Biology subjects

Smith, P.

Publications and source records attributed to Smith, P..

51 records · Page 3Linked to original sources

bioRxiv2021

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected Patients

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

microbiology↗

bioRxiv2021

Cholesterol Oxidation Modulates the Formation of Liquid-Ordered Domains in Model Membranes

7-ketocholesterol (KChol) is one of the most cytotoxic oxysterols found in the plasma membrane, and increased levels of KChol are associated with numerous pathologies. It is thought to induce apoptosis via inactivation of the phosphatidylinositol 3-kinase/Akt signaling pathway -- a pathway that depends on lipid-rafts as signaling platforms. By means of coarse-grained molecular dynamics simulations, we demonstrate that KChol disrupts the liquid-liquid phase separation seen in an equimolar mixture of (dipalmitoylphosphatidylcholine) DPPC, (dioleoylphosphatidylcholine) DOPC, and Cholesterol (Chol). This disruption occurs via two mechanisms: i) KChol adopts a wider range of orientations with the membrane, which disrupts the packing of neigh-boring lipids and ii) KChol has no preference for DPPC over DOPC, which is the main driving force for lateral demixing in DPPC/DOPC/Chol membranes. This provides a molecular description of the means by which KChol induces apoptosis, and illustrates that a single chemical substitution to cholesterol can have a profound impact on the lateral organization of lipid membranes. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/445501v3_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@10777a1org.highwire.dtl.DTLVardef@13ef4c9org.highwire.dtl.DTLVardef@38e0c4org.highwire.dtl.DTLVardef@2c7b91_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

The genomic landscape of early stage ovarian high grade serous carcinoma

PurposeHigh grade serous carcinoma (HGSC) is the commonest type of ovarian cancer. Nearly all HGSC cases are diagnosed at late stage and it is not clear whether early stage HGSC has unique characteristics compared to late stage tumours. Experimental DesignWe analysed samples from 45 patients with FIGO stage I - IIA HGSC - 40 from the pathology archives of three large UK cancer centres and 5 from the BriTROC-1 study. We performed shallow whole genome sequencing (sWGS) and targeted next generation sequencing to investigate somatic mutations and copy number alterations. We compared results to 51 stage IIIC/IV HGSC patients from the BriTROC-1 study. ResultsThere was no difference in median age between the early stage (median 61.3 years, range 40-84) and late stage (median 62.3 years, range 34-76) patients at diagnosis. TP53 mutations were near-universal (92% early stage, 100% late stage samples) and there were no significant differences in the rates of other somatic mutations, including BRCA1 and BRCA2, or focal copy number alterations between early- and late-stage cohorts. There were also no unique amplifications or deletions in either cohort. However, median ploidy was greater in late stage (median 3.1) than early stage (median 2.0) samples. In addition, there were higher numbers of breakpoints per 10MB and per chromosome arm and higher absolute copy number in late stage than early stage cohorts; early stage samples had longer segment length. Overall copy number signature exposures were significantly different between early and late stage samples with greater signature 3 exposure in early stage and greater signature 4 in late stage. Both simplex plot and unsupervised hierarchical clustering suggested that a subset of late stage samples retain early stage appearances with high signature 3 and co-clustering with the early stage samples ConclusionsThese data suggest that there are no unique mutations or focal copy number alterations in early stage HGSC. However, whole genome duplication is significantly more common in late-stage disease, suggesting evolution during disease progression. However, a subset of late stage HGSC retains early-stage features, which are associated with improved overall survival.

