Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses

Purpose: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. Methods: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. Results: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. Conclusions: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles

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

Molecular Characterization of a Rare Case of Bilateral Vitreoretinal T Cell Lymphoma through Vitreous Liquid Biopsy

Vitreoretinal lymphoma (VRL) is an uncommon eye malignancy, and VRLs of T cell origin are rare. They are difficult to treat, and their molecular underpinnings, including actionable genomic alterations, remain to be elucidated. At present, vitreous fluid liquid biopsies represent a valuable VRL sample for molecular analysis to study VRLs. In this study, we report the molecular diagnostic workup of a rare case of bilateral T cell VRL and characterize its genomic landscape, including identification of potentially targetable alterations. Using next-generation sequencing of vitreous-derived DNA with a pan-cancer 126-gene panel, we found a copy number gain of BRAF and copy number loss of tumor suppressor DNMT3A. To the best of our knowledge, this represents the first exploration of the T cell VRL cancer genome and supports vitreous liquid biopsy as a suitable approach for precision oncology treatments

Validação interna do questionário de estágio de prontidão para mudança do comportamento alimentar e de atividade física Internal validation of the stage of change questionnaire for alimentary and physical activity behaviors

OBJETIVO: Traduzir para a língua portuguesa e adaptar o conteúdo do questionário de avaliação dos estágios de prontidão para mudança do comportamento alimentar e de atividade física proposto por Sutton et al para a aplicação em adolescentes, além de verificar a consistência interna e a reprodutibilidade da nova versão do instrumento. MÉTODOS: A validação de conteúdo foi realizada por um grupo multiprofissional, foi consultado após a tradução e re-tradução do instrumento com o intuito de gerar uma versão coerente, seguida da adaptação cultural realizada por meio de escala Likert, que objetivou analisar a clareza e a relevância, avaliada pelos adolescentes para cada uma das afirmações. A reprodutibilidade e a consistência interna foram analisadas pelo preenchimento do questionário por 32 adolescentes obesos de ambos os sexos, ingressantes em programa de tratamento multiprofissional da obesidade, com idade média de 13,5 (±1,6) anos e peso médio de 86,1kg (±18,8). Para avaliar a consistência interna, aplicou-se o coeficiente Alpha de Cronbach e, para a reprodutibilidade, os coeficientes de correlação de Pearson, Spearman e intraclasse, além do teste t. RESULTADOS: Os resultados revelaram alta reprodutibilidade do instrumento e, no que diz respeito à consistência interna, foram demonstrados bons índices de confiabilidade para todos os domínios, tendo em vista que todos eles apresentaram valores superiores ou bem próximos a 0,70. CONCLUSÕES: O questionário apresentou boa consistência interna e reprodutibilidade para avaliar o estágio de prontidão de mudança do comportamento alimentar e de atividade física em adolescentes obesos. Contudo, para que a validade externa do instrumento seja confirmada, é oportuno que outros estudos sejam realizados.<br>OBJECTIVE: To translate into Portuguese the contents of the questionnaire for adaptation assessment of dietary and physical activity changes proposed by Sutton et al, to be used with adolescents; and to verify the internal consistency and reliability of this tool. METHODS: The content validation was accomplished by a multidisciplinary group, which was consulted after the translation and back translation of the instrument in order to generate a coherent version, followed by the cultural adaptation performed using a Likert's scale, from which adolescents assessed clarity and relevance of each statement. The reliability and internal consistency of the tool were assessed by a questionnaire applied to 32 obese adolescents of both genders from a multiprofessional program for obesity treatment. They had, on average, 13.5±1.59 years old and 86.1±18.8kg. The Cronbach's alpha coefficient was used to assess the internal consistency, as well as Pearson, Spearman and intraclass correlation coefficients. Also the t-test was used to assess reliability. RESULTS: The results of test and retests revealed the high reliability of the tool. Internal consistency showed good reliability rates for all areas, with all values higher than or near to 0.70. CONCLUSIONS: The questionnaire presented good internal consistency and reliability in obese adolescents. However, it is appropriate to carry out further studies in order to confirm the external validity of the instrument

Phylogenomics and the rise of the angiosperms.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5-7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade