Biology subjects

Graham, M.

Publications and source records attributed to Graham, M..

28 records · Page 2Linked to original sources

bioRxiv2020

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA

2.IntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 L of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD {+/-}7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2019

Genomic interrogation of the burden and transmission of multidrug-resistant pathogens within and across hospital networks

BackgroundMultidrug-resistant organisms (MDROs) disproportionately affect hospitalized patients due to the combination of comorbidities, frequent antimicrobial use, and in-hospital MDRO transmission. Identification of MDRO transmission by hospital microbiology laboratories is difficult due to limitations of existing typing methods.\n\nMethodsWe conducted a prospective multicenter genomics implementation study (8 hospitals, 2800 beds) from 24th April to 18th June 2017 in Melbourne, Australia. Clinical and screening isolates from hospital inpatients were collected for six MDROs (vanA VRE, MRSA, ESBL E. coli [ESBL-Ec] and Klebsiella pneumoniae [ESBL-Kp], and carbapenem-resistant Pseudomonas aeruginosa [CRPa] and Acinetobacter baumannii [CRAb]), sequenced (Illumina NextSeq) and analyzed using open-source tools. MDRO transmission was assessed by genomics (core SNP phylogeny, grouped by species and ST) and compared to epidemiologic data.\n\nResults408 isolates were collected from 358 patients; 47.5% were screening isolates. ESBL-Ec was most common (52.5%), then MRSA (21.6%), vanA VRE (15.7%) and ESBL-Kp (7.6%).\n\nWe define the transmission rate for each MDRO by genomics and epidemiology; 31.6% of all study patients had potential genomic links to other study isolates; 86% of these were confirmed by epidemiologic links (probable or possible transmission). The highest transmission rates occurred with vanA VRE (88.4% of patients).\n\nConclusionsCombining genomics with high-quality epidemiologic data gives substantial insights into the burden and distribution of critical MDROs in hospitals, including in-hospital transmission. By defining transmission rates by genomics, we hope to enable comparisons over time and between sites, and introduce this as a new outcome measure to assess the efficacy of infection control interventions.

genomics↗

bioRxiv2019

AMPK regulates ESCRT-dependent microautophagy of proteasomes concomitant with proteasome storage granule assembly during glucose starvation

The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in phase-separated proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.

cell biology↗

bioRxiv2019

TWIST1 homo- and heterodimers orchestrate specificity control in embryonic stem cell lineage differentiation and craniofacial development

The extensive array of bHLH transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human-TWIST1 expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimer and heterodimers with TCF3, TCF4 and TCF12 E-proteins are the predominant dimer combinations. Dimers formation or their balance are altered by disease-causing mutations in TWIST1 helix domains, which may account for the defective differentiation of the craniofacial mesenchyme observed in patients. Functional analyses of the loss and gain of TWIST1-E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1-E-protein heterodimers activate the differentiation of mesoderm and neural crest cells which is accompanied by epithelial-to-mesenchymal transition, while TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage.

developmental biology↗

bioRxiv2019

Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro

AimsFormation of a perfusable microvascular network (VN) is critical for tissue engineering of solid organs. Stromal cells can support endothelial cell (EC) self-assembly into a VN, but distinct stromal cell populations may play different roles in this process. Here we investigated the effects that two widely used stromal cells populations, fibroblasts (FBs) and pericytes (PCs), have on VN formation. Methods and resultsWe examined the effects of adding defined stromal cell populations on the self-assembly of ECs derived from human endothelial colony forming cells (ECFCs) into perfusable VNs in fibrin gels cast within a microfluidics chamber. ECs alone fail to fully assemble a perfusable VN. Human lung FBs stimulate the formation of EC lined VNs within microfluidic devices. RNA-seq analysis suggested that FBs produce high levels of hepatocyte growth factor (HGF), and addition of recombinant HGF improved VN formation within devices. Human placental PCs could not substitute for FBs, but in the presence of FBs, PCs closely associated with ECs, formed a common basement membrane, extended microfilaments intercellularly, and reduced microvessel diameters. ConclusionsDifferent stromal cell types provide different functions in microvessel assembly by ECs. FBs support VN formation by providing paracrine growth factors whereas PCs directly interact with ECs to modify microvascular morphology. Statement of ContributionNatalia Kosyakova, Derek Kao, William G. Chang were primarily responsible for the conception, design, interpretation of experiments, and drafting of the manuscript. Francesc Lopez-Giraldez carried out analysis of RNA-seq data. Susann Spindler and Gregory Tietjen assisted with microvessel analysis software. Morven Graham and Xinran Liu assisted with the electron microscopy. Kevin J. James and Jee Won Shin assisted with data collection. Jordan Pober assisted with a critical review of manuscript and experimental design.

