Biology subjects

Murray, J.

Publications and source records attributed to Murray, J..

36 records · Page 2Linked to original sources

bioRxiv2021

Structure, immunogenicity, and conformation-dependent receptor binding of the post-fusion human metapneumovirus F protein

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparan sulfate. A library of heparan sulfate (HS) oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparan binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on hMPV F. Overall, this study provides new characteristics of the hMPV F protein informative for vaccine and therapy development. ImportanceHuman metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the post-fusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of post-fusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced TH1/TH2 response and target two previously discovered neutralizing epitopes.

microbiology↗

bioRxiv2020

Disentangling the shark and ray trade in Indonesia to reconcile conservation with food security

Indonesian marine resources are among the richest on the planet, sustaining highly diverse fisheries and includes the largest elasmobranch landings in the world, making Indonesia one of the worlds largest exporters of shark and ray products. Socio-economic and food security considerations pertaining to Indonesian communities add further layers of complexity to the management and conservation of these vulnerable species. This study investigates how shark and ray trade flows in and out of Indonesia and attempts to examine patterns and drivers of the current scenario. We identify substantial discrepancies between reported landings and declared exports, and between Indonesian exports in shark fin and meat products and the corresponding figures reported by importing countries. These mismatches are estimated to amount to over $43.6M and $20.9M for fins and meat, respectively, for the period between 2012 and 2018. Although the declared exports are likely to be an underestimation because of significant unreported or illegal trading activities, we find that domestic consumption of shark and ray products plays a significant role in explaining these discrepancies due to the increasing local demand for meat. The study also unearths a general scenario of unsystematic data collection and lack of granularity of product terminology, which is inadequate to meet the challenges of over-exploitation, illegal trade and food security in Indonesia. We discuss how to improve data transparency to support trade regulations and governance actions, by improving inspection measures, and conserving elasmobranch populations without neglecting the socio-economic dimension of this complex system.

scientific communication and education↗

bioRxiv2020

Novel inhibitors of E. coli lipoprotein diacylglyceryl transferase are insensitive to resistance caused by lpp deletion

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify the first ever described Lgt inhibitors that potently inhibit Lgt biochemical activity in vitro and are bactericidal against wild-type Acinetobacter baumannii and E. coli strains. Unlike inhibition of other steps in lipoprotein biosynthesis, deletion of the major outer membrane lipoprotein, lpp, is not sufficient to rescue growth after Lgt depletion or provide resistance to Lgt inhibitors. Our data validate Lgt as a novel druggable antibacterial target and suggest that inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of downstream steps of bacterial lipoprotein biosynthesis and transport.

microbiology↗

bioRxiv2020

Coding de novo mutations identified by WGS reveal novel orofacial cleft genes

While de novo mutations (DNMs) are known to increase risk of congenital defects, DNMs have not been fully explored regarding orofacial clefts (OFCs), one of the most common human birth defects. Therefore, whole-genome sequencing of 756 case-parent trios of European, Colombian, and Taiwanese ancestry was performed to determine the contributions of coding DNMs to OFC risk. Overall, we identified a significant excess of loss-of-function DNMs in genes highly expressed in craniofacial tissues, as well as genes associated with known autosomal dominant OFC syndromes. This analysis also revealed roles for zinc-finger homeobox domain and SOX2-interacting genes in OFC etiology.

genetics↗

bioRxiv2020

Are there Physical Linkages between Genes that have Synergistic Fitness Effects?

Many of the effects on fitness in population genetics are due not to single locations in the genome, but to the interaction of genetic variants at multiple locations in the genome. Of particular interest are completely epistatic interactions, where a combination of genetic variants is required to produce an effect, and the effect cannot occur with any other combination. In diploids, epistasis is strongly connected to meiotic recombination, a process which can both assemble and destroy beneficial combinations of genetic variants. Additionally, epistatic interactions can be hard to detect in empirical studies, and mathematical models of epistasis and recombination are challenging to analyse, so despite their ubiquity epistatic interactions are regularly not considered. As a result, there is little consensus on when high levels of recombination might be expected, or how strongly recombination affects beneficial or deleterious fitness effects controlled by epistatic interactions. We address this question by conducting a meta-analysis and simulations. The meta-analysis used data drawn and curated from Drosophila melanogaster studies in Flybase. We extracted studies relating genetic combinations and phenotypically detectable effects on fitness, then analysed the relationship between the rate of recombination and effect on fitness with a statistical model. We also ran simulations under a two-locus Wright-Fisher model with recombination and epistatic selection. The results of both approaches indicated a tendency for genetic combinations with an epistatic effect on fitness to occur in an environment of reduced meiotic recombination. Two possible explanations for this are that the variants controlling such interactions are selected for in regions where there is little recombination, or that such interactions lead to selection for lower rates of recombination in the regions where those variants appear.

