Biology subjects

Johnson, N.

Publications and source records attributed to Johnson, N..

45 records · Page 3Linked to original sources

bioRxiv2020

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease, RHBDL2. We show that RHBDL2 prevents stochastic signalling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved, disease-linked proline residue is responsible for RHBDL2 recognising only the active conformation of Orai1, and cleavage by RHBDL2 is required to sharpen switch-like signalling triggered by store-operated calcium entry. Loss of RHBDL2 control of Orai1 causes severe dysregulation of CRAC channel effectors including transcription factor activation, inflammatory cytokine expression and T cell activation. We propose that this seek-and-destroy function may represent an ancient activity of rhomboid proteases in degrading unwanted signalling proteins.

biochemistry↗

bioRxiv2020

Population genomics of louping ill virus provide new insights into the evolution of tick-borne flaviviruses

BackgroundThe emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the UK. However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. Methodology/principal findingsWe sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. Conclusions/significanceThe 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors. Author SummaryTick-borne pathogens represent a major emerging threat to public health and in recent years have been expanding into new areas. LIV is a neglected virus endemic to the UK and Ireland (though it has been detected in Scandinavia and Russia) which is closely related to the major human pathogen TBEV, but predominantly causes disease in sheep and grouse. The recent detection of TBEV in the UK, which has also emerged elsewhere in Europe, requires more detailed understanding of the spread and sequence diversity of LIV. This could be important for diagnosis and vaccination, but also to improve our understanding of the evolution and emergence of these tick-borne viruses. Here we describe the sequencing of 22 LIV isolates which have been sampled from several host species across the past century. We have utilised this dataset to investigate the evolutionary pressures that LIV is subjected to and have explored the evolution of LIV using phylogenetic analysis. Crucially we were unable to estimate a reliable molecular clock rate for LIV and found that this problem also extends to a larger phylogeny of TBEV sequences. This work highlights a previously unknown caveat of tick-borne flavivirus evolutionary analysis which may be important for understanding the evolution of these important pathogens.

evolutionary biology↗

bioRxiv2020

Biotic and abiotic factors predict the biogeography of soil microbes in the Serengeti

Field-based observational research is the first step in understanding the factors that predict the biogeography and community structure of soil microbes. The Serengeti National Park in Tanzania is an ideal location for this type of research because active volcanoes generate strong environmental gradients due to ash deposition and a rain shadow. Also, as one of the last remaining naturally grazed ecosystems on Earth, the Serengeti provides insights about the influence of herbivory on microbial communities. We used 16S rRNA amplicons to characterize bacterial and archaeal communities in soils from a 13-year herbivore removal experiment to study the influence of environmental factors and grazing on the natural distribution of soil microbes. We collected soil samples from seven sites, each with three naturally grazed plots and three plots that were fenced to prevent grazing by large mammalian herbivores. Soil fertility (phosphorus, nitrogen, iron, calcium, organic matter), texture, and pH were measured at each plot. Beta diversity of bacterial and archaeal communities was most strongly correlated with soil texture (R2 = 32.4%). The abundance of many operational taxonomic units (OTUs) were correlated with soil texture, and the evenness of taxa within samples increased with fine-textured soil. Removal of grazing shifted community structure, with 31 OTUs that were significant indicator taxa of the ungrazed treatment and three OTUs that were significant indicators of the grazed treatment. ImportanceOur results show that in this regional scale study, soil texture was the best environmental predictor, and grazing by large mammals also structures bacterial and archaeal communities. When large mammals are removed, as humans have been doing for millenia, there are cascading effects into the microbial world that can influence ecosystem functions like carbon and nitrogen cycles. These empirical findings from a natural tropical savannah can help inform models of the global distribution and function of soil microbes.

microbiology↗

bioRxiv2019

Replication gaps underlie BRCA-deficiency and therapy response

Cancers that are deficient in BRCA1 or BRCA2 are hypersensitive to genotoxic agents, including platinums and other first-line chemotherapeutics. The established models propose that these cancers are hypersensitive because the chemotherapies block or degrade DNA replication forks and thereby create DNA double strand breaks, both of which require functional BRCA proteins to prevent or resolve by mechanisms termed fork protection (FP) or homologous recombination (HR). However, recent findings challenge this dogma because genotoxic agents do not initially cause DNA double strand breaks or stall replication forks. Here, we propose a new model for genotoxic chemotherapy in which ssDNA replication gaps underlie the hypersensitivity of BRCA deficient cancer, and we propose that defects in HR or FP do not. Specifically, we observed that ssDNA gaps develop in BRCA deficient cells because DNA replication is not effectively restrained in response to genotoxic stress. Moreover, we observe gap suppression (GS) by either restored fork restraint or by gap filling, both of which conferred resistance to therapy in tissue culture and BRCA patient tumors. In contrast, restored HR and FP were not sufficient to prevent hypersensitivity if ssDNA gaps were not eliminated. Together, these data suggest that ssDNA replication gaps underlie the BRCA cancer phenotype, \"BRCAness,\" and we propose are fundamental to the mechanism of action of genotoxic chemotherapies.

cancer biology↗

bioRxiv2019

PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation. We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions.

cancer biology↗

bioRxiv2019

Coding and regulatory drivers of mantle cell lymphoma identified through exome and genome sequencing

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes alongside previously published exome cohorts. To confirm our findings, we re-sequenced the genes identified in the exome cohort in 212 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations and further nominate two additional genes, EWSR1 and MEF2B, whose mutation respectively associated with poor and good outcome. Our sequencing revealed novel recurrent mutations including a unique missense hot spot in MEF2B and a pattern of non-coding mutations surrounding a single exon of the HNRNPH1 gene. We sequenced the whole genomes of 34 MCLs to confirm the focal nature of HNRNPH1 mutations. Using RNA-seq data from 110 of these cases, we identified a functional role for recurrent non-coding HNRNPH1 mutations in disrupting an auto-regulatory feedback mechanism. Overall, we identified three novel MCL-related genes with roles in RNA trafficking or splicing, namely DAZAP1, EWSR1, and HNRNPH1. Taken together, these data strongly implicate a role for aberrant regulation of splicing in MCL pathobiology.\n\nKey pointsO_LIRNA-binding proteins with roles in regulating alternative splicing, DAZAP1, EWSR1, HNRNPH1, are frequently mutated in MCL\nC_LIO_LIThe majority of recurrent somatic HNRNPH1 mutations are intronic and HNRNPH1 exhibits self-regulation through alternative splicing\nC_LI

genomics↗

bioRxiv2019

Impact of a caries preventive intervention in remote Indigenous Australian children

IMPORTANCEThe burden of dental caries in remote Indigenous communities in Australia is unacceptably high.\n\nOBJECTIVESWe tested the impact of an annual caries preventive intervention, delivered by a fly-in/fly-out professional team, on Indigenous children residing in a remote Australian community, involving selective fissure sealants, topical povidone iodine and fluoride varnish application. The outcome was caries increment at 12- and 24-month follow-up.\n\nDESIGN, SETTING, PARTICIPANTSAround 600 Indigenous children aged 5 to 17 years were invited to participate at baseline, of which 408 had caregiver consent provided. Of these, 196 consented to both the study and the treatment arm and comprised the experimental group. Two hundred and twelve consented to the epidemiological examination only, and constituted the comparison group.\n\nINTERVENTIONThe Big Bang intervention, which occurred annually, comprised placement of fissure sealants, and application of povidone-iodine and fluoride varnish, following completion of each childs dental treatment plan. Standard diet and oral hygiene advice was provided.\n\nMAIN OUTCOMES AND MEASURESCaries increment (number of tooth surfaces with new dental caries) in both primary and permanent dentitions at 12- and 24-month follow-up.\n\nRESULTSAt 12-month follow-up, children in the experimental group had, on average, 5.05 ({+/-}5.47) new carious lesions compared to 7.49 ({+/-}6.94) in the comparison group (p=0.001). The preventive fraction was 33%. At 24-month follow-up, children in the experimental group had, on average, 6.47 ({+/-}6.07) new carious lesions compared to 8.43 ({+/-}5.83) in the comparison group (p=0.002). The preventive fraction was 23%.\n\nCONCLUSIONS AND RELEVANCEIndigenous children exposed to the Big Bang caries intervention had significantly less increment in dental disease than those not exposed to the intervention. Benefits were demonstrated at both 12- and 24-month follow-ups, suggesting that the intervention is likely to be sustained if delivered across a childs life. The cost-effectiveness of this approach is being evaluated.\n\nQuestionDoes a quick, intensive, Big Bang program of intervention reduce dental disease among remote Indigenous Australian children?\n\nFindingsAt 12- and 24-month follow-ups, children in the experimental group had fewer new caries surfaces. At the 24-month follow-up, the average caries increment was 6.47 ({+/-}6.07) in the experimental group compared to 8.43 ({+/-}5.83) in the comparison group; a preventive fraction of 23%.\n\nMeaningAn intensive caries intervention delivered by a peripatetic team of professionals, involving fissure sealants, topical povidone iodine and fluoride varnish application reduces experience of dental disease among this vulnerable population.

