Subtelomeric I-scel-mediated double-strand breaks are repaired by homologous recombination in trypanosoma cruzi

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a I cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-Scel meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP306591/2015-411/51693-0; 11/51475-

Subtelomeric I-Scel-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a I cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-Scel meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Centroccidental Lisandro Alvarado, Lab Genet Mol Dr Yunis Turbay, Ciencias Salud, Barquisimeto, VenezuelaNIAID, Lab Malaria & Vector Res, NIH, Rockville, MD USAUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilConsejo Nacl Invest Cient & Tecn, Inst Invest Ingn Genet & Biol Mol, Lab Biol Mol Enfermedad Chagas, Buenos Aires, DF, ArgentinaJ Craig Venter Inst, Dept Infect Dis, Rockville, MD USAFdn Inst Estudios Avanzados, Ctr Biotecnol, Caracas, VenezuelaUniv Estadual Campinas, Fac Ciencias Med, Dept Patol Clin, Campinas, SP, BrazilDepartamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, BrazilFAPESP: 11/51693-0FAPESP: 11/51475-3CNPq: 306591/2015-4Web of Scienc

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Association between convalescent plasma treatment and mortality in COVID-19: a collaborative systematic review and meta-analysis of randomized clinical trials.

Funder: laura and john arnold foundationBACKGROUND: Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX ). METHODS: In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung-Knapp-Sidik-Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence. RESULTS: A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I2 = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis. CONCLUSIONS: Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Larval Diel Vertical Migration of the Marine Gastropod Kelletia kelletii

Documenting larval behavior is critical for building an understanding of larval dispersal dynamics and resultant population connectivity. Nocturnal diel vertical migration (DVM), a daily migration towards the surface of the water column at night and downward during the day, can profoundly influence dispersal outcomes. Via laboratory experiments we investigated whether marine gastropod Kelletia kelletii larvae undergo nocturnal DVM and whether the behavior was influenced by the presence of light, ontogeny, and laboratory culturing column height. Larvae exhibited a daily migration pattern consistent with nocturnal diel vertical migration with lower average vertical positioning (ZCM) during day-time hours and higher vertical positioning at night-time hours. ZCM patterns varied throughout ontogeny; larvae became more demersal as they approached competency. There was no effect of column height on larval ZCM. DVM behavior persisted in the absence of light, indicating a possible endogenous rhythm. Findings from field plankton tows corroborated laboratory nocturnal DVM findings; significantly more K. kelletii were found in surface waters at midnight compared to at noon. Unraveling the timing of and the cues initiating DVM behavior in K. kelletii larvae can help build predictive models of dispersal outcomes for this emerging fishery species

Genome Size, Karyotype Polymorphism and Chromosomal Evolution in Trypanosoma cruzi

Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites. Methodology/Principal Findings: The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (Jose-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively. Conclusions: Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo/FAPESPConselho Nacional de Desenvolvimento Cientifico e Tecnologico/CNPq (Brazil)FAPES

Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocyte-macrophage colony-stimulating factor or interleukin-3

Tyrosine kinase fusion oncogenes that occur as a result of chromosomal translocations have been shown to activate proliferative and antiapoptotic pathways in leukemic cells, but the importance of autocrine and paracrine expression of hematopoietic cytokines in leukemia pathogenesis is not understood. Evidence that leukemic transformation may be, at least in part, cytokine dependent includes data from primary human leukemia cells, cell culture experiments, and murine models of leukemia. This report demonstrates that interleukin (IL)-3 plasma levels are elevated in myeloproliferative disease (MPD) caused by the TEL/tyrosine kinase fusions TEL/platelet-derived growth factor beta receptor (PDGFbetaR), TEL/Janus kinase 2 (JAK2), and TEL/neurotrophin-3 receptor (TRKC). Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were elevated by TEL/PDGFbetaR and TEL/JAK2. However, all of the fusions tested efficiently induced MPD in mice genetically deficient for both GM-CSF and IL-3, demonstrating that these cytokines are not necessary for the development of disease in this model system. Furthermore, in experiments using normal marrow transduced with TEL/PDGFbetaR retrovirus mixed with marrow transduced with an enhanced green fluorescent protein (EGFP) retrovirus, the MPD induced in these mice demonstrated minimal stimulation of normal myelopoiesis by the TEL/PDGFbetaR-expressing cells. In contrast, recipients of mixed GM-CSF-transduced and EGFP-transduced marrow exhibited significant paracrine expansion of EGFP-expressing cells. Collectively, these data demonstrate that, although cytokine levels are elevated in murine bone marrow transplant models of leukemia using tyrosine kinase fusion oncogenes, GM-CSF and IL-3 are not required for myeloproliferation by any of the oncogenes tested