evolutionary biology↗

bioRxiv2020

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. Article SummaryScientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.

microbiology↗

bioRxiv2020

Protein-carbohydrate ingestion alters Vps34 cellular localization independent of changes in kinase activity in human skeletal muscle

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell size and growth in response to nutrients, however, the mechanisms by which nutrient levels are sensed by mTORC1 in human skeletal muscle are yet to be fully elucidated. The Class III PI3Kinase Vps34 has recently been proposed as a sensor essential for mTORC1 activation following nutrient stimulation. We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (age - 21 {+/-} 0.5yrs, mean {+/-} SEM) ingested a PRO-CHO beverage containing 20/44/1g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1h and 3h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean{+/-}SEM) - 0.273{+/-}0.021, 1h - 0.347{+/-}0.022, Pearsons Coefficient (r)) where it co-localized with mTOR (PRE - 0.312{+/-}0.018, 1h - 0.348{+/-}0.024, Pearsons Coefficient (r))). These alterations occurred in parallel to an increase in S6K1 kinase activity - 941{+/-}164% of PRE at 1h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle.

physiology↗

bioRxiv2019

Somatic genetics of CDR3 control H-bonds to MHC via V-domain rotational probability (dV) effecting germline CDR2 ‘scanning’ (dθ) of polymorphic MHC

The mechanism which adapts the T-cell antigen receptor (TCR) within a given major histocompatibility complex (MHC/HLA) genotype is essential for protection against pathogens. Historically attributed to relative affinity, genetically vast TCRs are surprisingly focused towards a micromolar affinity for their respective peptide (p) plus MHC (pMHC) ligands. Thus, the somatic diversity of the TCR with respect to MHC-restriction, and (ultimately) to pathogens, remains enigmatic. Here, we derive a triple integral solution (from fixed geometry) for any given V domain in TCR bound to pMHC. Solved complexes involving HLA-DR and HLA-DQ, where genetic linkage to the TCR is most profound, were examined in detail. Certain V domains displayed rare geometry within this panel--specifying a restricted rotational probability/volumetric density (dV). Remarkably, hydrogen (H) bond charge-relays distinguished these structures from the others; suggesting that CDR3 binding chemistry dictates CDR2 contacts on the opposite MHC-II alpha helix. Together, these data suggest that TCR recapitulate dV and specialise target pMHC recognition.

immunology↗

bioRxiv2019

Real brains in virtual worlds: Validating a novel oddball paradigm in virtual reality

Electroencephalography (EEG) research is typically conducted in controlled laboratory settings. This limits the generalizability to real-world situations. Virtual reality (VR) sits as a transitional tool that provides tight experimental control with more realistic stimuli. To test the validity of using VR for event-related potential (ERP) research we used a well-established paradigm, the oddball task. For our first study, we compared VR to traditional, monitor-based stimulus presentation using visual and auditory oddball tasks while EEG data was recorded. We were able to measure ERP waveforms typically associated with such oddball tasks, namely the P3 and earlier N2 components, in both conditions. Our results suggest that ERPs collected using VR head mounted displays and typical monitors were comparable on measures of latency, amplitude, and spectral composition. In a second study, we implemented a novel depth-based oddball task and we were able to measure the typical oddball-related ERPs elicited by the presentation of near and far stimuli. Interestingly, we observed significant differences in early ERPs components between near and far stimuli, even after controlling for the effects of the oddball task. Current results suggest that VR can serve as a valid means of stimulus presentation in novel or otherwise inaccessible environments for EEG experimentation. We demonstrated the capability of a depth-based oddball in reliably eliciting a P3 waveform. We also found an interaction between the depth at which objects are presented and early ERP responses. Further research is warranted to better explain this influence of depth on the EEG and ERP activity.

neuroscience↗

bioRxiv2018

A novel method for systematic genetic analysis and visualization of phenotypic heterogeneity applied to orofacial clefts

