Functional genomics in the regulation of the immune response

Genetics contribute substantially to the ability of the immune system to respond appropriately to a challenge. Consequently, many infectious and inflammatory diseases have a heritable component. As genome-wide association studies provide increasing data linking genetic variants to disease, we can leverage this information to gain insights into disease biology if we can elucidate the mechanisms underlying an observed association. Data-driven bioinformatic approaches, in vitro mechanistic studies and whole-organism approaches to study integrated pathophysiological systems provide complementary information to help establish causal links between specific variants and their effects on a target gene, and between that gene and disease pathogenesis. Cells of the mononuclear phagocyte system play key roles in regulation of innate and adaptive immune responses, either via secreted mediators or through direct cell-to-cell contacts. To advance understanding of how these regulatory processes, and genetic variations therein, shape the course of disease, I have combined experimental and bioinformatic approaches to explore novel genetic associations between genes for macrophage surface receptors and human disease. Specifically, functional follow-up of an association between adhesion G-protein-coupled receptor CD97 and severe influenza showed, using a mouse model, that deficiency of this receptor reduces the efficiency of the CD8+ T-lymphocyte response, a process critical to clearance of infected cells. Secondly, I addressed the question of how to identify causal variants in a disease-associated linkage disequilibrium block, for an association between macrophage regulatory receptor SIRPα and schizophrenia. To achieve this, I developed novel methodology for targeted locus screening using CRISPR/Cas9 mutagenesis, and identified a number of plausible causal regulatory variants that could affect expression of this receptor. Combining variant-level information with gene-level studies of disease pathophysiology can provide valuable insights into genetic causation of immune dys-regulation leading to disease, which may be harnessed for improved personalised disease risk prediction, or to identify therapeutically targetable pathways

TDP-43 expression in mouse models of amyotrophic lateral sclerosis and spinal muscular atrophy

<p>Abstract</p> <p>Background</p> <p>Redistribution of nuclear TAR DNA binding protein 43 (TDP-43) to the cytoplasm and ubiquitinated inclusions of spinal motor neurons and glial cells is characteristic of amyotrophic lateral sclerosis (ALS) pathology. Recent evidence suggests that TDP-43 pathology is common to sporadic ALS and familial ALS without SOD1 mutation, but not SOD1-related fALS cases. Furthermore, it remains unclear whether TDP-43 abnormalities occur in non-ALS forms of motor neuron disease. Here, we characterise TDP-43 localisation, expression levels and post-translational modifications in mouse models of ALS and spinal muscular atrophy (SMA).</p> <p>Results</p> <p>TDP-43 mislocalisation to ubiquitinated inclusions or cytoplasm was notably lacking in anterior horn cells from transgenic mutant SOD1^G93Amice. In addition, abnormally phosphorylated or truncated TDP-43 species were not detected in fractionated ALS mouse spinal cord or brain. Despite partial colocalisation of TDP-43 with SMN, depletion of SMN- and coilin-positive Cajal bodies in motor neurons of affected SMA mice did not alter nuclear TDP-43 distribution, expression or biochemistry in spinal cords.</p> <p>Conclusion</p> <p>These results emphasise that TDP-43 pathology characteristic of human sporadic ALS is not a core component of the neurodegenerative mechanisms caused by SOD1 mutation or SMN deficiency in mouse models of ALS and SMA, respectively.</p

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I.

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.This work was supported by an MRC research grant to J.P.L. (G0900113). M.D.J.P. and J.L.E. were MRC research students and S.P. a Wellcome Trust research student. K.B. was a British Heart Foundation Intermediate Fellow and P.J.L. is a Wellcome Trust Principal Fellow. The CIMR is supported by a Wellcome Trust Strategic Award 100140 and an electron microscope was purchased with Wellcome Trust grant 093026.This is the final version of the article. It first appeared from Portland Press via http://dx.doi.org/10.1042/BJ2015033

Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154665/1/fsb2fj078653com.pd

Systematic comparison of ranking aggregation methods for gene lists in experimental results

