Bacterial phase variation associated with repetitive DNA

Phase variation is mechanism of phenotypic switching used by many pathogenic bacterial species. This thesis describes work on three aspects of phase variation. Mathematical models are described which can be used to determine the rate of phase variation and subsequently the influence of variation rate and fitness differences associated with the altered phenotype on population structure. An approach to whole genome analysis has been developed which has been used to identify putative phase variable contingency genes in H. pylori, T. pallidum and N. meningitidis. This has identified many new contingency genes likely to be involved in host - bacterium and bacterium population interactions. Finally, a detailed molecular investigation of the promoter of the phase variable opc gene of N. meningitidis is presented. In this it is shown that the promoter located homopolymeric tract controls transcription by affecting the relative spacing and facing of promoter components, that this determines RNA polymerase binding to the promoter, and that this interaction involves direct contact of the α-subunit of RNA polymerase with the promoter. In addition it is shown that transcription is dependent upon an IHF consensus sequence in the opc promoter

Inter-species horizontal transfer resulting in core-genome and niche-adaptive variation within Helicobacter pylori

Background Horizontal gene transfer is central to evolution in most bacterial species. The detection of exchanged regions is often based upon analysis of compositional characteristics and their comparison to the organism as a whole. In this study we describe a new methodology combining aspects of established signature analysis with textual analysis approaches. This approach has been used to analyze the two available genome sequences of H. pylori. Results This gene-by-gene analysis reveals a wide range of genes related to both virulence behaviour and the strain differences that have been relatively recently acquired from other sequence backgrounds. These frequently involve single genes or small numbers of genes that are not associated with transposases or bacteriophage genes, nor with inverted repeats typically used as markers for horizontal transfer. In addition, clear examples of horizontal exchange in genes associated with 'core' metabolic functions were identified, supported by differences between the sequenced strains, including: ftsK, xerD and polA. In some cases it was possible to determine which strain represented the 'parent' and 'altered' states for insertion-deletion events. Different signature component lengths showed different sensitivities for the detection of some horizontally transferred genes, which may reflect different amelioration rates of sequence components. Conclusion New implementations of signature analysis that can be applied on a gene-by-gene basis for the identification of horizontally acquired sequences are described. These findings highlight the central role of the availability of homologous substrates in evolution mediated by horizontal exchange, and suggest that some components of the supposedly stable 'core genome' may actually be favoured targets for integration of foreign sequences because of their degree of conservation

Understanding the potential role of whole genome sequencing (WGS) in managing patients with gonorrhoea : a systematic review of WGS use on human pathogens in individual patient care

Objectives The utility of whole genome sequencing (WGS) to inform sexually transmitted infection (STI) patient management is unclear. Timely WGS data might support clinical management of STIs by characterising epidemiological links and antimicrobial resistance profiles. We conducted a systematic review of clinical application of WGS to any human pathogen that may be transposable to gonorrhoea. Methods We searched six databases for articles published between 01/01/2010-06/02/2023 that reported on real/near real-time human pathogen WGS to inform clinical intervention. All article types from all settings were included. Findings were analysed using narrative synthesis. Results We identified 12,179 articles, of which eight reported applications to inform tuberculosis (n=7) and gonorrhoea (n=1) clinical patient management. WGS data were successfully used as an adjunct to clinical and epidemiological data to enhance contact-tracing (n=2), inform antimicrobial therapy (n=5) and identify cross-contamination (n=1). WGS identified gonorrhoea transmission chains that were not established via partner notification. Future applications could include insights into pathogen exposure detected within sexual networks for targeted patient management. Conclusions While there was some evidence of WGS use to provide individualised tuberculosis and gonorrhoea treatment, the eight identified studies contained few participants. Future research should focus on testing WGS intervention effectiveness and examining ethical considerations of STI WGS use

Preparing for PrEP: estimating the size of the population eligible for HIV pre-exposure prophylaxis among men who have sex with men in England.

OBJECTIVES: The size of the population of men who have sex with men (MSM) who may be eligible for HIV pre-exposure prophylaxis (HIV-PrEP) in England remains unknown. To plan for a national PrEP implementation trial, we estimated the number of MSM attending sexual health clinics (SHCs) that may be eligible for HIV-PrEP in England. METHODS: Sexually transmitted infection (STI) surveillance data from 2010 to 2015 from the GUMCAD surveillance system were used to estimate the annual number of HIV-negative MSM who may be eligible for HIV-PrEP in England. Based on national eligibility criteria, we identified HIV-negative MSM attending SHCs with a HIV-negative test in the past year and used diagnosed bacterial STI (past year) in this group as a proxy for condomless sex and eligibility for HIV-PrEP. We estimated HIV incidence per 100 person-years (py) in these groups in 2014. RESULTS: During 2010-2015, the number of HIV-negative MSM attending SHCs with a HIV-negative test in the past year doubled from 14 643 to 29 023, and HIV incidence in this group was 1.9 (95% CI 1.6 to 2.2) per 100 py in 2014. In the same period, the subgroup with a bacterial STI diagnosis (past year), and therefore considered potentially eligible for HIV-PrEP in this analysis, increased from 4365 (30%) to 10 276 (35%). HIV incidence in this subgroup was 3.3 (95% CI 2.7 to 4.0) per 100 py in 2014. CONCLUSIONS: In 2015, approximately 10 000 HIV-negative MSM were considered potentially eligible for HIV-PrEP based on clinic history in GUMCAD. These data were used to inform the initial recruitment target for the PrEP Impact Trial and will inform future evaluations at a population level

Mitochondrial mutations and metabolic adaptation in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a five-year survival rate of ~8%, with characteristic molecular heterogeneity and restricted treatment options. Targeting metabolism has emerged as a potentially effective therapeutic strategy for cancers such as pancreatic cancer, which are driven by genetic alterations that are not tractable drug targets. Although somatic mitochondrial genome (mtDNA) mutations have been observed in various tumors types, understanding of metabolic genotype-phenotype relationships is limited. METHODS: We deployed an integrated approach combining genomics, metabolomics, and phenotypic analysis on a unique cohort of patient-derived pancreatic cancer cell lines (PDCLs). Genome analysis was performed via targeted sequencing of the mitochondrial genome (mtDNA) and nuclear genes encoding mitochondrial components and metabolic genes. Phenotypic characterization of PDCLs included measurement of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a Seahorse XF extracellular flux analyser, targeted metabolomics and pathway profiling, and radiolabelled glutamine tracing. RESULTS: We identified 24 somatic mutations in the mtDNA of 12 patient-derived pancreatic cancer cell lines (PDCLs). A further 18 mutations were identified in a targeted study of ~1000 nuclear genes important for mitochondrial function and metabolism. Comparison with reference datasets indicated a strong selection bias for non-synonymous mutants with predicted functional effects. Phenotypic analysis showed metabolic changes consistent with mitochondrial dysfunction, including reduced oxygen consumption and increased glycolysis. Metabolomics and radiolabeled substrate tracing indicated the initiation of reductive glutamine metabolism and lipid synthesis in tumours. CONCLUSIONS: The heterogeneous genomic landscape of pancreatic tumours may converge on a common metabolic phenotype, with individual tumours adapting to increased anabolic demands via different genetic mechanisms. Targeting resulting metabolic phenotypes may be a productive therapeutic strategy

Spatial-temporal modelling and analysis of bacterial colonies with phase variable genes

2015 Copyright is held by the owner/author(s). This article defines a novel spatial-temporal modelling and analysis methodology applied to a systems biology case study, namely phase variation patterning in bacterial colony growth. We employ coloured stochastic Petri nets to construct the model and run stochastic simulations to record the development of the circular colonies over time and space. The simulation output is visualised in 2D, and sector-like patterns are automatically detected and analysed. Space is modelled using 2.5 dimensions considering both a rectangular and circular geometry, and the effects of imposing different geometries on space are measured. We close by outlining an interpretation of the Petri net model in terms of finite difference approximations of partial differential equations (PDEs). One result is the derivation of the “best” nine-point diffusion model. Our multidimensional modelling and analysis approach is a precursor to potential future work on more complex multiscale modelling.EPSRC Research Grant EP I036168/1; German BMBF Research Grant 0315449H

SLAM-based Dense Surface Reconstruction in Monocular Minimally Invasive Surgery and its Application to Augmented Reality.

