Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

AbstractThe assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid–tegument protein interactions may represent initial events of the tegumentation process

(S)-(−)-Fluorenylethylchloroformate (FLEC) ; preparation using asymmetric transfer hydrogenation and application to the analysis and resolution of amines

Fluorenylethylchoroformate (FLEC) is a valuable chiral derivatisation reagent that is used for the resolution of a wide variety of chiral amines. Herein, we describe an improved preparation of (S)-(−)-FLEC using an efficient asymmetric catalytic transfer hydrogenation as the key step. We also demonstrate the application of FLEC as a chiral Fmoc equivalent for chiral resolution, with facile deprotection, of tetrahydroquinaldines, and its capacity for inducing regioselective outcomes in nitration reactions

Image-Based Cell Profiling Enables Quantitative Tissue Microscopy in Gastroenterology.

Immunofluorescence microscopy is an essential tool for tissue-based research, yet data reporting is almost always qualitative. Quantification of images, at the per-cell level, enables "flow cytometry-type" analyses with intact locational data but achieving this is complex. Gastrointestinal tissue, for example, is highly diverse: from mixed-cell epithelial layers through to discrete lymphoid patches. Moreover, different species (e.g., rat, mouse, and humans) and tissue preparations (paraffin/frozen) are all commonly studied. Here, using field-relevant examples, we develop open, user-friendly methodology that can encompass these variables to provide quantitative tissue microscopy for the field. Antibody-independent cell labeling approaches, compatible across preparation types and species, were optimized. Per-cell data were extracted from routine confocal micrographs, with semantic machine learning employed to tackle densely packed lymphoid tissues. Data analysis was achieved by flow cytometry-type analyses alongside visualization and statistical definition of cell locations, interactions and established microenvironments. First, quantification of Escherichia coli passage into human small bowel tissue, following Ussing chamber incubations exemplified objective quantification of rare events in the context of lumen-tissue crosstalk. Second, in rat jejenum, precise histological context revealed distinct populations of intraepithelial lymphocytes between and directly below enterocytes enabling quantification in context of total epithelial cell numbers. Finally, mouse mononuclear phagocyte-T cell interactions, cell expression and significant spatial cell congregations were mapped to shed light on cell-cell communication in lymphoid Peyer's patch. Accessible, quantitative tissue microscopy provides a new window-of-insight to diverse questions in gastroenterology. It can also help combat some of the data reproducibility crisis associated with antibody technologies and over-reliance on qualitative microscopy. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.UK Medical Research Council (grant number MR/R005699/1) UK Engineering and Physical Sciences Research Council (grant EP/H008683/1) UK Biotechnology and Biological Sciences Research Council (grant number BB/P026818/1

The experience of providing young people attending general practice with an online risk assessment tool to assess their own sexual health risk

<p>Abstract</p> <p>Background</p> <p>Targeted chlamydia screening has been advocated to reduce chlamydia associated reproductive sequelae. General practitioners are well positioned to play a major role in chlamydia control. The primary aim of this pilot study was to measure the effect of offering an online sexual health assessment tool, <it>Youth Check Your Risk</it>, on chlamydia testing rates among young people attending general practices. The secondary aim was to test the acceptability of the tool among general practitioners and young people.</p> <p>Methods</p> <p>General practitioners at three practices in Melbourne, Australia, referred patients aged 16 to 24 years to <it>Youth Check Your Risk </it><url>http://www.checkyourrisk.org.au</url> for use post-consultation between March to October 2007. The proportion of young people tested for chlamydia before and during the implementation of the tool was compared. Acceptability was assessed through a structured interviewer-administered questionnaire with general practitioners, and anonymous online data provided by <it>Youth Check Your Risk </it>users.</p> <p>Results</p> <p>The intervention did not result in any significant increases in the proportion of 16 to 24 year old males (2.7% to 3.0%) or females (6.3% to 6.4%) tested for chlamydia. A small increase in the proportion of 16 to 19 year old females tested was seen (4.1% to 7.2%). Of the 2997 patients seen during the intervention phase, 871 (29.1%) were referred to <it>Youth Check Your Risk </it>and 120 used it (13.8%). Major reasons for low referral rates reported by practitioners included lack of time, discomfort with raising the issue of testing, and difficulty in remembering to refer patients.</p> <p>Conclusion</p> <p>Offering an online sexual risk assessment tool in general practice did not significantly increase the proportion of young people tested for chlamydia, with GPs identifying a number of barriers to referring young people to <it>Youth Check Your Risk</it>. Future interventions aimed at increasing chlamydia screening in general practice with the aid of an online risk assessment tool need to identify and overcome barriers to testing.</p

