Systematic generation of in vivo G protein-coupled receptor mutants in the rat

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Protocolo de reabilitação acelerada após reconstrução de ligamento cruzado anterior - dados normativos

OBJETIVO: Avaliar os resultados obtidos com o protocolo de reabilitação acelerada, adaptado às condições de clínica, em pacientes submetidos à operação de reconstrução do ligamento cruzado anterior. MÉTODOS: Foram incluídos 30 pacientes, praticantes de atividade esportiva recreacional, submetidos à operação de reconstrução do ligamento cruzado anterior por meio do tendão patelar. Todos fizeram a reabilitação com o mesmo protocolo de tratamento e no mesmo local. A avaliação isocinética em diferentes ângulos foi realizada antes da operação e no 4° mês de pós-operatório utilizando dinamômetro isocinético computadorizado da marca Cybex Norm. RESULTADOS: As avaliações no pré-operatório em média demonstraram: pico de torque flexor 93% a 60°/s e 97,3% a 180°/s; extensor 87,3% a 60°/s e 94,7% a 180°/s; potência nos músculos flexores de 93,3% e nos extensores de 96,7%; trabalho muscular dos flexores de 91,7% e nos extensores de 90,3%; o ângulo do pico de torque flexor de 28,7°, na musculatura extensora o ângulo foi de 62,2°; pico de torque excêntrico nos flexores de 78,3% e nos extensores de 12,8%. Com quatro meses de pós-operatório os resultados obtidos em média foram: pico de torque flexor 95,4% a 60°/s e 97,1% 180°/s; extensor 70% a 60°/s e 75,7% a 180°/s; potência nos músculos flexores de 97,1% e nos extensores de 79,8%; trabalho muscular dos flexores de 94,2% e nos extensores de 94,2%; pico de torque excêntrico dos flexores de 84% e nos extensores de 24,2%; o ângulo do pico de torque flexor foi a 27,3°; na musculatura extensora o ângulo foi de 61,7°. CONCLUSÃO: Os resultados demonstraram que os pacientes tratados com o protocolo adaptado apresentam resultados semelhantes aos obtidos com o protocolo original em relação às condições musculares