cancer biology↗

bioRxiv2021

LiPyphilic: A Python toolkit for the analysis of lipid membrane simulations

Molecular dynamics simulations are now widely used to study emergent phenomena in lipid membranes with complex compositions. Here, we present LiPyphilic - a fast, fully tested, and easy to install Python package for analysing such simulations. Analysis tools in LiPyphilic include the identification of cholesterol flip-flop events, the classification of local lipid environments, and the degree of interleaflet registration. LiPyphilic is both force field and resolution agnostic, and thanks to the powerful atom selection language of MDAnalysis it can handle membranes with highly complex compositions. LiPyphilic also offers two on-the-fly trajectory transformations to i) fix membranes split across periodic boundaries and ii) perform nojump coordinate unwrapping. Our implementation of nojump unwrapping accounts for fluctuations in box volume under the NPT ensemble -- an issue that most current implementations have overlooked. The full documentation of LiPyphilic, including installation instructions, is available at https://lipyphilic.readthedocs.io/en/latest. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/442445v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@137f053org.highwire.dtl.DTLVardef@18302cdorg.highwire.dtl.DTLVardef@2cf919org.highwire.dtl.DTLVardef@95fa40_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Full species-wide leaf and seed ionomic diversity of Arabidopsis thaliana

O_LISoil is a heterogenous reservoir of essential elements needed for plant growth and development. Plants have evolved mechanisms to balance their nutritional needs based on availability of nutrients. This has led to genetically-based variation in the elemental composition ionome, of plants, both within and between species. C_LIO_LIWe explore this natural variation using a panel of wild-collected, geographically widespread Arabidopsis thaliana accessions from the 1001 Genomes Project including over 1,135 accessions, and the 19 parental accessions of the Multi-parent Advanced Generation Inter-Cross (MAGIC) panel, all with full-genome sequences available. C_LIO_LIWe present an experimental design pipeline for high-throughput ionomic screenings and analyses with improved normalisation procedures to account for errors and variability in conditions often encountered in large-scale, high-throughput data collection. We report quantification of the complete leaf and seed ionome of the entire collection using this pipeline and a digital tool-IonExplorer to interact with the dataset. C_LIO_LIWe describe the pattern of natural ionomic variation across the A. thaliana species and identify several accessions with extreme ionomic profiles. It forms a valuable resource for exploratory QTL, GWA studies to identify genes underlying natural variation in leaf and seed ionome and genetic adaptation of plants to soil conditions. C_LI

plant biology↗

bioRxiv2020

Asymmetric phospholipids impart novel biophysical properties to lipid bilayers allowing environmental adaptation

Phospholipids are a diverse group of biomolecules consisting of a hydrophilic head group and two hydrophobic acyl tails. The nature of the head and length and saturation of the acyl tails are important for defining the biophysical properties of lipid bilayers. It has recently been shown that the membranes of certain yeast species contain high levels of unusual asymmetric phospholipids, consisting of one long and one medium chain acyl moiety - a configuration not common in mammalian cells or other well studied model yeast species. This raises the possibility that structurally asymmetric phospholipids impart novel biophysical properties to the yeast membranes. Here, we use atomistic molecular dynamics simulations (MD) and environmentally-sensitive fluorescent membrane probes to characterize key biophysical parameters of membranes formed from asymmetric lipids for the first time. Interestingly, we show that saturated, but asymmetric phospholipids maintain membrane lipid order across a wider range of temperatures and do not require acyl tail unsaturation or sterols to maintain their properties. This may allow cells to maintain membrane fluidity even in environments which lack the oxygen required for the synthesis of unsaturated lipids and sterols.

biophysics↗

bioRxiv2020

Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-{beta}-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-{beta}-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-{beta}-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-{beta}-glucan content. (1,3;1,4)-{beta}-Glucan was absent in the grain of cslf6 knock-out lines whereas cslf9 knock-out lines had similar (1,3;1,4)-{beta}-glucan content to WT. However, cslf9 mutants showed changes in the abundance of other cell wall-related monosaccharides compared to WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knock-outs and to a lesser extent TGW in cslf9 knock-outs. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-{beta}-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-{beta}-glucan in mature grain.

plant biology↗

bioRxiv2020

Deep analysis of the USP18-dependent ISGylome and proteome unveils important roles for USP18 in tumour cell antigenicity and radiosensitivity

The deubiquitylating enzyme USP18 is a major negative regulator of the interferon (IFN) signalling cascade. IFN pathways contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy and are often deregulated in cancer. USP18 is the predominant human protease that cleaves interferon-stimulated gene ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line (HAP1) treated with type I IFN. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results suggest USP18 as a pharmacological target in cancer immunotherapy and radiotherapy.