MOTIVATION: A common experimental output in biomedical science is a list of genes implicated in a given biological process or disease. The gene lists resulting from a group of studies answering the same, or similar, questions can be combined by ranking aggregation methods to find a consensus or a more reliable answer. Evaluating a ranking aggregation method on a specific type of data before using it is required to support the reliability since the property of a dataset can influence the performance of an algorithm. Such evaluation on gene lists is usually based on a simulated database because of the lack of a known truth for real data. However, simulated datasets tend to be too small compared to experimental data and neglect key features, including heterogeneity of quality, relevance and the inclusion of unranked lists. RESULTS: In this study, a group of existing methods and their variations that are suitable for meta-analysis of gene lists are compared using simulated and real data. Simulated data were used to explore the performance of the aggregation methods as a function of emulating the common scenarios of real genomic data, with various heterogeneity of quality, noise level and a mix of unranked and ranked data using 20 000 possible entities. In addition to the evaluation with simulated data, a comparison using real genomic data on the SARS-CoV-2 virus, cancer (non-small cell lung cancer) and bacteria (macrophage apoptosis) was performed. We summarize the results of our evaluation in a simple flowchart to select a ranking aggregation method, and in an automated implementation using the meta-analysis by information content algorithm to infer heterogeneity of data quality across input datasets. AVAILABILITY AND IMPLEMENTATION: The code for simulated data generation and running edited version of algorithms: https://github.com/baillielab/comparison_of_RA_methods. Code to perform an optimal selection of methods based on the results of this review, using the MAIC algorithm to infer the characteristics of an input dataset, can be downloaded here: https://github.com/baillielab/maic. An online service for running MAIC: https://baillielab.net/maic. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Benchtop flow-NMR for rapid online monitoring of RAFT and free radical polymerisation in batch and continuous reactors

A “Benchtop” NMR spectrometer is used for detailed monitoring of controlled and free radical polymerisations performed in batch and continuous reactors both offline and in real-time. This allows detailed kinetic analysis with unprecedented temporal resolution for reactions which reach near completion in under five minutes

A genome-wide CRISPR/Cas9 screen reveals the requirement of host sphingomyelin synthase 1 for infection with Pseudorabies virus mutant gD–Pass

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked

Estimating the causal influence of body mass index on risk of Parkinson disease: A Mendelian randomisation study.

BACKGROUND: Both positive and negative associations between higher body mass index (BMI) and Parkinson disease (PD) have been reported in observational studies, but it has been difficult to establish causality because of the possibility of residual confounding or reverse causation. To our knowledge, Mendelian randomisation (MR)-the use of genetic instrumental variables (IVs) to explore causal effects-has not previously been used to test the effect of BMI on PD. METHODS AND FINDINGS: Two-sample MR was undertaken using genome-wide association (GWA) study data. The associations between the genetic instruments and BMI were obtained from the GIANT consortium and consisted of the per-allele difference in mean BMI for 77 independent variants that reached genome-wide significance. The per-allele difference in log-odds of PD for each of these variants was estimated from a recent meta-analysis, which included 13,708 cases of PD and 95,282 controls. The inverse-variance weighted method was used to estimate a pooled odds ratio (OR) for the effect of a 5-kg/m2 higher BMI on PD. Evidence of directional pleiotropy averaged across all variants was sought using MR-Egger regression. Frailty simulations were used to assess whether causal associations were affected by mortality selection. A combined genetic IV expected to confer a lifetime exposure of 5-kg/m2 higher BMI was associated with a lower risk of PD (OR 0.82, 95% CI 0.69-0.98). MR-Egger regression gave similar results, suggesting that directional pleiotropy was unlikely to be biasing the result (intercept 0.002; p = 0.654). However, the apparent protective influence of higher BMI could be at least partially induced by survival bias in the PD GWA study, as demonstrated by frailty simulations. Other important limitations of this application of MR include the inability to analyse non-linear associations, to undertake subgroup analyses, and to gain mechanistic insights. CONCLUSIONS: In this large study using two-sample MR, we found that variants known to influence BMI had effects on PD in a manner consistent with higher BMI leading to lower risk of PD. The mechanism underlying this apparent protective effect warrants further study