Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini

Translation initiation generally occurs at AUG codons in eukaryotes, although it has been shown that non-AUG or non-canonical translation initiation can also occur. However, the evidence for noncanonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling (Ribo-seq) studies. Here, using a strategy specifically designed to enrich N termini of proteins, we demonstrate that many human proteins are translated at noncanonical TISs. The large majority of TISs that mapped to 5' untranslated regions were noncanonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). It has been controversial whether the amino acid corresponding to the start codon is incorporated at the TIS or methionine is still incorporated. We found that methionine was incorporated at almost all noncanonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by the available ribosome profiling data. Sequence conservation across species and a higher abundance of noncanonical TISs than canonical ones in some cases suggests that the noncanonical TISs can have biological functions. Overall, this study provides evidence of protein translation initiation at noncanonical TISs and argues that further studies are required for elucidation of functional implications of such noncanonical translation initiation

Analytical and Clinical Performance of Two Point of Care Rapid Antibody Assays for SARS-CoV-2

Objective: The SARS-CoV-2 pandemic has reached to a state where populations across the world should ad-just to live with it like many other diseases. Regular serosurveys are essential for disease surveil-lance and policy decisions. In this study, we evaluated the analytical and clinical performance of two commercially available rapid antibody assays. Methods: SARS-CoV-2 PCR positive patients (N=104) were recruited for method evaluation study of two commercially available lateral flow Rapid IgM and IgG assays; Edinburgh Genetics ActivXpress+ COVID-19 IgG/IgM Immunoassay Complete Testing Kit (EGCV0092L) and Abchek COVID-19 IgM/IgG Antibody Rapid Test (NUL/COV-19/R&D/001). We have tested all the participants for SARS-CoV-2 with a Rapid Anti-gen Test (Abchek) on the day of sample collection. Additionally, we analyzed vaccinated people (N=187) for seroprevalence of IgG. EP Evaluator version 12 and GraphPad Prism 9.5.0 were used for statistical analysis. Results: The IgG seropositivity after 10-15 days of PCR positivity was 97.11% (Edinburg Genetics Assay) and 92.30% (Abchek Assay). The Rapid Antigen test was 100% negative with IgM negativity of 93.27% (Edinburg Genetics Assay) and 98.08% (Abchek Assay). The IgG seropositivity of vaccinated participants was 89.84% using both the assays. The IgG sero-positivity was 86.82% (Edinburg Genetics Assay, N=91) and 92.71% (Abchek Assay, N=96) in the study participants with post vaccination. Conclusions: These assays are robust and scalable. Both the assays can be used for serosurveys with desired scale and speed when a quick observation is needed for surveillance. These tests are cost effective, field deployable without need of any sophis-ticated instruments and large capital. Impact statement: During a public health emergency like the COVID-19 pandemic, regular sero-surveys are essential for disease surveillance and swift policy decisions. However, deployment of the gold standard methods like quantitative ELISA, neutralizing antibody assays for assessment of population based seroprevalence and immune status becomes logistically difficult, costly and time consuming. The point of care antibody assays are easily scalable, affordable and field deployable. The present study demonstrates that these tests are reliable in terms of analytical and clinical performance. These assays could be used when rapid observations are to be made by including large sample population. Keywords: LMICs; SARS-CoV-2; pandemic; point of care antibody tests; serosurvey

Comparative analysis of platelet depleted plasma prepared on the Roche 8100 automation line and manually centrifuged platelet poor plasma for routine coagulation assays

Objectives: To evaluate whether the routine coagulation tests can be performed using platelet depleted plasma (PDP, residual platelet count \u3c40000/μL) to achieve maximum efficiency of the automated workflow and compare results of these tests performed with platelet poor plasma (PPP residual platelet count \u3c10,000/μL) prepared manually \u27offline\u27. Design and methods: The PDP was obtained first following \u27on line\u27 centrifugation at 4150 RPM (3000g) for 7 min. The routine coagulation tests, Prothrombin Time (PT), Activated Partial Thromboplastin Clotting Time (aPTT), D-dimer (DD), Antithrombin III (AT3) and Fibrinogen (FBG) were performed. The PPP was obtained from an aliquot of PDP samples with additional \u27manual off line\u27 centrifugation at 7700 RPM (3314g) for 3 min (total 10 min, online + offline) and the same tests were performed. The statistical analysis was carried out using EP Evaluator v11 to compare results from both methods. Results: The results from both PPP and PDP samples demonstrated strong correlation. For example, PT (R = 0.9989; N = 55, and of Bias -0.12 (-0.67%), aPTT(R = 0.9957; N = 60, Bias 0.26 (0.58%)), AT3(R = 0.9800; N = 49, Bias -2.0 (-2.2%)), FBG (R = 0.9956; N = 57, Bias -1.9 (-0.5%)) and DD (R = 0.9981; N = 38, Bias 0.005 (0.373%)) with insignificant bias. Conclusions: The utilization of the Roche cobas® 8100 automated \u27online\u27 centrifugation helps achieve optimal workflow efficiency without impacting analytical performance of the PT, aPTT, DD, AT3 and FBG assays. The use of PDP can be superior method to PPP for routine coagulation tests. Keywords: Activated Partial Thromboplastin Clotting Time; Antithrombin; Coagulation; D-Dimer; Fibrinogen; Hematology; Laboratory automation; Pre-analytical automation; Prothrombin Time

Comparative analysis of platelet depleted plasma prepared on the Roche 8100 automation line and manually centrifuged platelet poor plasma for routine coagulation assays

Objectives: To evaluate whether the routine coagulation tests can be performed using platelet depleted plasma (PDP, residual platelet count <40000/μL) to achieve maximum efficiency of the automated workflow and compare results of these tests performed with platelet poor plasma (PPP residual platelet count <10,000/μL) prepared manually ‘offline’. Design and Methods: The PDP was obtained first following ‘on line’ centrifugation at 4150 RPM (3000g) for 7 min. The routine coagulation tests, Prothrombin Time (PT), Activated Partial Thromboplastin Clotting Time (aPTT), D-dimer (DD), Antithrombin III (AT3) and Fibrinogen (FBG) were performed. The PPP was obtained from an aliquot of PDP samples with additional ‘manual off line’ centrifugation at 7700 RPM (3314g) for 3 min (total 10 min, online + offline) and the same tests were performed. The statistical analysis was carried out using EP Evaluator v11 to compare results from both methods. Results: The results from both PPP and PDP samples demonstrated strong correlation. For example, PT (R = 0.9989; N = 55, and of Bias −0.12 (−0.67%), aPTT(R = 0.9957; N = 60, Bias 0.26 (0.58%)), AT3(R = 0.9800; N = 49, Bias −2.0 (−2.2%)), FBG (R = 0.9956; N = 57, Bias −1.9 (−0.5%)) and DD (R = 0.9981; N = 38, Bias 0.005 (0.373%)) with insignificant bias. Conclusions: The utilization of the Roche cobas® 8100 automated ‘online’ centrifugation helps achieve optimal workflow efficiency without impacting analytical performance of the PT, aPTT, DD, AT3 and FBG assays. The use of PDP can be superior method to PPP for routine coagulation tests

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors. In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies. In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05). The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%). Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development

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11 février 19401940/02/11 (A69).Appartient à l’ensemble documentaire : PoitouCh