Analysis of RNAs that interact with herpes simplex virus type 1 immediate early protein ICP27

ICP27 is an HSV-1 immediate early protein required for the switch from early to late gene expression. ICP27 binds viral and cellular RNAs, and recently a yeast three-hybrid (Y3-H) analysis has identified an array of viral RNA sequences that interact with ICP27. Presented here are analyses of functional assays using a selection of the Y3-H identified RNA sequences inserted into the 5’ untranslated region (UTR) of a chloramphenicol acetyl transferase (CAT) reporter plasmid. A set of plasmids was transfected into baby hamster kidney (BHK) cells and CAT assays carried out to analyse the effects of the sequences on gene expression. Results indicated that expression was increased when ICP27-binding sequences were present even though no viral proteins were present. Comparison of sequences revealed that no common activation code or RNA structure was present that could be responsible for the increase in CAT gene expression. The levels of expression were further determined in the presence of wild type (wt), ICP27-null or ICP27 mutant HSV-1 infection to investigate whether ICP27 had any affect on CAT expression when ICP27-binding sequences were present. Interestingly, enhanced expression was observed during wt HSV-1-infection when ICP27-binding sequences were present, whereas little to no enhancement was observed during ICP27-null or mutant virus infections. However, a higher fold increase in CAT gene expression was observed during a HSV-1 infection when ICP27-binding sequences were not present. This indicated that an inhibitory effect on CAT expression observed during wt HSV-1 infection when ICP27-binding sequences were present was ICP27-depdnent. As a control, a noncoding, protein binding regulatory HPV RNA sequence was inserted into the CAT reporter plasmid, transfected into BHK cells and then infected with wt HSV-1, ICP27-null or ICP27 mutant viruses. Surprisingly, CAT expression was increased, albeit to only a limited extent, indicating that the previously observed increase in gene expression was not HSV-1 sequence specific. However, upon transfection of plasmids with the HPV control sequence inserted in the reverse orientation and a subsequent infection of cells no increase in CAT expression was observed. Analysis of the control sequence in the reverse orientation identified a shortage in G residues, which led to the construction of CAT reporter plasmids containing homopolymer sequences to inserted into the 5’UTR. A series of transfections and subsequent mock, wt HSV-1 or ICP27-null virus infections were carried out using this set of constructs. CAT assay analysis revealed an increase in CAT expression, to levels similar to those observed when the HSV-1 sequences were present, when poly(G) homopolymers were used as inserts during wt HSV-1 infection, whereas poly(A), (C) and (T) gave low levels of expression

Evaluation of the Ecosystem Services and multiple benefits provided by SUDS and non SUDS ponds

This thesis explored the potential of four Sustainable Urban Drainage (SUDS) ponds, and four non-SUDS pond, to deliver key Ecosystem benefits, and services. Sustainable Urban Drainage Systems are designed to improve water quality, and provide amenity, biodiversity, and flood alleviation benefits. Ecosystem Services (ES) refer to the end user benefits associated with an ecosystem, and which are of direct use for humans. Within the context of this study, the key Ecosystem Service (ES) is water quality regulation which was considered using the proxies of macroinvertebrates and diatoms. An additional Ecosystem Service is wild species diversity which involved monitoring and evaluating flowering plants in relation to pollination. The project had four main research questions (RQ), and each was supported by its own research methodology. First the effectiveness of water quality regulation in SUDS and non-SUDS ponds using Average Score per Taxon was monitored (RQ1); secondly, nutrient removal services was observed between pond inlets and outlets, and potential indicators for reference, disturbed, and toxicity (RQ 2) for the ES of water quality regulation. RQ3 considered the potential for ponds to support flowering plants suitable for pollinators for the ES of wild species diversity. RQ4 focussed on the public perception survey, and willingness to pay for the multiple benefits of ponds and evaluated these in relation to the Whole Life Cost of the pond. RQ1 revealed that median values were higher for SUDS ponds than non SUDS ponds but not of statistical significance (p>0.05). For RQ2, median diatom counts were not statistically significant between SUDS and non SUDS ponds (p>0.05). There was a statistical difference between median observations for plants suitable for insect pollinators (p0.05). RQ4 revealed that habitat provision benefits outweighed costs for SUDS and non SUDS ponds in relation to Whole Life Cost analysis. The proposed framework, resulting from this research, may be used to inform local decision making and policy for SUDS design. This research aids the understanding of valuing ponds in relation to ecosystem benefits and services. It also highlights the significance of incorporating disadvantages into economic assessment; particularly where direct comparisons are made between ponds

A hepatitis C avidity test for determining recent and past infections in both plasma and dried blood spots

DBS testing has been used successfully to detect HCV antibody positive individuals. Determining how long someone has been infected is important for surveillance initiatives. Antibody avidity is a method that can be used to calculate recency of infection. A HCV avidity assay was evaluated for both plasma and DBS. Study design: To measure antibody avidity a commercial HCV ELISA was modified using 7 M urea. The plasma samples were split into: group 1 (recently infected N = 19), group 2 (chronic carrier N = 300) and group 3 (resolved infection N = 82). Mock DBS made from group 1 (N = 12), group 2 (N = 50), group 3 (N = 25) and two seroconverter panels were evaluated. 133 DBS taken from patients known to have a resolved infection or be a chronic carrier were also tested. The avidity assay cut-off was set at AI ≤ 30 for a recent infection. Using sequential samples the assay could detect a recent infection in the first 4–5 months from the point of infection. Most of the false positive results (AI < 30 among cases known not to have had recent infection) were detected among known resolved infections, in both the plasma and DBS; as a result, a testing algorithm has been designed incorporating both PCR and two dilution factors. The sensitivity and specificity of the assay on plasma was 100% and 99.3%, respectively, while DBS had 100% sensitivity and 98.3% specificity. The HCV avidity assay can be used to distinguish between chronic and recent infection using either plasma or DBS as the sample type

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission

Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals; FundRef: http://dx.doi.org/10.13039/501100002927Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

CK2 Protein Kinase Is Stimulated and Redistributed by Functional Herpes Simplex Virus ICP27 Protein

It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic α or α′ subunits and two regulatory β subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs

Effect of Modified Natural Filler O-Methylene Phosphonic κ-Carrageenan on Chitosan-Based Polymer Electrolytes

The potential for using O-methylene phosphonic &kappa;-carrageenan (OMPk) as a filler in the chitosan-based polymer electrolyte N-methylene phosphonic chitosan (NMPC) was investigated. OMPk, a derivative of &kappa;-carrageenan, was synthesized via phosphorylation and characterized using infrared spectroscopy (IR) and nuclear magnetic resonance (NMR). Both the IR and NMR results confirmed the phosphorylation of the parent carrageenan. The solid polymer electrolyte (SPE)-based NMPC was prepared by solution casting with different weight percentages of OMPk ranging from 2 to 8 wt %. The tensile strength of the polymer membrane increased from 18.02 to 38.95 MPa as the amount of OMPk increased to 6 wt %. However, the increase in the ionic conductivity did not match the increase in the tensile strength. The highest ionic conductivity was achieved with 4 wt % OMPk, which resulted in 1.43 &times; 10&minus;5 Scm&minus;1. The &kappa;-carrageenan-based OMPk filler strengthened the SPE while maintaining an acceptable level of ionic conductivity

Standmengenverbesserung von Schmiedegesenken Vortraege von Forschungsinstituten

TIB: RO 8931(16) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman