Isolation and Structural Analysis of Genomic Variants of Herpes Simplex Virus Type 2

The original aim of the project was to study recombination in HSV-2 strain HG52 using restriction enzyme sites as unselected markers. As no relevant DNA sequence data was available for HSV-2 it was decided to isolate site deletion mutants by enrichment selection of spontaneously occurring variants. The restriction enzyme Xba I was chosen as it makes only four staggered cuts in the HSV-2 genome and a virus, HG52X163X3X53, lacking all four Xba I sites was isolated following three rounds of enrichment selection. However, during the screening procedures involved in the isolation of HG52X163X3X53, a large number of variants with genomic alterations was identified and the work described in this thesis has concentrated on the analysis and characterization of these variants. Of the variants isolated after the initial enrichment selection of HG52 DNA five have been studied in detail. Three (HG52X85/4, HG52X85/5 and HG52X86) have deletions from IRL ranging in size from 2. 5x10e6 daltons to 6x10e6 daltons. Under immediate early conditions VmwIE64 production by HG52X85/5 and HG52X86 in which only the 3' part of IE1 is removed is reduced despite their deletions ending approximately 1kb downstream from the 3' end of UL54 which encodes this polypeptide. HG52X85/4 which also has a deletion ending approximately 1kb downstream of UL54 but in which an entire copy of IE1 is removed makes VmwIE64 in normal amounts. Of the two remaining variants one, HG52X192, has a deletion of approximately 1x10 6 daltons in each copy of RL. The final variant, HG52X19, has a deletion of approximately 9x10e6 daltons which removes the entire internal copy of the long repeat and half of the short repeat. The long segment of the genome is fixed in the prototype orientation whilst the short region inverts inefficiently through the undeleted part of the repeats (the internal copy of the 'a' sequence being deleted). The genome has non-HSV DNA inserted across the deletion. The inserted DNA has been reiterated to give different copy numbers and hence a heterogeneous genome population. It has been demonstrated that the inserted DNA is of bovine origin -presumably the result of recombination with the calf thymus DNA used as a carrier during transfection. To investigate if the enrichment selection procedure was responsible for the production of the unexpectedly high proportion of variants, isolates from untreated HSV stocks were studied. Of the fifty plaques of HG52 analysed, twelve differed significantly from the wild type structure. However, the plaques, five of HG52/5 and seven of HG52/10, represented only two variants with the same genome structures as HG52X86 and HG52X192 respectively. No significant variation was found in the stocks of other HSV strains examined (ie. HSV-2 strains 333 and 186 and HSV-1 strains 17 and KOS or plaque purified HG52 isolates (eg. tsl)). Variation was found in HSV-1 strain McKrae but its relevance was impossible to assess as the history of the stock is unknown. The conclusion has been drawn that variation has arisen in HG52 in the absence of enrichment selection although the structure of HG52X19 must have resulted from the transfection procedure. Three variants, HG52X163X12, HG52X163X14 and HG52X163X21, were isolated following the second round of enrichment selection using as the parent HG52X163 which lacks the 0.7m.c. Xba I site. HG52X163X12 has a deletion removing sequences from 0.94m.c. to 0.99m.c. (ie. part of U S and almost all of TRS) . The isolation of this variant demonstrates that genes US 10, 11, 12 and one copy of IE3 and one copy of oriS are non essential, at least in vitro. The two remaining variants, HG52X163X14 and HG52X163X21, both have deletions removing the same region as from HG52X163X12. However, the deleted sequences are replaced by sequences between 0.83m.c. -0.91m. c. in the inverted orientation thereby effectively deleting US between 0.94-0.96m.c. while extending the short repeats by 6kb. The only differences found between HG52X163X14 and HG52X163X21 were the loss of the 0.91m.c. Xba I site and a small insert containing a Hind III and EcoR I site at the 0.94m.c. Xba I site in the latter. It is possible that the enrichment selection procedure was responsible for the generation of these three variants as the rearrangements appear to be associated with the Xba I sites at 0.91 and 0.94m.c

Understanding the power of the "solo"

Solitude is a fundamental aspect of wilderness experiences and solo experiences, lasting 5 hours or more, have a strong 'survival' element and are a beneficial component to outdoor therapy programmes, sometimes marking life transitions for people. A shift has been made, however, towards solo experiences that centre on the natural environment rather than 'survival'. Shorter experiences of solitude in wilderness, e.g. quiet time or mini-solos, can offer the most "powerful" form of private reflection and can be self-initiated or prescribed. These 'shorter' solos have not, however, been extensively researched and neither have they been investigated in local green or semi-natural areas. This paper discusses a study that qualitatively explored people's experience, meaning-making and human-environment interaction during a mini-solo in a local green space and identified the impact of the experience on participants' sense of well-being. The mini-solo involved participants spending time in nature without distraction from technology or books and, if possible, people, lasting from 20 minutes to 1 hour. 12 older adults (55-74 years; 8 females and 4 males) recruited from walking groups and six younger adults (19-22 years; 4 females and 2 males) undertook a mini-solo. Pre- and post-experience interviews and journal writing (before, during and after the mini-solo) were analysed using thematic analysis. Four themes were created: ' Exerting Control', 'Aspects of Distraction' and 'Receptivity to Solo Experience'. Findings will be explained in relation to theory, literature and policy and may have implications for 'green prescriptions' as a short-term nature-based solution for well-being. Future directions for research are discussed. Keywords: mini-solo experience, wellbeing

Covid 19 – The impact of variable and ‘low normal’ pulse oximetry scores on Oximetry@Home services and clinical pathways: Confounding variables?

