Biology subjects

Smith, D. J.

Publications and source records attributed to Smith, D. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly1. The Omicron variant termed BA.2, which differs genetically substantially from BA.1, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed2,3. Here, we used antigenic cartography to quantify and visualize antigenic differences between SARS-CoV-2 variants using hamster sera obtained after primary infection. Whereas early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape (vaccine-induced) antibody responses as a result of different antigenic characteristics. Close monitoring of the antigenic changes of SARS-CoV-2 using antigenic cartography can be helpful in the selection of future vaccine strains.

microbiology↗

bioRxiv2022

Single cell analysis of gene expression in the substantia nigra pars compacta of a pesticide-induced mouse model of Parkinson's disease

Exposure to pesticides in humans increases the risk of Parkinsons disease (PD), but the mechanisms remain poorly understood. To elucidate these pathways, we dosed C57BL/6J mice with a combination of the pesticides maneb and paraquat (MNPQ). Behavioral analysis revealed motor deficits consistent with PD. Single cell RNA sequencing of substantia nigra pars compacta revealed both cell-type specific genes and genes expressed differentially between pesticide and control, including Fam241b, Emx2os, Bivm, Gm1439, Prdm15 and Rai2. Neurons had the largest number of significant differentially expressed genes, but comparable numbers were found in astrocytes and less so in oligodendrocytes. In addition, network analysis revealed enrichment in functions related to the extracellular matrix. These findings emphasize the importance of support cells in pesticide-induced PD and refocus our attention away from neurons as the sole agent of this disorder.

neuroscience↗

bioRxiv2022

Mapping SARS-CoV-2 antigenic relationships and serological responses

During the SARS-CoV-2 pandemic, multiple variants escaping pre-existing immunity emerged, causing concerns about continued protection. Here, we use antigenic cartography to analyze patterns of cross-reactivity among a panel of 21 variants and 15 groups of human sera obtained following primary infection with 10 different variants or after mRNA-1273 or mRNA-1273.351 vaccination. We find antigenic differences among pre-Omicron variants caused by substitutions at spike protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months post-2nd dose. We find changes in immunodominance of different spike regions depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine strain selection. One sentence summaryAntigenic Cartography of SARS-CoV-2 variants reveals amino acid substitutions governing immune escape and immunodominance patterns.

immunology↗

bioRxiv2022

Analysis of SARS-CoV-2 Omicron Neutralization Data up to 2021-12-22

The rapid spread of the Omicron BA.1 (B.1.1.529.1) SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) variant in 2021 resulted in international efforts to quickly assess its escape from immunity generated by vaccines and previous infections. Numerous laboratories published BA.1 neutralization data as preprints and reports. We collated this data in real time and regularly presented updates of the aggregated results in US, European and WHO research and advisory settings. Here, we retrospectively analyzed the accuracy of these aggregations from 85 different sources published during a time period from 2021/12/08 up to 2022/08/14. We found that the mean titer fold change from wild type-like variants to BA.1, a standard measure of a variants immune escape, remained stable after the first 15 days of data reporting in people who were twice vaccinated, and incoming data increased the confidence in this quantity. Further, it is possible to build reliable, stable antigenic maps from this collated data already after one month of incoming data. We here demonstrate that combining early reports from variable, independent sources can rapidly indicate a new virus variants immune escape and can therefore be of immense benefit for public health.

immunology↗

bioRxiv2021

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains

Although mRNA vaccines prevent COVID-19, variants jeopardize their efficacy as immunity wanes. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike) or modified (mRNA-1273.351, designed for B.1.351 spike) preclinical Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Immunization with high or low dose formulations of mRNA vaccines induced neutralizing antibodies in serum against ancestral SARS-CoV-2 and several variants, although levels were lower particularly against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. Nonetheless, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, and serve as a possible model for waning immunity, showed breakthrough lung infection and pneumonia with B.1.617.2. Thus, as levels of immunity induced by mRNA vaccines decline, breakthrough infection and disease likely will occur with some SARS-CoV-2 variants, suggesting a need for additional booster regimens.

microbiology↗

bioRxiv2021

Vaccination with B.1.1.7, B.1.351 and P.1 variants protects mice from challenge with wild type SARS-CoV-2