Phenotypic heterogeneity is a hallmark of complex traits, and genetic studies may focus on the trait as a whole or on individual subgroups. For example, in orofacial clefting (OFC), three subtypes - cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP) have been studied separately and in combination. It is more challenging, however, to dissect the genetic architecture and describe how a given locus may be contributing to distinct subtypes of a trait. We developed a framework for quantifying and interpreting evidence of subtype-specific or shared genetic effects in complex traits. We applied this technique to create a \"cleft map\" of the association of 30 genetic loci with three OFC subtypes. In addition to new associations, we found loci with subtype-specific effects (e.g., GRHL3 (CP), WNT5A (CLP)), as well as loci associated with two or all three subtypes. We cross-referenced these results with mouse craniofacial gene expression datasets, which identified promising candidate genes. However, we found no strong correlation between OFC subtypes and expression patterns. In aggregate, the cleft map revealed neither subtype-specific nor shared genetic effects operate in isolation in OFC architecture. Our approach can be easily applied to any complex trait with distinct phenotypic subgroups.

genetics↗

bioRxiv2018

Genome Wide Interaction Studies Identify Sex-Specific Risk Alleles for Nonsyndromic Orofacial Clefts

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans and is notable for its apparent sexual dimorphism where approximately twice as many males are affected as females. The sources of this disparity are largely unknown, but interactions between genetic and sex effects are likely contributors. We examined gene-by-sex (G x S) interactions in a worldwide sample of 2,142 NSCL/P cases and 1,700 controls recruited from 13 countries. First, we performed genome-wide joint tests of the genetic (G) and G x S effects genome-wide using logistic regression assuming an additive genetic model and adjusting for 18 principal components of ancestry. We further interrogated loci with suggestive results from the joint test (p < 1.00 x 10-5) by examining the G x S effects from the same model. Out of the 133 loci with suggestive results (p < 1.00 x 10-5) for the joint test, we observed one genome-wide significant G x S effect in the 10q21 locus (rs72804706; p = 6.69 x 10-9; OR = 2.62 [1.89, 3.62]) and 16 suggestive G x S effects. At the intergenic 10q21 locus, the risk of NSCL/P is estimated to increase with additional copies of the minor allele for females, but the opposite effect for males. Our observation that the impact of genetic variants on NSCL/P risk differs for males and females may further our understanding of the genetic architecture of NSCL/P and the sex differences underlying clefts and other birth defects.

genetics↗

bioRxiv2017

Gene Expression Distribution Deconvolution in Single Cell RNA Sequencing

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each genes expression distribution across cells, thus allowing the assessment of the dispersion, burstiness, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data is noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a re-examination of 9 public data sets, we propose a simple technical noise model for scRNA-seq data with Unique Molecular Identifiers (UMI). We develop DESCEND, a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and burstiness. DESCEND can adjust for cell-level covariates such as cell size, cell cycle and batch effects. DESCENDs noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations, and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially bursty genes, identifying cell types, and selecting differentiation markers.

genetics↗

bioRxiv2017

Lineage and stage-specific expressed CYCD7;1 coordinates the single symmetric division that creates stomatal guard cells

Plants, with cells fixed in place by rigid walls, often utilize spatial and temporally distinct cell division programs to organize and maintain organs. This leads to the question of how developmental regulators interact with the cell cycle machinery to link cell division events with particular developmental trajectories. In Arabidopsis leaves, the development of stomata, two-celled epidermal valves that mediate plant-atmosphere gas exchange, relies on a series of oriented stem-cell-like asymmetric divisions followed by a single symmetric division. The stomatal lineage is embedded in a tissue whose cells transition from proliferation to post-mitotic differentiation earlier, necessitating stomatal lineage-specific factors to prolong competence to divide. We show that the D-type cyclin, CYCD7;1 is specifically expressed just prior to the symmetric guard-cell forming division, and that it is limiting for this division. Further, we find that CYCD7;1 is capable of promoting divisions in multiple contexts, likely through RBR-dependent promotion of the G1/S transition, but that CYCD7;1 is regulated at the transcriptional level by cell-type specific transcription factors that confine its expression to the appropriate developmental window.

plant biology↗

bioRxiv2017

Stimulation of the final cell cycle in the stomatal lineage by the cyclin CYCD7;1 under regulation of the MYB transcription factor FOUR-LIPS

