Vaccination with DNA plasmids expressing Gn coupled to C3d or alphavirus replicons expressing Gn protects mice against rift valley fever virus

Background: Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent. Methodology/Principal Findings: We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12. Conclusion/Significance: These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use. © 2010 Bhardwaj et al

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Cyclosporin a disrupts notch signaling and vascular lumen maintenance.

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos

Notch inhibition partially rescues CSA induced vascular malfunction.

<p>(A) Effect of CSA and DAPT on vascular function in zebrafish embryos. Freshly laid Fli1-GFP / GATA1-RFP zebrafish embryos were incubated in 0.1% DMSO (Control), 10μM CSA, 15μM DAPT, or 10μM CSA + 15μM DAPT for 2 days. Bright field imaging revealed no gross morphological abnormalities in either CSA or DAPT treated fish, however CSA + DAPT treated fish experienced an acute curvature. GFP imaging revealed a lack of lumen structures in the ISV (white arrowheads) and aortic vessels (red arrowheads) of CSA treated fish. DAPT treated fish displayed normal luminal structure and blood flow. CSA + DAPT treated embryos had luminal structures (arrowheads) and blood flow similar to control or DAPT alone treated embryos. Shown are representative results from a single experiment that was performed five times in its entirety. (B) Quantitative analysis of blood flow in zebrafish treated with CSA, DAPT, or CSA + DAPT. Data shown represent the average +/− SE of five individual experiments. P-values were determined by student’s t-test.</p

Cyclosporin-A destabilizes vascular lumen structures in zebrafish embryos.

<p>Freshly laid Fli1-GFP/GATA1-RFP zebrafish embryos were incubated in 10μM CSA or DMSO vehicle control for one, two, or four days. Whole embryo brightfield imaging was used to monitored gross morphology. Development of the vascular system was monitored by fluorescent microscopy of endothelial specific GFP expression. Circulatory flow was monitored by fluorescent microscopy of red-blood cell specific RFP expression. (A) Effects of CSA on 1dpf embryos. 1 day after CSA treatment, brightfield imaging of zebrafish embryos (top panel) was unable to distinguish any significant developmental impact of CSA on gross embryo morphology. Microangiogram analysis revealed similar development of the primitive vascular system including sprouting intersegmental vessels. (B) Effects of CSA on 2 dpf embryos. Zebrafish embryos treated with CSA for two days displayed no obvious signs of developmental abnormality in bright field images. Low power GFP imaging revealed an apparently normal vascular system, however RFP imaging revealed a distinct lack of blood flow throughout the embryo. High power GFP imaging revealed a lack of vascular lumen structures in ISV structures (arrows). (C) Effect of CSA on 4 dpf embryos. After four days of CSA treatment, no vascular luminal structures (arrows) or blood flow was evident in CSA treated embryos.</p

CSA blocks Notch signaling.

<p>(A) Effect of CSA on Notch signaling <i>in vitro</i>. 293T cells were transfected with various combinations of myc-tagged murine Notch1 (N), JAG1 (J), or Delta-like 4 (D) and treated with either 0.1% DMSO or 10μM CSA. Whole cell lysates were fractionated through SDS-PAGE gels and western blotted with anti-Val1744 antibody to detect cleaved Notch1 NICD fragments (N1ICD). Stripped blots were re-blotted with β-actin or 9E10 anti-myc antibodies to control for protein loading and expression of various transfected cDNAs. Shown are representative western blots from a single experiment that was performed five times in its entirety. (B) Western blot quantitation comparing N1ICD levels in cells transfected with Notch1 alone to cells transfected with combinations of Notch and JAG1 or Dll4 in the presence or absence of CSA. Displayed data represent the mean +/− SE of five individual experiments. P-values were calculated with the Student’s t-test. (C) Effects of CSA on Notch activity <i>in</i> vivo. Tp1bglob:eGFP embryos which express GFP from a tandem array of 12 Notch responsive RBP-Jk binding sites were incubated in either 0.1% DMSO, 10μM DAPT, or 10μM CSA. 48 hours later, GFP signal intensity was quantified in whole, live embryos. Data shown represents the mean +/− SE of 4 individual experiments. P-values were determined by student’s t-test. (D) Representative pictures of Tp1bglob:eGFP zebrafish embryos incubated with 10M DAPT or 10M CSA and imaged by fluorescent microscopy.</p

Inhibition of cyclophilin A or calcineurin/NFAT destabilizes lumen structure.

<p>(A) Freshly laid Fli1-GFP / GATA1-RFP embryos were incubated in 0.1% DMSO, 2μM FK506, or 40μM <i>N</i>-MeVal-4-CsA (CSA-Analog) for two days. Fluorescent imaging was used to monitor overall vascular development (GFP) and blood flow (RFP). Similar to CSA, neither FK506 nor <i>N</i>-MeVal-4-CSA had an impact on gross morphology or overall vascular development however both drugs blocked blood flow.</p