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against coronavirus disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild type SARS-CoV-2 and from variants B.1.1.7, B.1.351 and P.1 for their immunogenicity and protective effect in vivo against challenge with wild type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7 vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild type SARS-CoV-2 in a mouse model. Author SummaryThe emergence of variants of SARS-CoV-2 has led to an urgency to study whether vaccines will lead to cross-protection against these variants. Here, we demonstrate that vaccination with spike proteins of various variants leads to cross-neutralizing responses, as well as protection in a mouse model against wild type SARS-CoV-2.

immunology↗

bioRxiv2021

Cost-effective mapping of genetic interactions in mammalian cells

Comprehensive maps of genetic interactions in mammalian cells are daunting to construct because of the large number of potential interactions, ~ 2 x 108 for protein coding genes. We previously used co-inheritance of distant genes from published radiation hybrid (RH) datasets to identify genetic interactions. However, it was necessary to combine six legacy datasets from four species to obtain adequate statistical power. Mapping resolution was also limited by the low density PCR genotyping. Here, we employ shallow sequencing of nascent human RH clones as an economical approach to constructing interaction maps. In this initial study, 15 clones were analyzed, enabling construction of a network with 225 genes and 2359 interactions (FDR < 0.05). Despite its small size, the network showed significant overlap with the previous RH network and with a protein-protein interaction network. Consumables were [lsim] $50 per clone, showing that affordable, high quality genetic interaction maps are feasible in mammalian cells.

genomics↗

bioRxiv2021

Extreme Phenotypic Diversity in Operant Responding for an Intravenous Cocaine or Saline Infusion in the Hybrid Mouse Diversity Panel

Cocaine self-administration is complexly determined trait, and a substantial proportion of individual differences in cocaine use is determined by genetic variation. Cocaine intravenous self-administration (IVSA) procedures in laboratory animals provide opportunities to prospectively investigate neurogenetic influences on the acquisition of voluntary cocaine use. Large and genetically diverse mouse populations, including the Hybrid Mouse Diversity Panel (HMDP), have been developed for forward genetic approaches that can reveal genetic variants that influence traits like cocaine IVSA. This population enables high resolution and well-powered genome wide association studies, as well as the discovery of genetic correlations. Here, we provide information on cocaine (or saline - as a control) IVSA in 65 strains of the HMDP. We found cocaine IVSA to be substantially heritable in this population, with strain-level intake ranging for near zero to >25 mg/kg/session. Though saline IVSA was also found to be heritable, a very modest genetic correlation between cocaine and saline IVSA indicates that operant responding for the cocaine reinforcer was influenced by a substantial proportion of unique genetic variants. These data indicate that the HMDP is suitable for forward genetic approaches for the analysis of cocaine IVSA, and this project has also led to the discovery of reference strains with extreme cocaine IVSA phenotypes, revealing them as polygenic models of risk and resilience to cocaine reinforcement. This is part of an ongoing effort to characterize genetic and genomic variation that moderates cocaine IVSA, which may, in turn, provide a more comprehensive understanding of cocaine risk genetics and neurobiology.

animal behavior and cognition↗

bioRxiv2020

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise short-lived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

biochemistry↗

bioRxiv2020

The impediment to replication at tRNA genes in S. cerevisiae does not require tRNA transcription, and is facilitated by topoisomerases and Rad18-dependent repair pathways

tRNA genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae. tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

molecular biology↗

bioRxiv2019

Reduced cellular levels of DNA polymerase delta alter replication dynamics and enzymology, and impair lagging-strand processing

DNA polymerase delta (Pol{delta} ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol{delta} is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol{delta} has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol{delta} subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol{delta} in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol{delta} impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol{delta} depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol{delta} is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol{delta} is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol{delta} is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability. SIGNIFICANCE STATEMENTDNA replication in eukaryotes is carried out by the replisome - a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol{delta} ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol{delta} cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol{delta} in budding yeast - a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol{delta} scarcity. The defects we observe provide insight into how destabilizing Pol{delta} mutations lead to genome instability.

molecular biology↗

bioRxiv2019

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ~2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci and 3 loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ~30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

genetics↗

bioRxiv2019

Light-induced polyp retraction and tissue rupture in the photosensitive, reef-building coral Acropora muricata