Incentive payments to general practitioners aimed at increasing opportunistic testing of young women for chlamydia: a pilot cluster randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>Financial incentives have been used for many years internationally to improve quality of care in general practice. The aim of this pilot study was to determine if offering general practitioners (GP) a small incentive payment per test would increase chlamydia testing in women aged 16 to 24 years, attending general practice.</p> <p>Methods</p> <p>General practice clinics (n = 12) across Victoria, Australia, were cluster randomized to receive either a $AUD5 payment per chlamydia test or no payment for testing 16 to 24 year old women for chlamydia. Data were collected on the number of chlamydia tests and patient consultations undertaken by each GP over two time periods: 12 month pre-trial and 6 month trial period. The impact of the intervention was assessed using a mixed effects logistic regression model, accommodating for clustering at GP level.</p> <p>Results</p> <p>Testing increased from 6.2% (95% CI: 4.2, 8.4) to 8.8% (95% CI: 4.8, 13.0) (p = 0.1) in the control group and from 11.5% (95% CI: 4.6, 18.5) to 13.4% (95% CI: 9.5, 17.5) (p = 0.4) in the intervention group. Overall, the intervention did not result in a significant increase in chlamydia testing in general practice. The odds ratio for an increase in testing in the intervention group compared to the control group was 0.9 (95% CI: 0.6, 1.2). Major barriers to increased chlamydia testing reported by GPs included a lack of time, difficulty in remembering to offer testing and a lack of patient awareness around testing.</p> <p>Conclusions</p> <p>A small financial incentive alone did not increase chlamydia testing among young women attending general practice. It is possible small incentive payments in conjunction with reminder and feedback systems may be effective, as may higher financial incentive payments. Further research is required to determine if financial incentives can increase testing in Australian general practice, the type and level of financial scheme required and whether incentives needs to be part of a multi-faceted package.</p> <p>Trial Registration</p> <p>Australian New Zealand Clinical Trial Registry ACTRN12608000499381.</p

Extrahepatic perfusion and incomplete hepatic perfusion after hepatic arterial infusion pump implantation:incidence and clinical implications

INTRODUCTION: This study investigates the incidence of extrahepatic perfusion and incomplete hepatic perfusion at intraoperative methylene blue testing and on postoperative nuclear imaging in patients undergoing hepatic arterial infusion pump (HAIP) chemotherapy.METHODS:The first 150 consecutive patients who underwent pump implantation in the Netherlands were included. All patients underwent surgical pump implantation with the catheter in the gastroduodenal artery. All patients underwent intraoperative methylene blue testing and postoperative nuclear imaging ( 99mTc-Macroaggregated albumin SPECT/CT) to determine perfusion via the pump. RESULTS: Patients were included between January-2018 and December-2021 across eight centers. During methylene blue testing, 29.3% had extrahepatic perfusion, all successfully managed intraoperatively. On nuclear imaging, no clinically relevant extrahepatic perfusion was detected (0%, 95%CI: 0.0-2.5%). During methylene blue testing, 2.0% had unresolved incomplete hepatic perfusion. On postoperative nuclear imaging, 8.1% had incomplete hepatic perfusion, leading to embolization in only 1.3%.CONCLUSION: Methylene blue testing during pump placement for intra-arterial chemotherapy identified extrahepatic perfusion in 29.3% of patients, but could be resolved intraoperatively in all patients. Postoperative nuclear imaging found no clinically relevant extrahepatic perfusion and led to embolization in only 1.3% of patients. The role of routine nuclear imaging after HAIP implantation should be studied in a larger cohort.</p

Extrahepatic perfusion and incomplete hepatic perfusion after hepatic arterial infusion pump implantation:incidence and clinical implications

