MS in South Asians in England: early disease onset and novel pattern of myelin autoimmunity.

BACKGROUND: Epidemiological studies describe a latitude gradient for increased MS prevalence and a preponderance of disease in Caucasian individuals. However, individuals from other ethnic backgrounds and low-risk regions can acquire a raised risk through migration. Nearly a fifth of the London population is of Asian/Asian-British origin and a significant proportion of referrals are from this group. METHODS: We investigated whether there were differences in timing, presentation, severity, and immunology of disease (with respect to CD4 myelin epitope recognition) between individuals in London with MS who were either of S. Asian or Caucasian origin. Individuals of S. Asian origin with MS were compared with healthy S. Asian controls, individuals with MS and of Caucasian origin and Caucasian controls. RESULTS: Age at MS onset is significantly lower in the S. Asian group, attributable to earlier onset specifically in UK-born individuals, though clinical presentation is similar. Analysis of CD4 autoimmunity to myelin antigens shows disease in S. Asian individuals to encompass recognition of novel epitopes; immunity to MBP116-130 in S. Asian individuals was highly disease-specific. CONCLUSIONS: These findings emphasize the need to define disease profiles across ethnicities and identify environmental triggers conferring acquired risk. Such findings must inform choices for immunotherapeutic interventions suitable for all, across ethnicities

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes

Azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis

<p>Abstract</p> <p>Background</p> <p>Viral bronchiolitis is the leading cause of hospitalization in young infants. It is associated with the development of childhood asthma and contributes to morbidity and mortality in the elderly. Currently no therapies effectively attenuate inflammation during the acute viral infection, or prevent the risk of post-viral asthma. We hypothesized that early treatment of a paramyxoviral bronchiolitis with azithromycin would attenuate acute and chronic airway inflammation.</p> <p>Methods</p> <p>Mice were inoculated with parainfluenza type 1, Sendai Virus (SeV), and treated daily with PBS or azithromycin for 7 days post-inoculation. On day 8 and 21 we assessed airway inflammation in lung tissue, and quantified immune cells and inflammatory mediators in bronchoalveolar lavage (BAL).</p> <p>Results</p> <p>Compared to treatment with PBS, azithromycin significantly attenuated post-viral weight loss. During the peak of acute inflammation (day 8), azithromycin decreased total leukocyte accumulation in the lung tissue and BAL, with the largest fold-reduction in BAL neutrophils. This decreased inflammation was independent of changes in viral load. Azithromycin significantly attenuated the concentration of BAL inflammatory mediators and enhanced resolution of chronic airway inflammation evident by decreased BAL inflammatory mediators on day 21.</p> <p>Conclusions</p> <p>In this mouse model of paramyxoviral bronchiolitis, azithromycin attenuated acute and chronic airway inflammation. These findings demonstrate anti-inflammatory effects of azithromycin that are not related to anti-viral activity. Our findings support the rationale for future prospective randomized clinical trials that will evaluate the effects of macrolides on acute viral bronchiolitis and their long-term consequences.</p

Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome.

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation

Next-generation sequencing: A challenge to meet the increasing demand for training workshops in Australia

The widespread adoption of high-throughput next-generation sequencing (NGS) technology among the Australian life science research community is highlighting an urgent need to up-skill biologists in tools required for handling and analysing their NGS data. There is currently a shortage of cutting-edge bioinformatics training courses in Australia as a consequence of a scarcity of skilled trainers with time and funding to develop and deliver training courses. To address this, a consortium of Australian research organizations, including Bioplatforms Australia, the Commonwealth Scientific and Industrial Research Organisation and the Australian Bioinformatics Network, have been collaborating with EMBL-EBI training team. A group of Australian bioinformaticians attended the train-the-trainer workshop to improve training skills in developing and delivering bioinformatics workshop curriculum. A 2-day NGS workshop was jointly developed to provide hands-on knowledge and understanding of typical NGS data analysis workflows. The road show–style workshop was successfully delivered at five geographically distant venues in Australia using the newly established Australian NeCTAR Research Cloud. We highlight the challenges we had to overcome at different stages from design to delivery, including the establishment of an Australian bioinformatics training network and the computing infrastructure and resource development. A virtual machine image, workshop materials and scripts for configuring a machine with workshop contents have all been made available under a Creative Commons Attribution 3.0 Unported License. This means participants continue to have convenient access to an environment they had become familiar and bioinformatics trainers are able to access and reuse these resources.Nathan S.Watson-Haigh, Catherine A. Shang, Matthias Haimel, Myrto Kostadima, Remco Loos, Nandan Deshpande, Konsta Duesing, Xi Li, Annette McGrath, Sean McWilliam, Simon Michnowicz, Paula Moolhuijzen, Steve Quenette, Jerico Nico De Leon Revote, SonikaTyagi and Maria V. Schneide

A dominant gain-of-function mutation in universal tyrosine kinase <i>SRC </i>causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies

The Src family kinase (SFK)member SRC is amajor target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, whichwe confirmedwith in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patientswith myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of a-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC formMKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC- positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets andMKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors. © 2016 by the American Association for the Advancement of Science; all rights reserved

A gain-of-function variant in <i>DIAPH1 </i>causes dominant macrothrombocytopenia and hearing loss

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MK). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping and similarity regression. We describe two unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 p.R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was associated with reduced proplatelet formation from cultured MKs, cell clustering and abnormal cortical filamentous actin. Similarly, in platelets there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Over-expression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insights into the autoregulation of DIAPH1 activity

SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data