RESUMO
Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).
Assuntos
Códon sem Sentido , Doenças Genéticas Inatas , Guanidinas , Quinazolinas , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Gentamicinas/farmacologia , Guanidinas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Quinazolinas/farmacologiaRESUMO
Glioma is one of the most important central nervous system tumors, ranked 15th in the most common cancer for men and women. Magnetic Resonance Imaging (MRI) represents a common tool for medical experts to the diagnosis of glioma. A set of multi-sequences from an MRI is selected according to the severity of the pathology. Our proposed approach aims moreto create a computer-aided system that is capable of helping morethe expert diagnose the brain gliomas. moreWe propose a supervised learning regime based on a convolutional neural network based framework and transfer learning techniques. Our research morefocuses on the performance of different pre-trained deep learning models with respect to different MRI sequences. We highlight the best combinations of such model-MRI sequence couple for our specific task of classifying healthy brain against brain with glioma. moreWe also propose to visually analyze the extracted deep features for studying the existing relation of the MRI sequences and models. This interpretability analysis gives some hints for medical expert to understand the diagnosis made by the models. Our study is based on the well-known BraTS datasets including multi-sequence images and expert diagnosis.
RESUMO
The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.
Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Genes Bacterianos/genética , Família Multigênica , Streptomyces/genética , Antibacterianos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Dicetopiperazinas/química , Hidroxilação , Modelos Químicos , Estrutura Molecular , Mutação , Streptomyces/metabolismoRESUMO
Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.
