R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.Fil: Mesquita, Fernanda C. P.. Universidade Federal do Rio de Janeiro; BrasilFil: Arantes, Paulo C.. Universidade Federal do Rio de Janeiro; BrasilFil: Kasai Brunswick, Tais H.. Universidade Federal do Rio de Janeiro; BrasilFil: Araujo, Dayana S.. Universidade Federal do Rio de Janeiro; BrasilFil: Gubert, Fernanda. Universidade Federal do Rio de Janeiro; BrasilFil: Monnerat, Gustavo. Universidade Federal do Rio de Janeiro; BrasilFil: Silva dos Santos, Danúbia. Universidade Federal do Rio de Janeiro; BrasilFil: Neiman, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Leitão, Isabela C.. Universidade Federal do Rio de Janeiro; BrasilFil: Barbosa, Raiana A. Q.. Universidade Federal do Rio de Janeiro; BrasilFil: Coutinho, Jorge L.. National Institute Of Cardiology; BrasilFil: Vaz, Isadora M.. Pontificia Universidad Catolica de Parana; BrasilFil: dos Santos, Marcus N.. Universidade Federal do Rio de Janeiro; BrasilFil: Borgonovo, Tamara. Pontificia Universidad Catolica de Parana; BrasilFil: Cruz, Fernando E. S.. National Institute of Cardiology; BrasilFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Medei, Emiliano H.. Universidade Federal do Rio de Janeiro; BrasilFil: Campos de Carvalho, Antonio C.. Universidade Federal do Rio de Janeiro; Brasil. National Institute of Cardiology; Brasil. National Institute for Science and Technology in Regenerative Medicine; BrasilFil: Carvalho, Adriana B.. Universidade Federal do Rio de Janeiro; Brasil. National Institute for Science and Technology in Regenerative Medicine; Brasi

Cholini (Coleoptera: Curculionidae, Molytinae) depositados na Coleção de Invertebrados do Instituto Nacional de Pesquisas da Amazônia

In Brazilian Amazonia, Cholini (Coleoptera, Curculionidae, Molytinae) is represented by 53 species distributed in seven generaAmeris Dejean, 1821; Cholus Germar, 1824; Homalinotus Sahlberg, 1823; Lobaspis Chevrolat, 1881; Odontoderes Sahlberg, 1823; Ozopherus Pascoe, 1872 and Rhinastus Schoenherr, 1825. This work documents the species of Cholini housed in the Invertebrate Collection of the Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil and gives the geographical and biological data associated with them. A total of 186 Cholini specimens were identified as belonging to 14 species (13 from Brazilian Amazonia) and five genera (Cholus, Homalinotus, Odontoderes, Ozopherus and Rhinastus). Only 24% of the Cholini species reported from Brazilian Amazonia are actually represented in the INPA collection, underscoring the need for a more systematical collecting based on available biological information. The known geographical distribution was expanded for the following speciesCholus granifer (Chevrolat, 1881) for Brazil; C. pantherinus (Olivier, 1790) for Manaus (Amazonas); Cholus parallelogrammus (Germar, 1824) for Piraquara (Paraná); Homalinotus depressus (Linnaeus, 1758) for lago Janauacá (Amazonas) and rio Tocantins (Pará); H. humeralis (Gyllenhal, 1836) for Novo Airão, Coari (Amazonas) and Porto Velho (Rondônia); H. nodipennis (Chevrolat, 1878) for Carauari, Lábrea (Amazonas) and Ariquemes (Rondônia); H. validus (Olivier, 1790) for rio Araguaia (Brasil), Manaus (Amazonas), rio Tocantins (Pará), Porto Velho and BR 364, Km 130 (Rondônia); Odontoderes carinatus (Guérin-Méneville, 1844) for Manaus (Amazonas); O. spinicollis (Boheman, 1836) for rio Uraricoera (Roraima); and Ozopherus muricatus Pascoe, 1872 for lago Janauacá (Amazonas). Homalinotus humeralis is reported for the first time from "urucuri" palm, Attalea phalerata Mart. ex Spreng.Na Amazônia brasileira, Cholini (Coleoptera, Curculionidae, Molytinae) é representada por 53 espécies, distribuídas em sete gêneros: Ameris Dejean, 1821; Cholus Germar, 1824; Homalinotus Sahlberg, 1823; Lobaspis Chevrolat, 1881; Odontoderes Sahlberg, 1823; Ozopherus Pascoe, 1872 e Rhinastus Schoenherr, 1825. Este trabalho documenta as espécies de Cholini depositadas na Coleção de Invertebrados do Instituto Nacional de Pesquisas da Amazônia, Manaus, Brasil, além de apresentar a distribuição geográfica e informações sobre a biologia dessas espécies. Foram identificados 186 espécimes de Cholini, pertencentes a 14 espécies (13 da Amazônia brasileira) e cinco gêneros (Cholus, Homalinotus, Odontoderes, Ozopherus e Rhinastus). Somente 24% das espécies de Cholini registradas para a Amazônia brasileira estão representadas na coleção do INPA, ressaltando a necessidade de um esforço de coleta sistemático baseado na informação biológica disponível. Foi ampliada a distribuição geográfica conhecida das seguintes espécies: Cholus granifer (Chevrolat, 1881) para Brasil; C. pantherinus (Olivier, 1790) para Manaus (Amazonas); Cholus parallelogrammus (Germar, 1824) para Piraquara (Paraná); Homalinotus depressus (Linnaeus, 1758) para lago Janauacá (Amazonas) e rio Tocantins (Pará); H. humeralis (Gyllenhal, 1836) para Novo Airão, Coari (Amazonas) e Porto Velho (Rondônia); H. nodipennis (Chevrolat, 1878) para Carauari, Lábrea (Amazonas) e Ariquemes (Rondônia); H. validus (Olivier, 1790) para rio Araguaia (Brasil), Manaus (Amazonas), rio Tocantins (Pará), Porto Velho e BR 364, Km 130 (Rondônia); Odontoderes carinatus (Guérin-Méneville, 1844) para Manaus (Amazonas); O. spinicollis (Boheman, 1836) para rio Uraricoera (Roraima) e Ozopherus muricatus Pascoe, 1872 para lago Janauacá (Amazonas). Homalinotus humeralis é associado pela primeira vez com a palmeira urucuri Attalea phalerata Mart. ex Spreng

Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants

Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance but increased high-affinity anti-spike antibodies

Summary: The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immune response of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic T cell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection

Accuracy and ease-of-use of seven point-of-care SARS-CoV-2 antigen-detecting tests: A multi-centre clinical evaluation.

BACKGROUND: Antigen-detecting rapid diagnostic tests (Ag-RDTs) for SARS-CoV-2 are important diagnostic tools. We assessed clinical performance and ease-of-use of seven Ag-RDTs in a prospective, manufacturer-independent, multi-centre cross-sectional diagnostic accuracy study to inform global decision makers. METHODS: Unvaccinated participants suspected of a first SARS-CoV-2 infection were recruited at six sites (Germany, Brazil). Ag-RDTs were evaluated sequentially, with collection of paired swabs for routine reverse transcription polymerase chain reaction (RT-PCR) testing and Ag-RDT testing. Performance was compared to RT-PCR overall and in sub-group analyses (viral load, symptoms, symptoms duration). To understandusability a System Usability Scale (SUS) questionnaire and ease-of-use (EoU) assessment were performed. FINDINGS: 7471 participants were included in the analysis. Sensitivities across Ag-RDTs ranged from 70·4%-90·1%, specificities were above 97·2% for all Ag-RDTs but one (93·1%).Ag-RDTs, Mologic, Bionote, Standard Q, showed diagnostic accuracy in line with WHO targets (> 80% sensitivity, > 97% specificity). All tests showed high sensitivity in the first three days after symptom onset (≥87·1%) and in individuals with viral loads≥ 6 log10SARS-CoV2 RNA copies/mL (≥ 88·7%). Usability varied, with Rapigen, Bionote and Standard Q reaching very good scores; 90, 88 and 84/100, respectively. INTERPRETATION: Variability in test performance is partially explained by variable viral loads in population evaluated over the course of the pandemic. All Ag-RDTs reach high sensitivity early in the disease and in individuals with high viral loads, supporting their role in identifying transmission relevant infections. For easy-to-use tests, performance shown will likely be maintained in routine implementation. FUNDING: Ministry of Science, Research and Arts, State of Baden-Wuerttemberg, Germany, internal funds from Heidelberg University Hospital, University Hospital Charité - Universitätsmedizin Berlin, UK Department of International Development, WHO, Unitaid

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p<0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p<0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status