INTRODUCTION: This study investigates the incidence of extrahepatic perfusion and incomplete hepatic perfusion at intraoperative methylene blue testing and on postoperative nuclear imaging in patients undergoing hepatic arterial infusion pump (HAIP) chemotherapy.METHODS:The first 150 consecutive patients who underwent pump implantation in the Netherlands were included. All patients underwent surgical pump implantation with the catheter in the gastroduodenal artery. All patients underwent intraoperative methylene blue testing and postoperative nuclear imaging ( 99mTc-Macroaggregated albumin SPECT/CT) to determine perfusion via the pump. RESULTS: Patients were included between January-2018 and December-2021 across eight centers. During methylene blue testing, 29.3% had extrahepatic perfusion, all successfully managed intraoperatively. On nuclear imaging, no clinically relevant extrahepatic perfusion was detected (0%, 95%CI: 0.0-2.5%). During methylene blue testing, 2.0% had unresolved incomplete hepatic perfusion. On postoperative nuclear imaging, 8.1% had incomplete hepatic perfusion, leading to embolization in only 1.3%.CONCLUSION: Methylene blue testing during pump placement for intra-arterial chemotherapy identified extrahepatic perfusion in 29.3% of patients, but could be resolved intraoperatively in all patients. Postoperative nuclear imaging found no clinically relevant extrahepatic perfusion and led to embolization in only 1.3% of patients. The role of routine nuclear imaging after HAIP implantation should be studied in a larger cohort.</p

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response

Whole blood-based measurement of SARS-CoV-2-specific T cells reveals asymptomatic infection and vaccine immunogenicity in healthy subjects and patients with solid-organ cancers.

Accurate assessment of SARS-CoV-2 immunity is critical in evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS-CoV-2-specific T-cell responses are a critical feature that will likely form a key correlate of protection against COVID-19. Here, we developed and optimized a high-throughput whole blood-based assay to determine the T-cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 231 healthy donors and 68 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were analysed for TH 1-type cytokines. Highly significant differential IFN-γ+ /IL-2+ SARS-CoV-2-specific T-cell responses were seen amongst previously infected COVID-19-positive healthy donors in comparison with unknown / naïve individuals (p < 0·0001). IFN-γ production was more effective at identifying asymptomatic donors, demonstrating higher sensitivity (96·0% vs. 83·3%) but lower specificity (84·4% vs. 92·5%) than measurement of IL-2. A single COVID-19 vaccine dose induced IFN-γ and/or IL-2 SARS-CoV-2-specific T-cell responses in 116 of 128 (90·6%) healthy donors, reducing significantly to 27 of 56 (48·2%) when measured in cancer patients (p < 0·0001). A second dose was sufficient to boost T-cell responses in the majority (90·6%) of cancer patients, albeit IFN-γ+ responses were still significantly lower overall than those induced in healthy donors (p = 0·034). Three-month post-vaccination T-cell responses also declined at a faster rate in cancer patients. Overall, this cost-effective standardizable test ensures accurate and comparable assessments of SARS-CoV-2-specific T-cell responses amenable to widespread population immunity testing, and identifies individuals at greater need of booster vaccinations

Whole blood-based measurement of SARS-CoV-2-specific T cell responses reveals asymptomatic infection and vaccine efficacy in healthy subjects and patients with solid organ cancers

Accurate assessment of SARS-CoV-2 immunity in the population is critical to evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS-CoV-2-specific T cell responses are a critical feature of the immune response that will likely form a key correlate of protection against COVID-19. Here, we developed and optimised a high-throughput whole blood-based assay to determine the T cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 156 healthy donors and 67 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were harvested and analysed for TH1-type effector cytokines (IFN-γ and IL-2). Amongst healthy donors, highly significant differential IFN-γ+/IL-2+ SARS-CoV-2-specific T cell responses were seen amongst vaccinated or previously infected COVID-19-positive individuals in comparison to unknown/naïve individuals (P < 0.0001). IL-2 production from T cells in response to SARS-CoV-2 derived antigens was a highly predictive diagnostic assay (P < 0.0001; 96.0% sensitivity, 93.9% specificity); measurement of IFN-γ+ SARS-CoV-2 specific T cell responses was equally effective at identifying asymptomatic (antibody and T cell positive) participants. A single dose of COVID-19 vaccine induced IFN-γ and/or IL-2 SARS-CoV-2-specific T cell responses in 28/29 (96.6%) of healthy donors, reducing significantly to 27/56 (48.2%) when measured in cancer patients (P = 0.0003). Overall, this cost-effective standardisable test ensures accurate and comparable assessments of SARS-CoV-2-specific T cell responses amenable to widespread population immunity testing