Abstract (180 words)Stomatal guard cells are formed through a sequence of asymmetric and symmetric divisions in the epidermis of the sporophyte of most land plants. We show that several D-type cyclins are consecutively activated in the stomatal linage in the epidermis of Arabidopsis thaliana. Whereas CYCD2;1 and CYCD3;2 are activated in the meristemoids early in the lineage, CYCD7;1 is activated before the final division. CYCD7;1 expression peaks in the guard mother cell, where its transcription is modulated by the FOUR-LIPS/MYB88 transcription factor. FOUR-LIPS/MYB88 interacts with the CYCD7;1 promoter and represses CYCD7;1 transcription. CYCD7;1 stimulates the final symmetric division in the stomatal lineage, since guard cell formation is delayed in the cycd7;1 mutant epidermis and guard mother cell (GMC) divisions in four-lips mutant guard mother cells are limited by loss of function of CYCD7;1. Hence, the precise activation of a specific D-type cyclin, CYCD7;1, is required for correct timing of the last symmetric division that creates the stomatal guards cells, and CYCD7;1 expression is regulated by the FLP/MYB pathway that ensures cell cycle arrest in the stomatal guard cells.\n\nSummary StatementThe formation of paired guard cells in the epidermis of the Arabidopsis thaliana shoot, requires the activity of the D-type cyclin CYCD7;1 for the normal timing of the final division.

plant biology↗

bioRxiv2017

A Comparison Between Single Cell RNA Sequencing And Single Molecule RNA FISH For Rare Cell Analysis

The development of single cell RNA sequencing technologies has emerged as a powerful means of profiling the transcriptional behavior of single cells, leveraging the breadth of sequencing measurements to make inferences about cell type. However, there is still little understanding of how well these methods perform at measuring single cell variability for small sets of genes and what \"transcriptome coverage\" (e.g. genes detected per cell) is needed for accurate measurements. Here, we use single molecule RNA FISH measurements of 26 genes in thousands of melanoma cells to provide an independent reference dataset to assess the performance of the DropSeq and Fluidigm single cell RNA sequencing platforms. We quantified the Gini coefficient, a measure of rare-cell expression variability, and find that the correspondence between RNA FISH and single cell RNA sequencing for Gini, unlike for mean, increases markedly with per-cell library complexity up to a threshold of [~]2000 genes detected. A similar complexity threshold also allows for robust assignment of multi-genic cell states such as cell cycle phase. Our results provide guidelines for selecting sequencing depth and complexity thresholds for single cell RNA sequencing. More generally, our results suggest that if the number of genes whose expression levels are required to answer any given biological question is small, then greater transcriptome complexity per cell is likely more important than obtaining very large numbers of cells.

genomics↗

bioRxiv2017

Gene Expression Recovery For Single Cell RNA Sequencing

Rapid advances in massively parallel single cell RNA sequencing (scRNA-seq) is paving the way for high-resolution single cell profiling of biological samples. In most scRNA-seq studies, only a small fraction of the transcripts present in each cell are sequenced. The efficiency, that is, the proportion of transcripts in the cell that are sequenced, can be especially low in highly parallelized experiments where the number of reads allocated for each cell is small. This leads to unreliable quantification of lowly and moderately expressed genes, resulting in extremely sparse data and hindering downstream analysis. To address this challenge, we introduce SAVER (Single-cell Analysis Via Expression Recovery), an expression recovery method for scRNA-seq that borrows information across genes and cells to impute the zeros as well as to improve the expression estimates for all genes. We show, by comparison to RNA fluorescence in situ hybridization (FISH) and by data down-sampling experiments, that SAVER reliably recovers cell-specific gene expression concentrations, cross-cell gene expression distributions, and gene-to-gene and cell-to-cell correlations. This improves the power and accuracy of any downstream analysis involving genes with low to moderate expression.

genomics↗

bioRxiv2017

Stability and function of a putative microtubule organizing center in the human parasite Toxoplasma gondii

The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here, we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and discovered that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 ({Delta}kinesinA{Delta}apr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live{Delta} kinesinA{Delta}apr1 parasites, and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and to invade host cells, as well as decreased secretion of effectors important for these processes. Together, the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

cell biology↗

bioRxiv2016

An icosahedral virus as a fluorescent calibration standard: methods for counting protein molecules in cells by fluorescence microscopy

The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report the construction, properties, and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.\n\nLay AbstractA broad goal of modern biology is to understand how the machines within living cells work. It is nowadays routine to identify the individual protein components of a machine, but not yet straightforward to tell how many copies of each component are needed to build a functional assembly. In many types of cells it is now possible to substitute for the native proteins within cells altered versions that are fluorescent. If one knew how much fluorescence is generated by a single molecule of the altered protein, then one could use a light microscope to count the number of copies of the protein in a cellular machine by simply measuring the total fluorescence coming from that part of the cell. This paper describes the construction and methods for using a set of fluorescent virus particles that can be used to determine how much fluorescence is contributed by one molecule of fluorescent protein. The virus particles were chosen for this role because the particular icosahedral symmetry of their structure guarantees that each particle contains exactly 240 copies of one fluorescent protein.

cell biology↗