Covid 19 Oximetry@Home services have been commissioned nationally. This allows higher risk patients with mild Covid 19 symptoms to remain at home, being supplied with a Pulse Oximeter to measure their oxygen saturation (SpO2) two to three times daily for two weeks. Patients record their readings manually or electronically which are monitored by a clinical team. Clinical decisions, using an algorithm, are based on SpO2 readings in a narrow range with 1-2 point changes potentially affecting care. In this article we discuss the problem that multiple factors affect SpO2 readings, and that some ‘normal’ individuals will have ‘low normal’ scores at the threshold of clinical management, without any known respiratory problem. We discuss the potential magnitude of this problem based on the associated literature and consider how this will impact on the use of the Oximetry@home services, potentially partially confounding their purpose; to reduce face to face medical care

An exploration of student satisfaction using photographic elicitation: differences between undergraduate and postgraduate students

Student satisfaction is both an important, yet controversial issue within the Higher Education sector, which is typically measured through policy-driven metrics such as the National Student Survey (NSS). The aim of the current paper is to report findings of two studies conducted that explored student satisfaction amongst two samples: undergraduates and postgraduates. Many existing studies and national metrics are largely quantitative in design; the current study utilises photographic elicitation and a student-led interview approach to explore this much researched topic with a novel methodology. Results are discussed in line with the implications for current practice and Higher Education Institutions

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.We would like to acknowledge Michal Maj and Line Ericsen, and Kevin Clark in the flow cytometry facilities at the Dunn School and WIMM respectively for providing cell sorting services. The WIMM facility is supported by the MRC HIU; MRC MHU (MC_UU_12009); NIHR Oxford BRC and John Fell Fund (131/030 and 101/517), the EPA fund (CF182 and CF170) and by the WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We thank Neil Ashley for his help on 10x sample preparation and sequencing. The WIMM Single Cell Core Facility was supported by the MRC MHU (MC_UU_12009), the Oxford Single Cell Biology Consortium (MR/M00919X/1) and the WT ISSF (097813/Z/11/B#) funding. The facility was supported by WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We also thank the High-Throughput Genomics Group (Wellcome Trust (WT) Centre for Human Genetics, funded by WT 090532/Z/09/Z), for generating sequencing data. We thank Valerie Kouskoff for providing the iRunx1 ES cell line, Supat Thongjuea and Guanlin Wang for advice with the scRNA-Seq analysis, Joey Riepsaame for advice with CRISP-R experiments, and Doug Higgs, Hedia Chagraoui, Dominic Owens, Andrew Nelson and Arne Mould for helpful discussions. M.D.B and C.P are supported by programmes in the MRC Molecular Hematology Unit Core award (Grant number: MC_UU_12009/2 M.D.B. and MC_UU_12009/9 C.P.). L.G. was supported by a Clarendon PhD studentship and the MRC Molecular Haematology Unit. The work was supported by grants from the Wellcome Trust (214175/Z/18/Z E.J.R, 10281/Z/13/Z L.T.G.H). L.T.G.H was supported by a Clarendon Fund Scholarship and Trinity College Titley Scholarship. E.J.R. is a Wellcome Trust Principal Fellow

The clinical profile of tuberous sclerosis complex (TSC) in the United Kingdom: A retrospective cohort study in the Clinical Practice Research Datalink (CPRD)

AbstractBackgroundTuberous Sclerosis Complex (TSC) is a multi-system genetic disorder characterised by the development of benign growths and diverse clinical manifestations, varying in severity, age at onset and with high clinical burden.AimsThis longitudinal study aims to describe the broad spectrum of clinical manifestation profiles in a large, representative cohort of TSC patients in the UK in order to better understand disease complexity.MethodsTSC patients in the Clinical Practice Research Datalink (CPRD) and linked Hospital Episodes Statistics (CPRD-HES) were retrospectively identified between 1987 and 2013. Available history was extracted for each patient and clinical diagnosis, procedure and medication records reviewed. A random selection of patients from the CPRD-HES was used as a Comparator cohort.ResultsThree hundred and thirty-four TSC patients with a mean (SD) age of 30.3 (18.6) years were identified (53% female). TSC was diagnosed at mean age 3.2 (4.2) years. Epilepsy and psychiatric manifestations were reported frequently in paediatric (77% and 55%, respectively) and adult patients (66% and 68%, respectively). The prevalence of manifestations in the TSC cohort was markedly higher versus the Comparator cohort. The majority of paediatric (46%) and adult TSC patients (62%) developed clinical manifestations affecting at least three organ systems and forty-nine distinctive organ system manifestation profiles were identified.ConclusionsTSC patients present with multiple and complex clinical manifestations and profiles that necessitate the co-ordinated action of a multidisciplinary team in order to improve the quality and efficiency of care

