RESUMO
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Duchenne , Animais , Sistemas CRISPR-Cas , Distrofina/genética , Distrofina/metabolismo , Éxons , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Deleção de SequênciaRESUMO
Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3ß as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.
Assuntos
Diferenciação Celular , Células Musculares/citologia , Proteínas Musculares/imunologia , Músculo Esquelético/citologia , NF-kappa B/imunologia , Proteínas Nucleares/imunologia , Proteínas Repressoras/imunologia , Animais , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Musculares/imunologia , Proteínas Musculares/genética , Músculo Esquelético/imunologia , NF-kappa B/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Repressoras/genéticaAssuntos
Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/radioterapia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Combinada , Humanos , Imunoterapia , Cuidados Pós-Operatórios , Cuidados Pré-Operatórios , Estudos Prospectivos , Dosagem Radioterapêutica , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias Retais/mortalidadeRESUMO
A specific radioimmunoassay of estriol has been developed using an antiserum obtained by immunization of rabbits against 1,3,5 (10)-Estratrien -3, 16 alpha 17 beta-triol-6 one 6 carboximethyl oxime: BAS (Estriol 6-CMO-BSA). The tracer used was Estriol (2, 4, 6, 4, 3H (N)). This assay does not require prior hydrolysis, extraction or purification. Either plasma, serum or urine can be used. Urine only requires 100 microliter (1:5) dilution and 100 microliter in serum or plasma. An incubation time of 60 minutes is requires; a linear standard curve is obtained by logit-log extrapolation and a good correlation was obtained (r = 0.063) with estriol determination by comparison to a generally accepted colorimetric method. The detection range is from 100 pg to 50,000 pg in plasma or serum and urine. The specificity of the antibody was determined by studies of cross reactivity with other steroids. The sensitivity (100 pg) and accuracy were proven to be highly satisfactory. This method is simple, rapid and accurate.
Assuntos
Estriol/análise , Animais , Estriol/sangue , Estriol/urina , Feminino , Humanos , Indicadores e Reagentes , Gravidez , RadioimunoensaioRESUMO
The authors report their experience of 30 patients with colorectal anastomotic stenosis treated by 62 dilatation sessions in order to evaluate which anastomotic characteristics could influence the success of dilatation therapy. Patients were subdivided into group A (dilatation successful) and group B (dilatation unsuccessful). Overall, dilatation was successful in 73.3% of cases, with only one important complication. The prognostic factors considered were anastomotic dehiscence, adjuvant radiotherapy, presence of colostomy at dilatation, site, morphology and length of the stenosis, presence of neoplastic recurrence, type of anastomosis and type of dilatation. Radiotherapy, local neoplastic recurrence and large anastomotic dehiscence were the more important independent prognostic factors. If present together, they were associated with an almost 100% probability of failure and, vice versa, if they were absent this probability was 5%.