Biology subjects

Thomas, S.

Publications and source records attributed to Thomas, S..

68 records · Page 4Linked to original sources

bioRxiv2020

Comparison of Rhesus and Cynomolgus macaques as an authentic model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques, resembling the mild clinical cases of COVID-19 in humans. Immune responses against SARS-CoV-2 were also similar in both species and equivalent to those reported in milder infections and convalescent human patients. Importantly, we have devised a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the optimal study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of novel and repurposed interventions against SARS-CoV-2. Accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

microbiology↗

bioRxiv2020

Temporally integrated single cell RNA sequencing analysis of controlled and natural primary human DENV-1 infections

Controlled dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To bridge this knowledge gap, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 controlled human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both controlled and natural DENV infection resulted in overlapping patterns of inflammatory gene upregulation, natural DENV infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by controlled and natural primary DENV infection are similar, but that natural DENV infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state

immunology↗

bioRxiv2020

A pan-genome method to determine core regions of the Bacillus subtilis and Escherichia coli genomes

Synthetic engineering of bacteria to produce industrial products is a burgeoning field of research and application. In order to optimize genome design, designers need to understand which genes are essential, which are optimal for growth, and locations in the genome that will be tolerated by the organism when inserting engineered cassettes. We present a pan-genome based method for the identification of core regions in a genome that are strongly conserved at the species level. We show that these core regions are very likely to contain all or almost all essential genes. We assert that synthetic engineers should avoid deleting or inserting into these core regions unless they understand and are manipulating the function of the genes in that region. Similarly, if the designer wishes to streamline the genome, non-core regions and in particular low penetrance genes would be good targets for deletion. Care should be taken to remove entire cassettes with similar penetrance of the genes within cassettes as they may harbor toxin/antitoxin genes which need to be removed in tandem. The bioinformatic approach introduced here saves considerable time and effort relative to knockout studies on single isolates of a given species and captures a broad understanding of the conservation of genes that are core to a species. ImportanceThe pan-genome approach presented in this paper can be used to determine core regions of a genome and has many possible applications. Synthetic engineering design can be informed by which genes/regions are more conserved (core) versus less conserved. The level of conservation of adjacent non-core genes tends to define cassettes of genes which may be part of a pathway or system that can inform researchers about possible functional significance. The pattern of gene presence across the different genomes of a species can inform the understanding of evolution and horizontal gene acquisition. The approach saves considerable time and effort relative to laboratory methods used to identify essential genes in species.

evolutionary biology↗

bioRxiv2020

Dose-dependent response to infection with SARS-CoV-2 in the ferret model: evidence of protection to re-challenge

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions. Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5x106 pfu) and medium (5x104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5x102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

microbiology↗

bioRxiv2020

Toward a single dose cure for Buruli ulcer

A single dose of TELACEBEC (Q203), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks post treatment. Clinical use of Q203 could dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.

microbiology↗

bioRxiv2020

BioLaboro: A bioinformatics system for detecting molecular assay signature erosion and designing new assays in response to emerging and reemerging pathogens

BackgroundEmerging and reemerging infectious diseases such as the novel Coronavirus disease, COVID-19 and Ebola pose a significant threat to global society and test the public health communitys preparedness to rapidly respond to an outbreak with effective diagnostics and therapeutics. Recent advances in next generation sequencing technologies enable rapid generation of pathogen genome sequence data, within 24 hours of obtaining a sample in some instances. With these data, one can quickly evaluate the effectiveness of existing diagnostics and therapeutics using in silico approaches. The propensity of some viruses to rapidly accumulate mutations can lead to the failure of molecular detection assays creating the need for redesigned or newly designed assays. ResultsHere we describe a bioinformatics system named BioLaboro to identify signature regions in a given pathogen genome, design PCR assays targeting those regions, and then test the PCR assays in silico to determine their sensitivity and specificity. We demonstrate BioLaboro with two use cases: Bombali Ebolavirus (BOMV) and the novel Coronavirus 2019 (SARS-CoV-2). For the BOMV, we analyzed 30 currently available real-time reverse transcription-PCR assays against the three available complete genome sequences of BOMV. Only two met our in silico criteria for successful detection and neither had perfect matches to the primer/probe sequences. We designed five new primer sets against BOMV signatures and all had true positive hits to the three BOMV genomes and no false positive hits to any other sequence. Four assays are closely clustered in the nucleoprotein gene and one is located in the glycoprotein gene. Similarly, for the SARS-CoV-2, we designed five highly specific primer sets that hit all 145 whole genomes (available as of February 28, 2020) and none of the near neighbors. ConclusionsHere we applied BioLaboro in two real-world use cases to demonstrate its capability; 1) to identify signature regions, 2) to assess the efficacy of existing PCR assays to detect pathogens as they evolve over time, and 3) to design new assays with perfect in silico detection accuracy, all within hours, for further development and deployment. BioLaboro is designed with a user-friendly graphical user interface for biologists with limited bioinformatics experience.