epidemiology↗

bioRxiv2019

Coordinated development of the mouse extrahepatic bile duct: implications for neonatal susceptibility to biliary injury

Background & AimsThe extrahepatic bile duct is the primary tissue initially affected by the cholangiopathy biliary atresia. Biliary atresia affects neonates exclusively and current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts.\n\nMethodsWe studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes and isolated extrahepatic bile ducts, and a collagen reporter mouse. Methods included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix.\n\nResultsThe cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen.\n\nConclusionWe identified four key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis.\n\nLay SummaryBiliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction with resulting liver injury. We identify four key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.\n\nHighlightsO_LIThe apical glycocalyx is thin and patchy in neonatal compared to adult cholangiocytes\nC_LIO_LINeonatal cholangiocytes have immature cell-cell junctions and increased permeability\nC_LIO_LIThe neonatal submucosal space has minimal collagen I or elastin\nC_LIO_LIThe neonatal submucosal space contains many actively collagen-secreting cells\nC_LI\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC=\"FIGDIR/small/576256_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (27K):\norg.highwire.dtl.DTLVardef@1685e52org.highwire.dtl.DTLVardef@1086478org.highwire.dtl.DTLVardef@1fc3f55org.highwire.dtl.DTLVardef@1a20a5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2018

Determining rewiring effects of alternatively spliced isoforms on protein-protein interactions using a computational approach

The critical role of alternative splicing (AS) in cell functioning has recently become apparent, whether in studying tissue-or cell-specific regulation, or understanding molecular mechanisms governing a complex disorder. Studying the rewiring, or edgetic, effects of alternatively spliced isoforms on protein interactome can provide system-wide insights into these questions. Unfortunately, high-throughput experiments for such studies are expensive and time-consuming, hence the need to develop an in-silico approach. Here, we formulated the problem of characterization the edgetic effects of AS on protein-protein interactions (PPIs) as a binary classification problem and introduced a first computational approach to solve it. We first developed a supervised feature-based classifier that benefited from the traditional features describing a PPI, the problem-specific features that characterized the difference between the reference and alternative isoforms, and a novel domain interaction potential that allowed pinpointing the domains employed during a specific PPI. We then expanded this approach by including a large set of unlabeled interactomics data and developing a semi-supervised learning method. Our method called AS-IN (Alternatively Splicing INteraction prediction) Tool was compared with the state-of-the-art PPI prediction tools and showed a superior performance, achieving 0.92 in precision and recall. We demonstrated the utility of AS-IN Tool by applying it to the transcriptomic data obtained from the brain and liver tissues of a healthy mouse and western diet fed mouse that developed type two diabetes. We showed that the edgetic effects of differentially expressed transcripts associated with the disease condition are system-wide and unlikely to be detected by looking only at the gene-specific expression levels.

bioinformatics↗