Coral reefs are in alarming decline due to climate emergency, pollution and other man-made disturbances. The numerous ecosystem services derived from coral reefs are underpinned by the growth and physical complexity of reef-forming corals. Our knowledge of their fundamental biology is limited by available technology. We need a better understanding of larval settlement and development, skeletogenesis, interactions with pathogens and symbionts, and how this biology interacts with environmental factors such as light exposure, temperature, and ocean acidification. We here focus on a fast-growing key coloniser, Acropora muricata. To enable dynamic imaging of the photosensitive organism at different scales, we developed light-sheet illumination for fluorescence microscopy of small coral colonies. Our approach reveals live polyps in previously unseen detail. An imaging range for Acropora muricata with no measurable photodamage is defined based upon polyp expansion, coral tissue reaction, and photobleaching. We quantify polyp retraction as a photosensitive behavioural response and show sparse zooxanthellar expulsion and coral tissue rupture at higher intensities of blue light. The simple and flexible technique enables non-invasive continuous dynamic imaging of highly photosensitive organisms with sizes between 1 mm3 and 5 cm3, for eight hours, at high temporal resolution, on a scale from multiple polyps down to cellular resolution. This live imaging tool opens a new window into the dynamics of reef-building corals.

developmental biology↗

bioRxiv2019

Carotid intima-media thickness in UK Biobank: Identification of novel genome-wide loci, sex-specific effects and genetic correlations with obesity and glucometabolic traits.

ObjectivesAtherosclerosis is the underlying cause of most cardiovascular disease, but mechanisms underlying atherosclerosis are incompletely understood. Ultra-sound measurement of the carotid artery intima-media thickness (cIMT) can be used to measure vascular remodelling, which is indicative of atherosclerosis. Genome-wide association studies have identified a number of genetic loci associated with cIMT, but heterogeneity of measurements collected by many small cohorts have been a major limitation in these efforts. Here we conducted genome-wide association analyses in UK Biobank (N=22,179), the largest single study with consistent cIMT measurements.\n\nApproach and resultsWe used BOLT-LMM to run linear regression of cIMT in UK Biobank, adjusted for age, sex, genotyping platform and population structure. In white British participants, we identified 4 novel loci associated with cIMT and replicated most previously reported loci. In the first sex-specific analyses of cIMT, we identified a female-specific locus on Chromosome 5, associated with cIMT in women only and highlight VCAN as a good candidate gene at this locus. Genetic correlations with body-mass index and glucometabolic traits were also observed.\n\nConclusionThese findings replicate previously reported associations, highlight novel biology and provide new directions for investigating the sex differences observed in cardiovascular disease presentation and progression.

genetics↗

bioRxiv2019

Understanding Cognitive Impairment in Mood Disorders: Mediation Analyses in the UK Biobank Cohort

BackgroundCognitive impairment is strongly linked with persistent disability in people with mood disorders, but the factors that explain cognitive impairment in this population are unclear.\n\nAimsWe aimed to estimate the total effect of (i) bipolar disorder (BD) and (ii) major depression on cognitive function, and the magnitude of the effect that was explained by potentially modifiable intermediate factors.\n\nMethodCross-sectional study using baseline data from the UK Biobank cohort. Participants were categorised as BD (N=2,709), major depression (N=50,975), or no mood disorder (N=102,931 to 105,284). The outcomes were computerised tests of reasoning, reaction time and memory. The potential mediators were cardiometabolic disease and psychotropic medication. Analyses were informed by graphical methods, and controlled for confounding using regression, propensity score-based methods, and G-computation.\n\nResultsGroup differences of small magnitude were found on a visuospatial memory test. Z-score differences for BD were in the range -0.23 to -0.17 (95% CI range -0.39 to -0.03) across different estimation methods, and approximately -0.07 (95% CI -0.10 to -0.03) for major depression. One-quarter of the effect was mediated via psychotropic medication in the BD group (-0.05; 95% CI -0.09 to -0.01). No evidence was found for mediation via cardiometabolic disease.\n\nConclusionsIn a large community-based sample in middle to early old age, BD and depression were associated with lower visuospatial memory performance, in part potentially due to psychotropic medication use. Mood disorders and their treatments will have increasing importance for population cognitive health as the proportion of older adults continues to grow.

epidemiology↗

bioRxiv2019

Novel genome-wide associations for anhedonia, genetic correlation with psychiatric disorders, and polygenic association with brain structure.

