Biology subjects

Thomas, M.

Publications and source records attributed to Thomas, M..

10 recordsLinked to original sources

bioRxiv2018

GRID - Genomics of Rare Immune Disorders: a highly sensitive and specific diagnostic gene panel for patients with primary immunodeficiencies

Primary Immune disorders affect 15,000 new patients every year in Europe. Genetic tests are usually performed on a single or very limited number of genes leaving the majority of patients without a genetic diagnosis. We designed, optimised and validated a new clinical diagnostic platform called GRID, Genomics of Rare Immune Disorders, to screen in parallel 279 genes, including 2015 IUIS genes, known to be causative of Primary Immune disorders (PID). Validation to clinical standard using more than 58,000 variants in 176 PID patients shows an excellent sensitivity, specificity. The customised and automated bioinformatics pipeline prioritises and reports pertinent Single Nucleotide Variants (SNVs), INsertions and DELetions (INDELs) as well as Copy Number Variants (CNVs). An example of the clinical utility of the GRID panel, is represented by a patient initially diagnosed with X-linked agammaglobulinemia due to a missense variant in the BTK gene with severe inflammatory bowel disease. GRID results identified two additional compound heterozygous variants in IL17RC, potentially driving the altered phenotype.

genomics↗

bioRxiv2018

Cryogenically preserved RBCs support gametocytogenesis of Plasmodium falciparum in vitro and gametogenesis in mosquitoes.

BackgroundThe malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is small membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of \"infectious\" gametocytes requires 10-12 days with considerable physico-chemical demands imposed on host RBCs and thus, \"fresh\" RBCs that are [≤]1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs (cryo-preserved RBCs herein) is approved by the European and US FDAs as an alternative to refrigeration (4{degrees}C) for preserving RBC quality and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for >4 days.\n\nResultsUsing the standard laboratory strain, P. falciparum NF54, we first demonstrate that cryo-preserved RBCs preserved in the gaseous phase of liquid nitrogen and thawed after storage for 1, 4, 8 and 12 weeks, supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Using data from 11 SMFAs and RBCs from 4 separate donors with 3 donors re-tested following various periods of cryo-preservation, we show that overall levels of sporogony in the mosquito, as measured by oocyst prevalence and burdens in the midguts and sporozoites in salivary glands, were similar or better than using [≤]1-week old refrigerated RBCs. Additionally, the potential for cryo-preserved RBCs to serve as a universal substrate for SMFAs is shown for a Cambodian isolate of P. falciparum.\n\nConclusionsConsidering the suitability of cryo-preserved RBCs for P. falciparum SMFAs, we suggest guidelines for their use and how they can be integrated into an existing laboratory/insectary framework with the potential to significantly reduce running costs and provide greater reliability. Finally, we discuss scenarios where cryo-preserved RBCs may be especially useful in enhancing our understanding and/or providing novel insights into the patterns and process underlying human-to-mosquito transmission.

microbiology↗

bioRxiv2018

Gut Microbial succession during conventionalization of germfree chicken

A gnotobiotic chicken model was developed to study the succession of intestinal microflora from hatching to 18 days of age. Intestinal samples were collected from a local population of feral chickens and administered orally to germ-free 3 day old chicks. Animals were enthanized on 0, 9 and 18 days of age and intestinal samples were collected and subjected to genomic analysis. The five most prevalent phyla were Bacteroidetes (45.73{+/-}3.35%), Firmicutes (36.47{+/-}2.60%), Proteobacteria (8.28{+/-}0.91%), Actinobacteria (5.09{+/-}0.52%), and Spriochetes (2.10{+/-}0.38%). Principle coordinate analysis indicated the 0, 9 day and 18 day variables clustered together and the microbial communities changed temporally. The Morista-Horn index values ranged from 0.72 to 1, indicating the communities at 0, 9 or 18 days were more similar than dissimilar. The predicted functional profiles of the microbiomes of 0, 9 and 18 days were also similar. These results indicate the gnotobiotic chicks stably maintain the phylogentic diversity and predicted metabolic functionality of the inoculum community.\n\nImportanceThe domestic chicken is the cornerstone of animal agriculture worldwide with a flock population exceeding 40 billion birds/year. It serves as the economically valuable source of protein globally. Microbiome of poultry has important effects on chicken growth, feed conversion, immune status and pathogen resistance. The significance of our research is in developing a gnotobiotic chicken model to study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse set of gut bacteria. Therefore, besides using this model to study mechanisms of gut microbiota interactions in the chicken gut, our model could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota.

microbiology↗

bioRxiv2018

NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody dependent cellular cytotoxicity

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite-host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

immunology↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗

bioRxiv2018

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

Introduction Introduction Results and Discussion Conclusion Author Contributions Competing Financial Interests 1. gRNA choices and... 2. Zygote injection 3. DNA extraction from... 4. Amplicon sequencing and... 5. Whole Genome Sequencing 6. Probability of detecting... 7. Variant calling, Trio... 8. Intersection of all... 9. Comparison of SNVs... 10. Validation with PCR... 11. Re-analysis of De... Supplementary Figure Supplementary tables References CRISPR-Cas technologies have transformed genome-editing of experimental organisms and have ...