Polymorphisms in Cyclooxygenase, Lipoxygenase and TP53 genes predict colorectal polyp risk reduction by aspirin in the seAFOod polyp prevention trial

Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin E2. We investigated whether 35 single nucleotide polymorphisms (SNPs) in oxylipin metabolism genes such as cyclooxygenase [PTGS] and lipoxygenase [ALOX], as well as 7 SNPs already associated with colorectal cancer (CRC) risk reduction by aspirin (eg. TP53; rs104522), modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomised 2x2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. Five hundred and forty-two (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with non-aspirin users was restricted to rs4837960 (PTGS1) common homozygotes (IRR 0.69 [95%CI 0.53,0.90]; q=0.06), rs2745557 (PTGS2) compound heterozygote-rare homozygotes (IRR 0.60 [0.41,0.88]; q=0.06), rs7090328 (ALOX5) rare homozygotes (IRR 0.27 [0.11,0.64]; q=0.05), rs2073438 (ALOX12) common homozygotes (IRR 0.57 [0.41,0.80]; q=0.05), and rs104522 (TP53) rare homozygotes (IRR 0.37 [0.17,0.79]; q=0.06). No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalised, predictive models of CRC chemoprevention by aspirin

Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27Xic1 is expressed in the developing kidney in the nephrostomal regions. Using over-expression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27Xic1, and reveal its differentiation function is not universally utilised in all developing tissues

Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases.

Inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, affect several million individuals worldwide. Crohn's disease and ulcerative colitis are complex diseases that are heterogeneous at the clinical, immunological, molecular, genetic, and microbial levels. Individual contributing factors have been the focus of extensive research. As part of the Integrative Human Microbiome Project (HMP2 or iHMP), we followed 132 subjects for one year each to generate integrated longitudinal molecular profiles of host and microbial activity during disease (up to 24 time points each; in total 2,965 stool, biopsy, and blood specimens). Here we present the results, which provide a comprehensive view of functional dysbiosis in the gut microbiome during inflammatory bowel disease activity. We demonstrate a characteristic increase in facultative anaerobes at the expense of obligate anaerobes, as well as molecular disruptions in microbial transcription (for example, among clostridia), metabolite pools (acylcarnitines, bile acids, and short-chain fatty acids), and levels of antibodies in host serum. Periods of disease activity were also marked by increases in temporal variability, with characteristic taxonomic, functional, and biochemical shifts. Finally, integrative analysis identified microbial, biochemical, and host factors central to this dysregulation. The study's infrastructure resources, results, and data, which are available through the Inflammatory Bowel Disease Multi'omics Database ( http://ibdmdb.org ), provide the most comprehensive description to date of host and microbial activities in inflammatory bowel diseases

A randomised controlled trial and cost-effectiveness evaluation of "booster" interventions to sustain increases in physical activity in middle-aged adults in deprived urban neighbourhoods

Background: Systematic reviews have identified a range of brief interventions which increase physical activity in previously sedentary people. There is an absence of evidence about whether follow up beyond three months can maintain long term physical activity. This study assesses whether it is worth providing motivational interviews, three months after giving initial advice, to those who have become more active. Methods/Design: Study candidates (n = 1500) will initially be given an interactive DVD and receive two telephone follow ups at monthly intervals checking on receipt and use of the DVD. Only those that have increased their physical activity after three months (n = 600) will be randomised into the study. These participants will receive either a "mini booster" (n = 200), "full booster" (n = 200) or no booster (n = 200). The "mini booster" consists of two telephone calls one month apart to discuss physical activity and maintenance strategies. The "full booster" consists of a face-to-face meeting with the facilitator at the same intervals. The purpose of these booster sessions is to help the individual maintain their increase in physical activity. Differences in physical activity, quality of life and costs associated with the booster interventions, will be measured three and nine months from randomisation. The research will be conducted in 20 of the most deprived neighbourhoods in Sheffield, which have large, ethnically diverse populations, high levels of economic deprivation, low levels of physical activity, poorer health and shorter life expectancy. Participants will be recruited through general practices and community groups, as well as by postal invitation, to ensure the participation of minority ethnic groups and those with lower levels of literacy. Sheffield City Council and Primary Care Trust fund a range of facilities and activities to promote physical activity and variations in access to these between neighbourhoods will make it possible to examine whether the effectiveness of the intervention is modified by access to community facilities. A one-year integrated feasibility study will confirm that recruitment targets are achievable based on a 10% sample.Discussion: The choice of study population, study interventions, brief intervention preceding the study, and outcome measure are discussed