107 research outputs found
Investigation of the extent and role of N-linked glycosylation in the human scavenger receptor CD36
No full textHuman CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidised low density lipoprotein (oxLDL) and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation and ten putative N-linked glycosylation sites situated in the large extracellular loop of the protein have been identified, however their utilisation and role in the folding and function of the protein have not been characterised. Using mass spectrometry on purified and PNGaseF-deglycosylated CD36, and also by comparing the electrophoretic mobility of different glycosylation-site mutants, this study determined that nine of the ten sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells, established that glycosylation is necessary for trafficking to the plasma membrane. Minimally-glycosylated mutants that supported trafficking were identified and indicated the importance of carboxy-terminal sites N247, N321 and N417 and amino-terminal sites N102 and N205. However, unlike the related mouse scavenger receptor SR-BI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally-glycosylated mutants appear to be predominantly core glycosylated indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified LDL
Investigation of the extent and role of N-linked glycosylation in the human scavenger receptor CD36
No full textHuman CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidised low density lipoprotein (oxLDL) and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation and ten putative N-linked glycosylation sites situated in the large extracellular loop of the protein have been identified, however their utilisation and role in the folding and function of the protein have not been characterised. Using mass spectrometry on purified and PNGaseF-deglycosylated CD36, and also by comparing the electrophoretic mobility of different glycosylation-site mutants, this study determined that nine of the ten sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells, established that glycosylation is necessary for trafficking to the plasma membrane. Minimally-glycosylated mutants that supported trafficking were identified and indicated the importance of carboxy-terminal sites N247, N321 and N417 and amino-terminal sites N102 and N205. However, unlike the related mouse scavenger receptor SR-BI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally-glycosylated mutants appear to be predominantly core glycosylated indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified LDL.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex.
No full textThe LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1
A crowd of BashTheBug volunteers reproducibly and accurately measure the minimum inhibitory concentrations of 13 antitubercular drugs from photographs of 96-well broth microdilution plates
No full textTuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally, this is done by culturing a clinical sample and then exposing aliquots to a panel of antibiotics, each being present at a pre-determined concentration, thereby determining if the sample isresistant or susceptible to each sample. The minimum inhibitory concentration (MIC) of a drug is the lowestconcentration that inhibits growth and is a more useful quantity but requires each sample to be tested at a range ofconcentrations for each drug. Using 96-well broth micro dilution plates with each well containing a lyophilised pre-determined amount of an antibiotic is a convenient and cost-effective way to measure the MICs of several drugs at once for a clinical sample. Although accurate, this is still an expensive and slow process that requires highly-skilled and experienced laboratory scientists. Here we show that, through the BashTheBug project hosted on the Zooniverse citizen science platform, a crowd of volunteers can reproducibly and accurately determine the MICs for 13 drugs and that simply taking the median or mode of 11–17 independent classifications is sufficient. There is therefore a potential role for crowds to support (but not supplant) the role of experts in antibiotic susceptibility testing
Scalable Pathogen Pipeline Platform (SP^3): enabling unified genomic data analysis with elastic cloud computing
No full textPathogen genomic data analysis can be extremely bespoke and diverse. This paper presents our plan and progress towards creating a Scalable Pathogen Pipeline Platform (SP3) providing an efficient and unified process of collecting, analysing and comparing genomic data analysis with the benefit of elastic cloud computing. SP3 enables container-centric bioinformatic workflows run on personal computers, High-performance computing (HPC) clusters and cloud platforms. We have deployed and tested SP3 on local HPC, Google Cloud Platform (GCP), Microsoft Azure and OpenStack Platforms. SP3 allows users to fetch genomic sequencing data from European Nucleotide Archive (ENA) and conduct analysis with open-source bioinformatic pipelines. We believe SP3 will promote common standards around pathogen genomic data quality, data processing and data analysis, helping answer the challenges of tools divergence and leveraging a pool of public genomic data repository and cloud resources