cell biology↗

bioRxiv2018

Microbiota-metabolites interactions in non-human primate gastrointestinal tract

BackgroundThe microbiota has been recognised as an important part for maintaining human health. Perturbation to its structure has been implicated in many diseases, such as obesity and cancers. The microbiota is highly metabolically active and fills in many niche metabolic pathways absent from the human host. Diseases such as obesity, cardiovascular disease and colorectal cancer has been linked to altered microbiota metabolism. However, there is a gap in the current knowledge on how mucosal-associated microbiota and colon mucosa interact. Here we performed an integrated analysis between the mucosal-associated microbiota and the mucosal tissue metabolites in healthy non-human primates.\n\nResultsWe found that the overall microbiota composition is influenced by both the tissue location as well as the host. We also identified bacteria signatures for different intestinal locations. The distal colon bacterial signature includes Ruminococcaceae, Bacteroidales, Christensenellaceae, Clostridiales, Sphaerochaeta, Victivallaceae, GMD14H09, CF231, ML615J-28, RF39 and R4-45B taxa. In the cecum, the signatures include Prevotella, Anaerovibrio, Roseburia, and Anaerostipes. Desulfovibrionaceae family is the only taxon that may be a signature for the duodenum. We also found an intricate global relationship between the microbiota and the host tissue metabolome that is mainly driven by the distal colon. Most importantly, we found microbial-centric tissue metabolites clusters that may have potential implications to studying host-microbiota metabolic interactions.

genomics↗

bioRxiv2018

An integrated workflow for cross-linking/mass spectrometry

We present a concise workflow to enhance the mass spectrometric detection of cross-linked peptides by introducing sequential digestion and the cross-link identification software Xi. Sequential digestion enhances peptide detection by shortening long tryptic peptides while avoiding over-digestion. We demonstrate our simple 12-fraction protocol for cross-linked multi-protein complexes and cell lysates, quantitative analysis, and high-density cross-linking, without requiring specific cross-linker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

biochemistry↗

bioRxiv2018

ALOXE3 is a hepatic fasting-responsive lipoxygenase that enhances insulin sensitivity via hepatic PPARγ

ABSTARCTThe hepatic glucose fasting response is gaining traction as a therapeutic pathway to enhance hepatic and whole-host metabolism. However, the mechanisms underlying these metabolic effects remain unclear. Here, we demonstrate the lipoxygenase, ALOXE3, is a novel effector of the thepatic fasting response. We show that ALOXE3 is activated during fasting, glucose withdrawal, and trehalose/trehalose analogue treatment. Hepatocyte-specific ALOXE3 expression reduced weight gain and hepatic steatosis in dietaryand genetically obese (db/db) models. ALOXE3 expression moreover enhanced basal thermogenesis and abrogated insulin resistance in db/db diabetic mice. Targeted metabolomics demonstrated accumulation of the PPAR{gamma} ligand, 12-KETE in hepatocytes overexpressing ALOXE3. Strikingly, PPAR{gamma} inhibition reversed hepatic ALOXE3-mediated insulin sensitization, suppression of hepatocellular ATP production and oxygen consumption, and gene induction of PPAR{gamma} coactivator-1a (PGC1) expression. Moreover, hepatocyte-specific PPAR{gamma} deletion reversed the therapeutic effect of hepatic ALOXE3 expression on diet-induced insulin intolerance. ALOXE3 is therefore a novel effector of the hepatocellular fasting response that leverages both PPAR{gamma}-mediated and pleiotropic effects to augment hepatic and whole-host metabolism, and is thus a promising target to ameliorate metabolic disease.

physiology↗

bioRxiv2017

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

genetics↗

bioRxiv2016

SNVPhyl: A Single Nucleotide Variant Phylogenomics pipeline for microbial genomic epidemiology

MotivationThe recent widespread application of whole-genome sequencing (WGS) for microbial disease investigations has spurred the development of new bioinformatics tools, including a notable proliferation of phylogenomics pipelines designed for infectious disease surveillance and outbreak investigation. Transitioning the use of WGS data out of the research lab and into the front lines of surveillance and outbreak response requires user-friendly, reproducible, and scalable pipelines that have been well validated.\n\nResultsSNVPhyl (Single Nucleotide Variant Phylogenomics) is a bioinformatics pipeline for identifying high-quality SNVs and constructing a whole genome phylogeny from a collection of WGS reads and a reference genome. Individual pipeline components are integrated into the Galaxy bioinformatics framework, enabling data analysis in a user-friendly, reproducible, and scalable environment. We show that SNVPhyl can detect SNVs with high sensitivity and specificity and identify and remove regions of high SNV density (indicative of recombination). SNVPhyl is able to correctly distinguish outbreak from non-outbreak isolates across a range of variant-calling settings, sequencing-coverage thresholds, or in the presence of contamination.\n\nAvailabilitySNVPhyl is available as a Galaxy workflow, Docker and virtual machine images, and a Unix-based command-line application. SNVPhyl is released under the Apache 2.0 license and available at http://snvphyl.readthedocs.io/ or at https://github.com/phac-nml/snvphyl-galaxy.

bioinformatics↗