While Minimally Invasive Surgery (MIS) offers considerable benefits to patients, it also imposes big challenges on a surgeon's performance due to well-known issues and restrictions associated with the field of view (FOV), hand-eye misalignment and disorientation, as well as the lack of stereoscopic depth perception in monocular endoscopy. Augmented Reality (AR) technology can help to overcome these limitations by augmenting the real scene with annotations, labels, tumour measurements or even a 3D reconstruction of anatomy structures at the target surgical locations. However, previous research attempts of using AR technology in monocular MIS surgical scenes have been mainly focused on the information overlay without addressing correct spatial calibrations, which could lead to incorrect localization of annotations and labels, and inaccurate depth cues and tumour measurements. In this paper, we present a novel intra-operative dense surface reconstruction framework that is capable of providing geometry information from only monocular MIS videos for geometry-aware AR applications such as site measurements and depth cues. We address a number of compelling issues in augmenting a scene for a monocular MIS environment, such as drifting and inaccurate planar mapping. Methods A state-of-the-art Simultaneous Localization And Mapping (SLAM) algorithm used in robotics has been extended to deal with monocular MIS surgical scenes for reliable endoscopic camera tracking and salient point mapping. A robust global 3D surface reconstruction framework has been developed for building a dense surface using only unorganized sparse point clouds extracted from the SLAM. The 3D surface reconstruction framework employs the Moving Least Squares (MLS) smoothing algorithm and the Poisson surface reconstruction framework for real time processing of the point clouds data set. Finally, the 3D geometric information of the surgical scene allows better understanding and accurate placement AR augmentations based on a robust 3D calibration. Results We demonstrate the clinical relevance of our proposed system through two examples: a) measurement of the surface; b) depth cues in monocular endoscopy. The performance and accuracy evaluations of the proposed framework consist of two steps. First, we have created a computer-generated endoscopy simulation video to quantify the accuracy of the camera tracking by comparing the results of the video camera tracking with the recorded ground-truth camera trajectories. The accuracy of the surface reconstruction is assessed by evaluating the Root Mean Square Distance (RMSD) of surface vertices of the reconstructed mesh with that of the ground truth 3D models. An error of 1.24mm for the camera trajectories has been obtained and the RMSD for surface reconstruction is 2.54mm, which compare favourably with previous approaches. Second, \textit{in vivo} laparoscopic videos are used to examine the quality of accurate AR based annotation and measurement, and the creation of depth cues. These results show the potential promise of our geometry-aware AR technology to be used in MIS surgical scenes. Conclusions The results show that the new framework is robust and accurate in dealing with challenging situations such as the rapid endoscopy camera movements in monocular MIS scenes. Both camera tracking and surface reconstruction based on a sparse point cloud are effective and operated in real-time. This demonstrates the potential of our algorithm for accurate AR localization and depth augmentation with geometric cues and correct surface measurements in MIS with monocular endoscopes

Combined point of care nucleic acid and antibody testing for SARS-CoV-2 following emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in hospital is essential, though complicated by 30-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant now dominates the pandemic and it is unclear how serological tests designed to detect anti-Spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95CI 57.8-92.9%) by rapid NAAT alone. Combined point of care antibody test and rapid NAAT is not impacted by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Diving into the vertical dimension of elasmobranch movement ecology

Knowledge of the three-dimensional movement patterns of elasmobranchs is vital to understand their ecological roles and exposure to anthropogenic pressures. To date, comparative studies among species at global scales have mostly focused on horizontal movements. Our study addresses the knowledge gap of vertical movements by compiling the first global synthesis of vertical habitat use by elasmobranchs from data obtained by deployment of 989 biotelemetry tags on 38 elasmobranch species. Elasmobranchs displayed high intra- and interspecific variability in vertical movement patterns. Substantial vertical overlap was observed for many epipelagic elasmobranchs, indicating an increased likelihood to display spatial overlap, biologically interact, and share similar risk to anthropogenic threats that vary on a vertical gradient. We highlight the critical next steps toward incorporating vertical movement into global management and monitoring strategies for elasmobranchs, emphasizing the need to address geographic and taxonomic biases in deployments and to concurrently consider both horizontal and vertical movements