Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme

Pediatricians and nutritionists knowledge about treatment of cow milk allergy in infants

OBJECTIVE: Evaluate the knowledge of pediatricians and nutritionists regarding the exclusion diet of cow milk and derivates, with emphasis on questions related to the nutrition of children submitted to such diet. METHODS: Cross-sectional study that enrolled pediatricians (n=53) and nutritionists (n=29) from public hospitals in São Paulo, Brazil, during 2005. Data was collected through self-administered questionnaires. RESULTS: The age of the professionals varied from 21 to 50 years old. Regarding professional experience, 41.2% were graduated for less than five years and 91.6% had a specialization course, masters and/or PhD degree. The vast majority of professionals (97.5%) confirmed that they regularly evaluated the diet of children that needed exclusion of cow milk. However, only 48% of the professionals conducted a more detailed evaluation of the diet, including calculations of food ingestion. Only 38.7% of the professionals compared child s food ingestion with some recommended pattern. Recommendations for daily ingestion of calcium by children up to the age of 36 months were properly mentioned by 22% of the pediatricians and 60.7% of the nutritionists (p=0.001). Inadequate cow milk substitute products were recommended by 66% of the pediatricians and by 48.3% of the nutritionists. Regarding labels of industrialized products, 81.6% of the pediatricians and 96.4% of the nutritionists advised the parents to look for all terms that could indicate the presence of cow milk protein. CONCLUSIONS: Pediatricians and nutritionists made conceptual errors in their main recommendations regarding the treatment of cow milk protein allergy.OBJETIVO: Avaliar o conhecimento de pediatras e nutricionistas sobre a dieta de exclusão do leite de vaca e seus derivados, com ênfase em questões relacionadas à nutrição da criança. MÉTODOS: Estudo transversal descritivo, do qual participaram pediatras (n=53) e nutricionistas (n=29), vinculados a hospitais públicos do Município de São Paulo, no ano de 2005. Os dados foram coletados por questionário auto-administrado. RESULTADOS: A idade dos profissionais variou de 21 a 50 anos. Quanto ao tempo de graduação, 41,2% eram formados a menos de cinco anos e 91,6% possuíam especialização, mestrado e/ou doutorado. A maioria (97,5%) afirmou avaliar a dieta de crianças submetidas à exclusão do leite de vaca, entretanto, somente 48% o faziam de forma mais detalhadas, incluindo o cálculo da ingestão alimentar. Apenas 38,7% comparam a ingestão alimentar da criança com algum padrão de recomendação. A recomendação diária da ingestão de cálcio para crianças com até 36 meses foi corretamente assinalada por 22% dos pediatras e 60,7% dos nutricionistas (p=0,001). Produtos não adequados como substitutos do leite de vaca seriam recomendados por 66% dos pediatras e 48,3% dos nutricionistas. Com relação à leitura de rótulos de produtos industrializados, 81,6% dos pediatras e 96,4% dos nutricionistas orientam os pais a ler todos os termos que indicam a presença das proteínas do leite de vaca. CONCLUSÕES: Os pediatras e nutricionista demonstraram erro conceitual no que se refere às principais recomendações terapêuticas na alergia às proteínas do leite de vaca.Universidade Federal de São Paulo (UNIFESP)UNIFESPUNIFESPSciEL

Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

Trypanosoma cruzi, is the etiological agent of a neglected tropical malady known as Chagas' disease, which affects about 8 million people in Latin America. 30–40% of affected individuals develop a symptomatic chronic infection, with cardiomyopathy being the most prevalent condition. T. cruzi utilizes an interesting strategy for entering cells: T. cruzi enhances intracellular calcium levels, which in turn trigger the exocytosis of lysosomal contents. Lysosomes then donate their membrane for the formation of the parasitophorous vacuole. Membrane rafts, cholesterol-enriched microdomains in the host cell plasma membrane, have also been implicated in T. cruzi invasion process. Since both plasma membrane and lysosomes collaborate in parasite invasion, we decided to study the importance of these membrane domains for lysosomal recruitment and fusion during T. cruzi invasion into host cells. Our results show that drug dependent depletion of plasma membrane cholesterol changes raft organization and induces excessive lysosome exocytosis in the earlier stages of treatment, leading to a depletion of lysosomes near the cell cortex, which in turn compromises T. cruzi invasion. Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events of pre-docked lysosomes, reducing lysosome availability at the cell cortex and consequently compromising T. cruzi infection

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

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

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