SARS-CoV-2 antibody trajectories after a single COVID-19 vaccination with and without prior infection
No full textGiven high SARS-CoV-2 incidence, coupled with slow and inequitable vaccine roll-out in many settings, there is a need for evidence to underpin optimum vaccine deployment, aiming to maximise global population immunity. We evaluate whether a single vaccination in individuals who have already been infected with SARS-CoV-2 generates similar initial and subsequent antibody responses to two vaccinations in those without prior infection. We compared anti-spike IgG antibody responses after a single vaccination with ChAdOx1, BNT162b2, or mRNA-1273 SARS-CoV-2 vaccines in the COVID-19 Infection Survey in the UK general population. In 100,849 adults median (50 (IQR: 37–63) years) receiving at least one vaccination, 13,404 (13.3%) had serological/PCR evidence of prior infection. Prior infection significantly boosted antibody responses, producing higher peak levels and/or longer half-lives after one dose of all three vaccines than those without prior infection receiving one or two vaccinations. In those with prior infection, the median time above the positivity threshold was >1 year after the first vaccination. Single-dose vaccination targeted to those previously infected may provide at least as good protection to two-dose vaccination among those without previous infection
DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing
No full textPurpose Speed of bloodstream infection diagnosis is vital to reduce morbidity and mortality. Whole genome sequencing (WGS) performed directly from liquid blood culture could provide single-assay species and antibiotic susceptibility prediction; however, high inhibitor and human cell/DNA concentrations limit pathogen recovery. We develop a method for the preparation of bacterial DNA for WGS-based diagnostics direct from liquid blood culture. Methodology We evaluate three commercial DNA extraction kits: BiOstic Bacteraemia, Amplex Hyplex and MolYsis Plus. Differential centrifugation, filtration, selective lysis and solid-phase reversible immobilization bead clean-up are tested to improve human cells/DNA and inhibitor removal. Using WGS (Illumina/MinION), we assess human DNA removal, pathogen recovery, and predict species and antibiotic susceptibility inpositive blood cultures of 44 Gram-negative and 54 Staphylococcus species. Results/Key findings BiOstic kit extractions yield the greatest mean DNA concentration, 94–301 ng µl−1, versus 0–2.5 ng µl−1 using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9–2.1) and human DNA (0.0–4.4×106 copies) in BiOstic extracts. Differential centrifugation (2000 g , 1 min) prior to BiOstic extraction reduces human DNA by 63–89 % with selective lysis minimizing by a further 62 %. Post-extraction bead clean-up lowers inhibition. Overall, 67 % of sequenced samples (Illumina MiSeq) contain 93 % concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If >60 % of sequencing reads are human (7/98 samples) susceptibility prediction becomes compromised. Novel MinION-based WGS (n=9) currently gives rapid species identification but not susceptibility prediction. Conclusion Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay
GenomegaMap: Within-species genome-wide dN/dS estimation from over 10,000 genomes
No full textThe dN/dS ratio provides evidence of adaptation or functional constraint in protein-coding genes by quantifying the relative excess or deficit of amino acid-replacing versus silent nucleotide variation. Inexpensive sequencing promises a better understanding of parameters, such as dN/dS, but analyzing very large data sets poses a major statistical challenge. Here, I introduce genomegaMap for estimating within-species genome-wide variation in dN/dS, and I apply it to 3,979 genes across 10,209 tuberculosis genomes to characterize the selection pressures shaping this global pathogen. GenomegaMap is a phylogeny-free method that addresses two major problems with existing approaches: 1) It is fast no matter how large the sample size and 2) it is robust to recombination, which causes phylogenetic methods to report artefactual signals of adaptation. GenomegaMap uses population genetics theory to approximate the distribution of allele frequencies under general, parent-dependent mutation models. Coalescent simulations show that substitution parameters are well estimated even when genomegaMap’s simplifying assumption of independence among sites is violated. I demonstrate the ability of genomegaMap to detect genuine signatures of selection at antimicrobial resistance-conferring substitutions in Mycobacterium tuberculosis and describe a novel signature of selection in the cold-shock DEAD-box protein A gene deaD/csdA. The genomegaMap approach helps accelerate the exploitation of big data for gaining new insights into evolution within species
Structural basis for LMO2-driven recruitment of the SCL:E47bHLH heterodimer to hematopoietic-specific transcriptional targets.
No full textCell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces T cell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL's reported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes
High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing
No full textThe rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection
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