bioinformatics↗

bioRxiv2019

Effective Combination Immunotherapy using Oncolytic Viruses to Deliver CAR Targets to Solid Tumors

Chimeric antigen receptor (CAR)-engineered T cell therapy for solid tumors is limited by the lack of tumor-restricted and homogeneous expression of tumor antigens1,2. Therefore, we engineered an oncolytic virus to express a non-signaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-specific CAR T cells. Infecting tumor cells with a chimeric oncolytic vaccinia virus coding for CD19t (OV19t) produced de novo CD19 surface-antigen expression prior to virus-mediated tumor lysis. Co-cultured CD19-CAR T cells secreted cytokines and elicited potent cytolytic activity against infected tumors. Using multiple mouse tumor models, intratumoral delivery of OV19t induced tumor expression of CD19t and improved tumor control following CD19-CAR T cell administration. CAR T cell-mediated tumor killing also promoted release of virus from dying tumor cells, which propogated tumor expression of CD19t. These data demonstrate a novel immunotherapy approach utilizing oncolytic viruses to promote de novo CAR T cell targeting of solid tumors. One Sentence SummaryWe describe a novel and effective combination immunotherapy utilizing oncolytic viruses to deliver de novo cell surface expression of CD19 antigen promoting CD19-CAR T cell anti-tumor responses against solid tumors.

cancer biology↗

bioRxiv2019

Genomic epidemiology of a complex, multi-species plasmid-borne blaKPC carbapenemase outbreak in Enterobacterales in the UK, 2009-2014

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the commonest transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC has historically been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the UK, blaKPC has caused a large-scale, persistent outbreak focused on hospitals in North-West England. This outbreak has evolved to be polyclonal and poly-species, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterisation. We observed the dissemination of blaKPC (predominantly blaKPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 (97%) isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments amongst plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates) and IncR replicons (252/604 isolates). The subset of reconstructed plasmid sequences also highlighted modular exchange amongst non-blaKPC and blaKPC plasmids, and the common presence of multiple replicons within blaKPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales, for the implementation of adequate surveillance approaches, and for control.\n\nIMPORTANCEAntimicrobial resistance is a major threat to the management of infections, and resistance to carbapenems, one of the \"last line\" antibiotics available for managing drug-resistant infections, is a significant problem. This study used large-scale whole genome sequencing over a five-year period in the UK to highlight the complexity of genetic structures facilitating the spread of an important carbapenem resistance gene (blaKPC) amongst a number of bacterial species that cause disease in humans. In contrast to a recent pan-European study from 2012-2013(1), which demonstrated the major role of spread of clonal blaKPC-Klebsiella pneumoniae lineages in continental Europe, our study highlights the substantial plasticity in genetic mechanisms underpinning the dissemination of blaKPC. This genetic flux has important implications for: the surveillance of drug resistance (i.e. making surveillance more difficult); detection of outbreaks and tracking hospital transmission; generalizability of surveillance findings over time and for different regions; and for the implementation and evaluation of control interventions.

genomics↗

bioRxiv2019

Identifying epigenetic biomarkers of established prognostic factors and survival in a clinical cohort of individuals with oropharyngeal cancer

Smoking status, alcohol consumption and HPV infection (acquired through sexual activity) are the predominant risk factors for oropharyngeal cancer and are thought to alter the prognosis of the disease. Here, we conduct epigenome-wide association studies (EWAS) of these factors and [~]3-year survival using Illumina Methylation EPIC blood DNA methylation profiles from 409 individuals in the Head and Neck 5000 (HN5000) study. CpG site associations below our multiple-testing threshold (PBonferroni < 0.05) with both a prognostic factor and with survival were observed in four gene regions: SPEG (smoking), GFI1 (smoking), PPT2 (smoking), and KHD3CL (alcohol consumption). These were further analysed using 2-step Mendelian randomization to assess whether methylation may be a causal mediator of cancer survival. Evidence for mediation was observed only in the SPEG gene region, showing an association with decreased survival (mortality HR: 1.28, 95% CI: 1.14 to 1.43, P: 2.12x10-05). Replication in data from independent datasets, and from HN5000 participants with longer follow-up times is needed to confirm these findings.

epidemiology↗

bioRxiv2019

Individual differences in stereotypy and neuron subtype translatome with TrkB deletion