Anhedonia is a core feature of several psychiatric disorders but its biological underpinnings are poorly understood. We performed a genome-wide association study of anhedonia in 375,275 UK Biobank participants and assessed for genetic correlation between anhedonia and neuropsychiatric conditions (major depressive disorder, schizophrenia, bipolar disorder, obsessive compulsive disorder and Parkinsons Disease). We then used a polygenic risk score approach to test for association between genetic loading for anhedonia and both brain structure and brain function. This included: magnetic resonance imaging (MRI) assessments of total grey matter volume, white matter volume, cerebrospinal fluid volume, and 15 cortical/subcortical regions of interest; diffusion tensor imaging (DTI) measures of white matter tract integrity; and functional MRI activity during an emotion processing task. We identified 11 novel loci associated at genome-wide significance with anhedonia, with a SNP heritability estimate (h2SNP) of 5.6%. Strong positive genetic correlations were found between anhedonia and major depressive disorder, schizophrenia and bipolar disorder; but not with obsessive compulsive disorder or Parkinsons Disease. Polygenic risk for anhedonia was associated with poorer brain white matter integrity, smaller total grey matter volume, and smaller volumes of brain regions linked to reward and pleasure processing, including nucleus accumbens, caudate and medial frontal cortex. In summary, the identification of novel anhedonia-associated loci substantially expands our current understanding of the biological basis of anhedonia and genetic correlations with several psychiatric disorders confirm the utility of this trait as a transdiagnostic marker of vulnerability to mental illness. We also provide the first evidence that genetic risk for anhedonia influences brain structure, particularly in regions associated with reward and pleasure processing.

genetics↗

bioRxiv2019

Simulations of particle tracking in the oligociliated mouse node and implications for left-right symmetry breaking mechanics

The concept of internal anatomical asymmetry is familiar; usually in humans the heart is on the left and the liver is on the right, however how does the developing embryo know to produce this consistent laterality? Symmetry breaking initiates with left-right asymmetric cilia-driven fluid mechanics in a small fluid-filled structure called the ventral node in mice. However the question of what converts this flow into left-right asymmetric development remains unanswered. A leading hypotheses is that flow transports morphogen containing vesicles within the node, the absorption of which results in asymmetrical gene expression. To investigate how vesicle transport might result in the situs patterns observed in wildtype and mutant experiments, we extend the open source Stokes flow package, NEAREST, to consider the hydrodynamic and Brownian motion of particles in a mouse model with flow driven by one, two, and 112 beating cilia.Three models for morphogen-containing particle released are simulated to assess their compatibility with observed results in oligociliated and wildtype mouse embryos: uniformly random release, localised cilium stress induced release, and localised release from motile cilia themselves. Only the uniformly random release model appears consistent with the data, with neither localised-release model resulting in significant transport in the oligociliated embryo.

developmental biology↗

bioRxiv2019

Chromatin activity at GWAS loci identifies T cell states driving complex immune diseases

Complex immune disease variants are enriched in active chromatin regions of T cells and macrophages. However, whether these variants function in specific cell states or stages of cell activation is unknown. We stimulated T cells and macrophages in the presence of thirteen different cytokine cocktails linked to immune diseases and profiled active enhancers and promoters together with regions of open chromatin. We observed that T cell activation induced major chromatin remodelling, while additional exposure to cytokines fine-tuned the magnitude of these changes. Therefore, we developed a new statistical method that accounts for subtle changes in chromatin landscape to identify SNP enrichment across cell states. Our results point towards the role of immune disease variants in early rather than late activation of memory CD4+ T cells, and with limited differences across polarizing cytokines. Furthermore, we demonstrate that inflammatory bowel disease variants are enriched in chromatin regions active in Th1 cells, while asthma variants overlap regions active in Th2 cells. We also show that Alzheimers disease variants are enriched in different macrophage cell states. Our results represent the first in-depth analysis of immune disease variants across a comprehensive panel of activation states of T cells and macrophages.

genetics↗