ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα−, PR−, and Her2−, named accordingly “Triple-Negative” (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5–12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygroR) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50–100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships

Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library

Tixagevimab–cilgavimab for treatment of patients hospitalised with COVID-19: a randomised, double-blind, phase 3 trial

Background: Tixagevimab–cilgavimab is a neutralising monoclonal antibody combination hypothesised to improve outcomes for patients hospitalised with COVID-19. We aimed to compare tixagevimab–cilgavimab versus placebo, in patients receiving remdesivir and other standard care. Methods: In a randomised, double-blind, phase 3, placebo-controlled trial, adults with symptoms for up to 12 days and hospitalised for COVID-19 at 81 sites in the USA, Europe, Uganda, and Singapore were randomly assigned in a 1:1 ratio to receive intravenous tixagevimab 300 mg–cilgavimab 300 mg or placebo, in addition to remdesivir and other standard care. Patients were excluded if they had acute organ failure including receipt of invasive mechanical ventilation, extracorporeal membrane oxygenation, vasopressor therapy, mechanical circulatory support, or new renal replacement therapy. The study drug was prepared by an unmasked pharmacist; study participants, site study staff, investigators, and clinical providers were masked to study assignment. The primary outcome was time to sustained recovery up to day 90, defined as 14 consecutive days at home after hospital discharge, with co-primary analyses for the full cohort and for participants who were neutralising antibody-negative at baseline. Efficacy and safety analyses were done in the modified intention-to-treat population, defined as participants who received a complete or partial infusion of tixagevimab–cilgavimab or placebo. This study is registered with ClinicalTrials.gov, NCT04501978 and the participant follow-up is ongoing. Findings: From Feb 10 to Sept 30, 2021, 1455 patients were randomly assigned and 1417 in the primary modified intention-to-treat population were infused with tixagevimab–cilgavimab (n=710) or placebo (n=707). The estimated cumulative incidence of sustained recovery was 89% for tixagevimab–cilgavimab and 86% for placebo group participants at day 90 in the full cohort (recovery rate ratio [RRR] 1·08 [95% CI 0·97–1·20]; p=0·21). Results were similar in the seronegative subgroup (RRR 1·14 [0·97–1·34]; p=0·13). Mortality was lower in the tixagevimab–cilgavimab group (61 [9%]) versus placebo group (86 [12%]; hazard ratio [HR] 0·70 [95% CI 0·50–0·97]; p=0·032). The composite safety outcome occurred in 178 (25%) tixagevimab–cilgavimab and 212 (30%) placebo group participants (HR 0·83 [0·68–1·01]; p=0·059). Serious adverse events occurred in 34 (5%) participants in the tixagevimab–cilgavimab group and 38 (5%) in the placebo group. Interpretation: Among patients hospitalised with COVID-19 receiving remdesivir and other standard care, tixagevimab–cilgavimab did not improve the primary outcome of time to sustained recovery but was safe and mortality was lower. Funding: US National Institutes of Health (NIH) and Operation Warp Speed

Configuration errors analysis in SRAM-based FPGAs: software tool and practical results

Special Issue on 18th European Symposium on Reliability of Electron Devices, Failure Physics and Analysis Bordeaux, France, 08-12 October 2007 Edited by Nathalie Labat and André TouboulInternational audienceThe reconfigurability of SRAM-based FPGAs has also some drawbacks, especially when used in systems requiring a high level of safety and/or dependability. Dealing with single-event effects is an important issue in these systems. This paper presents a software tool to analyze a bit-stream and the functional effects of errors in it. Results of analyses ar

Configuration errors analysis in SRAM-based FPGAs

International audienc

Dynamic Testing of an SRAM-Based FPGA by Time-Resolved Laser Fault Injection

International audienceThis paper presents principles and results of dynamic testing of an SRAM-based FPGA using time- resolved fault injection with a pulsed laser. The synchronization setup and experimental procedure are detailed. Fault injection results obtained with a DES crypto-core application implemented on a Xilinx Virtex II are discussed

Approach for Variability Management of Legal Rights in Human Resources Software Product Lines

International audienceThis work concerns software product lines (SPL); it comes from the experience gained collaborating with Berger-Levrault, a French society leader in Human Resources systems. This enterprise serves many French and European territorial communities. They had a variability problem associated to the differences of applicable legal rights in different countries or territories, and this activity was performed manually at a high cost. On the other hand, functionalities were common and mandatory and did not very much. The crucial issue in SPL development and practice is to manage the correct selection of variants. However, no standard methods have been developed yet, and industry builds SPL using on-the-market or in-house techniques and methods, aware of the benefits a product line can provide; nevertheless, this development must return the investment, and this is not always the case. In this work an approach to variability management in case of legal rights applicability to different entities is proposed. This architecture-centric and quality-based approach uses a reference architecture that has been built with a bottom-up strategy. Variability is incorporated to the reference architecture at abstract level considering non-functional properties. A "production plan" to reduce the gap between abstraction and implementation levels is defined

CaMKII activity in the inflammatory response of cardiac diseases

Inflammation is a physiological process by which the body responds to external insults and stress conditions, and it is characterized by the production of pro-inflammatory mediators such as cytokines. The acute inflammatory response is solved by removing the threat. Conversely, a chronic inflammatory state is established due to a prolonged inflammatory response and may lead to tissue damage. Based on the evidence of a reciprocal regulation between inflammation process and calcium unbalance, here we described the involvement of a calcium sensor in cardiac diseases with inflammatory drift. Indeed, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in several diseases with an inflammatory component, such as myocardial infarction, ischemia/reperfusion injury, pressure overload/hypertrophy, and arrhythmic syndromes, in which it actively regulates pro-inflammatory signaling, among which includes nuclear factor kappa-B (NF-κB), thus contributing to pathological cardiac remodeling. Thus, CaMKII may represent a key target to modulate the severity of the inflammatory-driven degeneration