Motor stereotypies occurring in early-onset neuropsychiatric diseases are associated with dysregulated basal ganglia direct-pathway activity. Disruptions in network connectivity through impaired neuronal structure have been implicated in both rodents and humans. However, the neurobiological mechanisms leading to direct-pathway neuron disconnectivity in stereotypy remain poorly understood. We have a mouse line with Tropomyosin receptor kinase B (TrkB) receptor deletion from D1-expressing cells (D1-Cre-flTrkB) in which a subset of animals shows repetitive rotations and head tics with juvenile onset. Here we demonstrate these behaviors may be associated with abnormal direct-pathway activity by reducing rotations using chemogenetic inhibition of dorsal striatum D1-medium spiny neurons (D1-MSNs) in both juvenile and young adult mice. Taking advantage of phenotypical differences in animals with similar genotype, we then interrogated the D1-MSN specific translatome associated with repetitive behavior by using RNA-sequencing of ribosome-associated mRNA. Detailed translatome analysis followed by multiplexed gene expression assessment revealed profound alterations in neuronal projection and synaptic structure related genes in stereotypy mice. Examination of neuronal morphology demonstrated dendritic atrophy and dendritic spine loss in dorsal striatum D1-MSNs from mice with repetitive behavior. Together, our results uncover phenotype-specific molecular alterations in D1-MSNs that relate to morphological adaptations in mice displaying stereotypy behavior.

neuroscience↗

bioRxiv2019

Immune profiling in M. tuberculosis infection enables stratification of patients with active disease

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem with an estimated 1.7 billion persons infected worldwide. Clinical challenges in TB include the lack of a blood-based test for active disease, and the absence of prognostic biomarkers for early treatment response. Current blood based tests, such as QuantiFERON-TB Gold (QFT), are based on an IFN{gamma} readout following Mtb antigen stimulation. However, they do not distinguish active TB disease from asymptomatic Mtb infection. We hypothesized that the use of TruCulture, an improved immunomonitoring method for whole blood collection and immune stimulation, could improve the discrimination of active disease from latent Mtb infection. To test our hypothesis, we stimulated whole blood from active TB patients (before and after successful treatment), comparing them to asymptomatic latently infected individuals. Mtb-specific antigens (ESAT-6, CFP-10, TB7.7) and live bacillus Calmette-Guerin (BCG) were used for TruCulture stimulation conditions, with direct comparison to QFT. Protein analyses were performed on the culture supernatants using ELISA and Luminex multi-analyte profiling. TruCulture showed an ability to discriminate active TB cases from latent controls (p < 0.0001, AUC = 0.81, 95% CI: 0.69-0.93) as compared to QFT (p = 0.47 AUC = 0.56, 95% CI: 0.40-0.72), based on an IFN{gamma} readout after Mtb antigen stimulation. The stratification of the two groups could be further improved by using the Mtb Ag/BCG IFN{gamma} ratio response (p < 0.0001, AUC = 0.918, 95% CI: 0.84-0.98). We also identified additional cytokines that distinguished latent infection from TB disease; and show that the primary differences between the TruCulture and QFT systems were a result of higher levels of non-specific innate immune activation in QFT tubes, due to the lack of a buffering solution in the latter. We conclude that TruCulture offers a next-generation solution for whole blood stimulation and immunomonitoring with the possibility to discriminate active and latently infected persons.

immunology↗

bioRxiv2018

Validation of a low-cost, carbon dioxide-based cryoablation system for percutaneous tumor ablation

Breast cancer rates are rising in low- and middle-income countries (LMICs), yet there is a lack of accessible and cost-effective treatment. As a result, the cancer burden and death rates are highest in LMICs. In an effort to meet this need, our work presents the design and feasibility of a low-cost cryoablation system using widely-available carbon dioxide as the only consumable. This system uses an 8-gauge outer-diameter needle and Joule-Thomson expansion to percutaneously necrose tissue with cryoablation. Bench top experiments characterized temperature dynamics in ultrasound gel demonstrated that isotherms greater than 2 cm were formed. Further, this system was applied to mammary tumors in an in vivo rat model and necrosis was verified by histopathology. Finally, freezing capacity under a large heat load was assessed with an in vivo porcine study, where volumes of necrosis greater than 1.5 cm in diameter confirmed by histopathology were induced in a highly perfused liver after two 7-minute freeze cycles. These results demonstrate the feasibility of a carbon-dioxide based cryoablation system for improving solid tumor treatment options in resource-constrained environments.

bioengineering↗

bioRxiv2018

Massively parallel dissection of human accelerated regions in human and chimpanzee neural progenitors

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 Human Accelerated Regions (HARs), some of the fastest evolving regions of the human genome. We predicted that 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in human and chimpanzee neural progenitor cells. The species-specific enhancer activity of assayed HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. These findings suggest that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly.

evolutionary biology↗

bioRxiv2017

The Epstein-Barr virus episome maneuvers between nuclear chromatin compartments during reactivation

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposis sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts localize on the EBV side to OriP, the latent origin of replication. This genetic element, and the EBNA1 protein that binds there, are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts localize on the human side to gene-poor regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of a myriad of interchromosomal associations also suggests a role for this type of long - range association in gene regulation.\n\nIMPORTANCEThe human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double stranded DNA virus, Epstein-Barr virus (EBV), uses similar mechanisms as the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and active compartments during reactivation. This study is advances our knowledge of the EBV life cycle, adding three-dimensional re-localization as a novel component to the molecular events that occur during reactivation. Furthermore, the data adds to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

microbiology↗