Research progress in mitochondrial gene editing technology / 浙江大学学报·医学版

Mitochondrial DNA (mtDNA) mutations result in a variety of genetic diseases. As an emerging therapeutic method, mtDNA editing technology recognizes targets more based on the protein and less on the nucleic acid. Although the protein recognition type mtDNA editing technology represented by zinc finger nuclease technology, transcription activator like effector nuclease technology and base editing technology has made some progress, the disadvantages of complex recognition sequence design hinder further popularization. Gene editing based on nucleic acid recognition by the CRISPR system shows superiority due to the simple structure, easy design and modification. However, the lack of effective means to deliver nucleic acids into mitochondria limits application in the field of mtDNA editing. With the advances in the study of endogenous and exogenous import pathways and the deepening understanding of DNA repair mechanisms, growing evidence shows the feasibility of nucleic acid delivery and the broad application prospects of nucleic acid recognition type mtDNA editing technology. Based on the classification of recognition elements, this article summarizes the current principles and development of mitochondrial gene editing technology, and discusses its application prospects.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2\'-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

The impact of health education on stress reaction condition and quality of life of patients with sudden deafness / 中国现代医生

Objective To investigate the impact of health education on stress reaction condition and quality of life of patients with sudden deafness. Methods Form February 2011 to November 2013 in our hospital cured unilateral sud-den deafness 110 patients were randomly divided to the intervention group and the control group each of 55 cases, the intervention group received cognitive behavioral education, psychological guidance before and after relaxation training guidance to the main content of health education, more intervention groups at SASRQ, WHOQOL-BREF, PSQI score and efficacy. Results The intervention group SASRQ total score were significantly lower than the control group (P<0.05). After the intervention group WHOQOL-BREF psychological domain, social relations, health status and overall quality of life scores were significantly higher than the control group(P<0.05). After the intervention group dimensions PSQI scores were significantly lower than the control group(P<0.05). The intervention group and control significant ef-ficiency difference were statistically significant(P<0.05). Conclusion Patients with sudden deafness obvious physiolog-ical and psychological stress affect the quality of life, health education helps alleviate patient stress, improve sleep,improve quality of life and promote improved treatment of sudden deafness.

Effect of healthy education on quality of life and disease cognitive level of patients with sudden hearing loss / 中国现代医生

Objective To discuss effect of healthy education on quality of life and disease cognitive level of patients with sudden hearing loss. Methods Selected 60 cases with sudden hearing loss and received healthy education. Related knowledge, quality of life were compared. Results After healthy education, disease cognitive levels of patients increased apparently compared with than before education (P<0.01). After education, listening level, psychological states, social function were improved apparently than before education (P<0.01). Conclusion Healthy education can improve quality of life and disease cognitive level of patients with sudden hearing loss.

Effect of health education intervention on efficacy and quality of life for patients with Meniere's / 中国现代医生

Objective To investigate the effect of improving the symptoms of Meniere's disease and quality of life of health education intervention. Methods From May 2012 to August 2013 in our hospital treated 87 cases of Meniere's disease were divided into intervention group (44 cases) and control group (43 cases), intervention group treated with Meniere's disease health education,vestibular symptom Index (VSI),dizziness handicap inventory (DHI),self-rating anxiety scale(SDS),self-rating depression scale(SAS)to improve the situation were compared. Results The intervention group after the intervention equilibrium,vertigo,dizziness,nausea,visual sensitivity,headache,and scores were signifi cantly lower than the control group(P<0.05). Intervention group after the intervention DHI emotional,functional,phys-ical sexual scores were significantly lower than the control group (P<0.05). After the intervention group SDS and SAS scores were significantly lower than the control group(P<0.05). Conclusion Meniere's disease has a significant impact on the vestibular function,psychological and quality of life of patients, health education helps to improve vestibular symptoms,reduce negative emotions,improve quality of life.