cancer biology↗

bioRxiv2020

A Grain of Salt

We quantified grain sodium (Na+) content across a barley GWAS panel grown under optimal conditions. We identified a strong association with a region containing two low and one high Na+ accumulating haplotypes of a Class 1 HIGH-AFFINITY POTASSIUM TRANSPORTER (HKT1;5) known to be involved in regulating plant Na+ homeostasis. The haplotypes exhibited an average 1.8-fold difference in grain Na+ content. We show that an L189P substitution disrupts Na+ transport in the high Na+ lines, disturbs the plasma membrane localisation typical of HKT1;5 and induces a conformational change in the protein predicted to compromise function. Under NaCl stress, lines containing P189 accumulate high levels of Na+, but show no significant difference in biomass. P189 increases in frequency from wild-species to elite cultivars leading us to speculate that the compromised haplotype is undergoing directional selection possibly due to the value of Na+ as a functional nutrient in non-saline environments.

genetics↗

bioRxiv2019

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: Q22T, W123R, F155G, Y169M, W438D, V401A

A key goal of protein engineering is to accurately model the stability and catalytic activity of enzymes. However, the limitations of functional predictive abilities pose a major challenge for modeling algorithm design, and can be attributed to the lack of large data sets quantifying the functional properties of enzymes. Here, the thermal stability (TM) and Michaelis-Menten constants (kcat, KM, and kcat/KM) of six new variants of the {beta}-glucosidase B (BglB) protein are quantitatively characterized. Molecular stability of the enzyme variants were hypothesized using the Foldit software and BglB was synthesized in E. coli cells. Testing was done through a colorimetric kinetic assay and thermal stability fluorescence-based protein unfolding assay. Results from the assays suggest that all mutations, with the exception of variant Y169M, all experienced reduced catalytic efficiency compared to the wildtype. Assay results indicate that variant W123R is more thermally stable compared to the wildtype, while the differences in thermal stability between the other variants, and the wildtype are negligible. The collected thermal stability and catalytic efficiency data has been added to a data set with the aim of improving Rosetta algorithms for modeling and predicting the functional interactions between biomolecules through a machine learning algorithm and facilitate the precise engineering of protein catalysts.

biochemistry↗

bioRxiv2019

Neutrophils mediate the capture of peritoneal contaminants by fat-associated lymphoid clusters of the omentum

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs), which collects peritoneal contaminants and provides a first layer of immunological defence within the abdomen. Using single-cell RNA sequencing and spatial analysis of omental stromal cells, we reveal that the surface of FALCs are covered with specialised mesothelial cells, which we name FALC cover cells. We demonstrate that CXCL1 is expressed by FALC cover cells and that CXCL1 is critical for the retention and accumulation of neutrophils within FALCs during peritonitis. We show that protein arginine deiminase 4 mediates the formation of dense neutrophil aggregates, which are required for the neutralisation of particles present in the peritoneal cavity. Finally, we provide confirmatory evidence in humans with acute appendicitis, that the omentum is also a site of neutrophil recruitment and bacterial capture, and is thus an important component of the immunological defence against the propagation of peritoneal contaminants.

immunology↗

bioRxiv2019

Coupling multiplex pre-amplification and droplet digital PCR for longitudinal monitoring of ESR1 and PIK3CA mutations from plasma cell-free DNA

Here we report on the development of a sensitive and cost-effective method to longitudinally track ESR1 and PIK3CA mutations from cfDNA in patients with metastatic breast cancer (MBC) using a streamlined and de-centralized workflow. Hotspot mutations in ESR1 have been shown to cause resistance to aromatase inhibitor-based and anti-estrogenic therapies, while PIK3CA mutations have high prevalence in MBC. As a result, their utility as circulating biomarkers to predict or monitor response in the clinical development of investigational compounds has been the focus of many studies. Six regions in ESR1 and PIK3CA genes containing 20 hotspot mutations were pre-amplified, followed by optimized singleplex ddPCR assays to detect allele frequencies of individual mutations. Without pre-amplification, the limit of detection (LOD) and limit of linearity (LOL) of individual ddPCR assays were at 0.05-0.1% and 0.25% level, respectively. With pre-amplification, the LOD and LOL were slightly elevated at 0.1-0.25% and 0.25-0.5% levels, respectively. High concordance was achieved to the BEAMing assay (Sysmex Inostics) for mutation positive assays (r=0.98, P<0.0001). In conclusion, coupling pre-amplification and ddPCR assays allowed us for the detection of up to 20 hot spot mutations in ESR1 and PIK3CA with high sensitivity and reproducibility.

