find any statistically significant evidence that rs74796499 genotype exerts any effects the expression of either CD25, CD69 or LacCer in ex-vivo cells or stimulated and unstimulated cells cultured under a range of pH conditions

Transcript specific regulation of expression influences susceptibility to multiple sclerosis.

Genome-wide association studies (GWAS) have identified over 100 loci containing single nucleotide variants (SNVs) that influence the risk of developing multiple sclerosis (MS). Most of these loci lie in non-coding regulatory regions of the genome that are active in immune cells and are therefore thought to modify risk by altering the expression of key immune genes. To explore this hypothesis we screened genes flanking MS-associated variants for evidence of allele specific expression (ASE) by quantifying the transcription of coding variants in linkage disequilibrium with MS-associated SNVs. In total, we were able to identify and successfully analyse 200 such coding variants (from 112 genes) in both CD4+ and CD8+ T cells from 106 MS patients and 105 controls. Fifty-six of these coding variants (from 43 genes) showed statistically significant evidence of ASE in one or both cell types. In the Lck interacting transmembrane adaptor 1 gene (LIME1), for example, we were able to show that in both cell types, the MS-associated variant rs2256814 increased the expression of some transcripts while simultaneously reducing the expression of other transcripts. In CD4+ cells from an additional independent set of 96 cases and 93 controls we were able to replicate the effect of this SNV on the balance of alternate LIME1 transcripts using qPCR (p = 5 × 10-24). Our data thus indicate that some of the MS-associated SNVs identified by GWAS likely exert their effects on risk by distorting the balance of alternate transcripts rather than by changing the overall level of gene expression

Multiple sclerosis risk variants alter expression of co-stimulatory genes in B cells.

The increasing evidence supporting a role for B cells in the pathogenesis of multiple sclerosis prompted us to investigate the influence of known susceptibility variants on the surface expression of co-stimulatory molecules in these cells. Using flow cytometry we measured surface expression of CD40 and CD86 in B cells from 68 patients and 162 healthy controls that were genotyped for the multiple sclerosis associated single nucleotide polymorphisms (SNPs) rs4810485, which maps within the CD40 gene, and rs9282641, which maps within the CD86 gene. We found that carrying the risk allele rs4810485*T lowered the cell-surface expression of CD40 in all tested B cell subtypes (in total B cells P ≤ 5.10 × 10-5 in patients and ≤4.09 × 10-6 in controls), while carrying the risk allele rs9282641*G increased the expression of CD86, with this effect primarily seen in the naïve B cell subset (P = 0.048 in patients and 5.38 × 10-5 in controls). In concordance with these results, analysis of RNA expression demonstrated that the risk allele rs4810485*T resulted in lower total CD40 expression (P = 0.057) but with an increased proportion of alternative splice-forms leading to decoy receptors (P = 4.00 × 10-7). Finally, we also observed that the risk allele rs4810485*T was associated with decreased levels of interleukin-10 (P = 0.020), which is considered to have an immunoregulatory function downstream of CD40. Given the importance of these co-stimulatory molecules in determining the immune reaction that appears in response to antigen our data suggest that B cells might have an important antigen presentation and immunoregulatory role in the pathogenesis of multiple sclerosis

Estimating the Reproducibility of Experimental Philosophy

Responding to recent concerns about the reliability of the published literature in psychology and other disciplines, we formed the X-Phi Replicability Project (XRP) to estimate the reproducibility of experimental philosophy (osf.io/dvkpr). Drawing on a representative sample of 40 x-phi studies published between 2003 and 2015, we enlisted 20 research teams across 8 countries to conduct a high-quality replication of each study in order to compare the results to the original published findings. We found that x-phi studies – as represented in our sample – successfully replicated about 70% of the time. We discuss possible reasons for this relatively high replication rate in the field of experimental philosophy and offer suggestions for best research practices going forward

Acetylome and Succinylome Profiling of <i>Edwardsiella tarda</i> Reveals Key Roles of Both Lysine Acylations in Bacterial Antibiotic Resistance

The antibiotic resistance of Edwardsiella tarda is becoming increasingly prevalent, and thus novel antimicrobial strategies are being sought. Lysine acylation has been demonstrated to play an important role in bacterial physiological functions, while its role in bacterial antibiotic resistance remains largely unclear. In this study, we investigated the lysine acetylation and succinylation profiles of E. tarda strain EIB202 using affinity antibody purification combined with LC-MS/MS. A total of 1511 lysine-acetylation sites were identified on 589 proteins, and 2346 lysine-succinylation sites were further identified on 692 proteins of this pathogen. Further bioinformatic analysis showed that both post-translational modifications (PTMs) were enriched in the tricarboxylic acid (TCA) cycle, pyruvate metabolism, biosynthesis, and carbon metabolism. In addition, 948 peptides of 437 proteins had overlapping associations with multiple metabolic pathways. Moreover, both acetylation and succinylation were found in many antimicrobial resistance (AMR) proteins, suggesting their potentially vital roles in antibiotic resistance. In general, our work provides insights into the acetylome and succinylome features responsible for the antibiotic resistance mechanism of E. tarda, and the results may facilitate future investigations into the pathogenesis of this bacterium

Seagrass meadows provide multiple benefits to adjacent coral reefs through various microhabitat functions

Tropical seagrass meadows and coral reefs often function as interconnected marine habitats, but they are often studied and managed as homogenous units. As macrohabitats, seagrass meadows provide important benefits to adjacent reef ecosystems by acting as natural filters of sediments and nutrients, and by providing critical feeding, nursery, and refuge habitats for reef fishes and other fauna. Whilst the macrohabitat functions of seagrass meadows have been often acknowledged, their microhabitats functions have largely been neglected. The purpose of the study is to explore how seagrass meadows provide multiple benefits to adjacent coral reefs through various microhabitat functions. The paper reveals some of the diversity of microhabitats that seagrass meadows contain, such as macroalgal mats, rubble cavities, sand patches with sparse seagrass, anemone gardens, hard substratum, and sponges mixed with seagrass. We highlight the ways in which reef creatures have diversified and specialized in using these different microhabitats, and postulate that seagrass microhabitat diversity enhances the habitat function and faunal diversity of seagrass meadows