bioengineering↗

bioRxiv2018

Ostreococcus tauri is a high-lipid content green algae that extrudes clustered lipid droplets

Lipid droplet biogenesis, accumulation and secretion is an important field of research spanning biofuel feedstock production in algae and yeast to plant-microbe symbiosis or human metabolic disorders and other diseases. Here we evaluate the critical elements that influence lipid accumulation in the highly simplified and smallest known eukaryote Ostreococcus tauri and identify several conditions that satisfy its classification as an oleaginous green alga. In addition, these experiments revealed the release of excess lipids in pea-pod like structures where many dense lipid droplets are clustered in a linear fashion surrounded by an enveloping membrane which contrasts with known mechanisms from other eukaryotes. These results highlight the potential for Ostreococcus tauri to probe the evolution of lipid droplet dynamics as an emerging model organism with a compacted eukaryotic genome and also to impact lipid feedstock bioproduction applications either directly or using synthetic biology.\n\nOne Sentence SummaryThe smallest known eukaryote Ostreococcus tauri is oleaginous and sheds lipid droplets as pea-pod like membrane enclosed clusters.

cell biology↗

bioRxiv2018

Synchronous diversification of Sulawesi’s iconic artiodactyls driven by recent geological events

The high degree of endemism on Sulawesi has previously been suggested to have vicariant origins, dating back 40 Myr ago. Recent studies, however, suggest that much of Sulawesis fauna assembled over the last 15 Myr. Here, we test the hypothesis that recent uplift of previously submerged portions of land on Sulawesi promoted diversification, and that much of the its faunal assemblage is much younger than the island itself. To do so, we combined palaeogeographical reconstructions with genetic and morphometric data sets derived from Sulawesis three largest mammals: the Babirusa, Anoa, and Sulawesi warty pig. Our results indicate that although these species most likely colonized the area that is now Sulawesi at different times (14 Myr ago to 2-3 Myr ago), they experienced an almost synchronous expansion from the central part of the island. Geological reconstructions indicate that this area was above sea level for most of the last 4 Myr, unlike most parts of the island. We conclude that recent emergence of land on Sulawesi (~1-2 Myr) may have allowed species to expand synchronously. Altogether, our results indicates that the establishment of the highly endemic faunal assemblage on Sulawesi was driven by geological events over the last few million years.

evolutionary biology↗

bioRxiv2017

Whole genome sequencing-based detection of antimicrobial resistance and virulence in non-typhoidal Salmonella enterica isolated from wildlife

The aim of this study was to generate a reference set of Salmonella enterica genomes isolated from wildlife from the United States and to determine the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. We sequenced the whole genomes of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pet cases that were submitted to the Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, Oklahoma. Among 103 isolates, 50.48% were from wild birds, 0.9% was from fish, 24.27% each were from reptiles and mammals. 50.48% isolates showed resistance to at least one antibiotic. Resistance against the aminoglycoside streptomycin was most common while 9 isolates were found to be multi-drug resistant having resistance against more than three antibiotics. Determination of virulence gene profile revealed that the genes belonging to csg operons, the fim genes that encode for type 1 fimbriae and the genes belonging to type III secretion system were predominant among the isolates. The universal presence of fimbrial genes and the genes encoded by pathogenicity islands 1-2 among the isolates we report here indicates that these isolates could potentially cause disease in humans. Therefore, the genomes we report here could be a valuable reference point for future traceback investigations when wildlife is considered to be the potential source of human Salmonellosis.

microbiology↗

bioRxiv2017

Metagenomic characterization of the effect of feed additives on the gut microbiome and antibiotic resistome of feedlot cattle

In North America, antibiotic feed additives such as monensin and tylosin are added to the finishing diets of feedlot cattle to counter the ill-effects of feeding diets with rapidly digestible carbohydrates. While these feed additives have been proven to improve feed efficiency, and reduce liver abscess incidence, how these products impact the gastrointestinal microbiota is not completely understood. Furthermore, there are concerns that antibiotic feed additives may expand the antibiotic resistome of feedlot cattle by enriching antimicrobial resistance genes in pathogenic and nonpathogenic bacteria in the gut microbiota. In this study, we analyzed the impact of providing antibiotic feed additives to feedlot cattle using metagenome sequencing of treated and untreated animals. Our results indicate that use of antibiotic feed additives does not produce discernable changes at the phylum level however treated cattle had reduced the abundance of gram-positive bacteria at the genus level. The abundance of Ruminococcus, Erysipelotrichaceae and Lachanospira in the gut of treated steers was reduced. This may impact the ability of these animals to exclude pathogens from the gut. However, our results did not show any correlation between the presence of antimicrobial resistance genes in the gut microbiota and the administration of antibiotic feed additives.

microbiology↗