genetics↗

bioRxiv2017

Novel Compensatory Mechanisms Enable the Mutant KCNT1 Channels to Induce Seizures

Mutations in the sodium-activated potassium channel (KCNT1) gene are linked to epilepsy. Surprisingly, all KCNT1 mutations examined to date increase K+ current amplitude. These findings present a major neurophysiological paradox: how do gain-of-function KCNT1 mutations expected to silence neurons cause epilepsy? Here, we use Drosophila to show that expressing mutant KCNT1 in GABAergic neurons leads to seizures, consistent with the notion that silencing inhibitory neurons tips the balance towards hyperexcitation. Unexpectedly, mutant KCNT1 expressed in motoneurons also causes seizures. One striking observation is that mutant KCNT1 causes abnormally large and spontaneous EJPs (sEJPs). Our data suggest that these sEJPs result from local depolarization of synaptic terminals due to a reduction in Shaker channel levels and more active Na+ channels. Hence, we provide the first in vivo evidence that both disinhibition of inhibitory neurons and compensatory plasticity in motoneurons can account for the paradoxical effects of gain-of-function mutant KCNT1 in epilepsy.

neuroscience↗

bioRxiv2017

Regulation of Life Span by the Gut Microbiota in The Short-Lived African Turquoise Killifish

Gut bacteria occupy the interface between the organism and the external environment, contributing to homeostasis and disease. Yet, the causal role of the gut microbiota during host aging is largely unexplored. Here, using the African turquoise killifish (Nothobranchius furzeri), a naturally short-lived vertebrate, we show that the gut microbiota plays a key role in modulating vertebrate life span. Recolonizing the gut of middle-age individuals with bacteria from young donors resulted in life span extension and delayed behavioral decline. This intervention prevented the decrease in microbial diversity associated with host aging and maintained a young-like gut bacterial community, characterized by overrepresentation of the key genera Exiguobacterium, Planococcus, Propionigenium and Psychrobacter. Our findings demonstrate that the natural microbial gut community of young individuals can causally induce long-lasting beneficial systemic effects that lead to life span extension in a vertebrate model.

genomics↗

bioRxiv2016

Genome-wide meta-analysis of cognitive empathy: heritability, and correlates with sex, neuropsychiatric conditions and brain anatomy

We conducted a genome-wide meta-analysis of cognitive empathy using the Reading the Mind in the Eyes Test (Eyes Test) in 88,056 research volunteers of European Ancestry (44,574 females and 43,482 males) from 23andMe Inc., and an additional 1,497 research volunteers of European Ancestry (891 females and 606 males) from the Brisbane Longitudinal Twin Study (BLTS). We confirmed a female advantage on the Eyes Test (Cohens d = 0.21, P < 2.2x10-16), and identified a locus in 3p26.1 that is associated with scores on the Eyes Test in females (rs7641347, Pmeta = 1.58 x 10-8). Common single nucleotide polymorphisms (SNPs) explained 5.8% (95% CI: 0.45 - 0.72; P = 1.00 x 10-17) of the total trait variance in both sexes, and we identified a twin heritability of 0.28 (95% CI: 0.13-0.42). Finally, we identified significant genetic correlation between the Eyes Test and anorexia nervosa, measures of empathy (the Empathy Quotient), openness (NEO-Five Factor Inventory), and different measures of educational attainment and cognitive aptitude, and show that the genetic determinants of volumes of the dorsal striatum (caudate nucleus and putamen) are positively correlated with the genetic determinants of performance on the Eyes Test.

genetics↗