Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

起始密码下游序列与16srRNA3′端序列的匹配性对基因表达效率的影响

目的 :研究起始密码下游序列与 16srRNA 3′端 +146 9——— +1482局部序列间的匹配关系对基因表达效率的影响。方法 :我们一方面对人IL_2、人IL_8、人IL_6 ,人GM_CSF、人G_CSF、人IL_1ra、人IL_9等全长cDNA序列及 5′末端局部序列与 16srRNA 3′端 +146 9——— +1482序列进行匹配性进行分析 ,寻找序列匹配性与表达效率间的关系 ;另一方面 ,对人IL_6、人GM_CSFcDNA 5′端序列进行沉默突变 ,增强其与 16srRNA 3′端 +146 9——— +1482序列间的匹配性 ,并克隆入pKpL4表达型质粒载体 ,通过大肠杆菌进行表达 ,实验验证序列匹配性与基因表达效率的关系。结果 :分析发现起始密码下游局部序列与 16srRNA 3′端 +146 9——— +1482核酸匹配性越好 ,则目的蛋白表达效率越高 ;目的基因编码区内与 16srRNA 3′ +146 9——— +1482间的最大匹配区域越靠近 5′末端 ,目的蛋白表达效率越高。通过对人IL_6、人GM_CSFcDNA 5′末端序列进行沉默突变 ,改善其与 16srRNA 3′端 +146 9——— +1482序列的匹配性后 ,目的蛋白的表达量均呈不同程度地提高。结论 :提高起始密码下游序列与 16srRNA 3′端 +146 9——— +1482局部序列间的匹配性 ,可增强目的基因的表达效率。     

DNA测序建立甘肃当归、大黄种子rRNA基因图谱的研究

目的：对甘肃地道性中药材当归、大黄种子中rRNA基因内转录间隔区进行碱基序列测定，以期建立从分子水平对中草药种子进行鉴定的一种新方法。方法：提取当归、大黄种子中核DNA，利用特异性引物对其rRNA基因内转录间隔区进行套式PCR扩增，并将扩增产物进行碱基序列测定。结果：经琼脂糖凝胶电泳证实：以上述方法可获得当归、大黄种子DNA中rRNA基因内转录间隔区PCR扩增产物；并经测序后得到了当归、大黄种子rRNA基因内转录间隔区的碱基序列，且具有明显的差异和可对比性。结论：不同植物性中药材种子rRNA基因内转录间隔区碱基序列可做为从分子水平进行鉴定的标记。     

基于化学成分与肠道菌群探讨消癌解毒方中黄连-乌梅药对的配伍机制

目的 探讨黄连-乌梅药对与消癌解毒方水煎液中部分生物碱类成分的含量差异,并比较药对与全方对小鼠肠道菌群的影响。方法 采用Extend-C₁₈(100 mm×2.1 mm,1.8 μm)色谱柱,以0.1%甲酸水（A）-乙腈（B）进行梯度洗脱,流速0.3 mL/min,柱温35 ℃,进样量2 μL;离子化模式为电喷雾离子化（ESI）,以正离子模式检测,开展方法学验证和含量测定研究。按照给药不同将小鼠分为正常组、全方高浓度组、全方低浓度组、药对高浓度组和药对低浓度组,连续灌胃给药7 d,每组分别收集粪便样本,进行16S rRNA基因测序。结果 建立的含量测定方法在一定浓度范围内线性关系良好,方法学验证结果均符合相关要求,木兰花碱与非洲防己碱在黄连、药对以及全方中的含量分别为:(4.433±0.133)(8.905±0.154) mg/g,（3.545±0.033）（9.170±0.051） mg/g,（5.287±0.038）（13.861±0.690） mg/g。全方组和药对组中拟杆菌门Bacteroides的丰度均高于正常组,而全方组和药对组中厚壁菌门Firmicutes的丰度均低于正常组。药对高浓度组中的疣微菌门Verrucomicrobia和变形菌门Proteobacteria的丰度高于其他组。结论 液质联用方法和相关参数简便、可靠,可用于有关成分的含量检测。与单味药中含量相比,经过药对配伍后,非洲防己碱含量增加,而木兰花碱含量则略有降低;经过全方配伍后,2种成分的含量均增加。此外,肠道菌群实验表明无论是药对还是全方在给药后,会不同程度地影响肠道菌群内部占比改变,其可能对调节肠道系统平衡,具有一定帮助作用,从而为进一步阐释黄连-乌梅在消癌解毒方中配伍机制提供了参考和依据。      

Dietary Vitamin B1 Intake Influences Gut Microbial Community and the Consequent Production of Short-Chain Fatty Acids

The gut microbiota is closely related to good health; thus, there have been extensive efforts dedicated to improving health by controlling the gut microbial environment. Probiotics and prebiotics are being developed to support a healthier intestinal environment. However, much work remains to be performed to provide effective solutions to overcome individual differences in the gut microbial community. This study examined the importance of nutrients, other than dietary fiber, on the survival of gut bacteria in high-health-conscious populations. We found that vitamin B1, which is an essential nutrient for humans, had a significant effect on the survival and competition of bacteria in the symbiotic gut microbiota. In particular, sufficient dietary vitamin B1 intake affects the relative abundance of Ruminococcaceae, and these bacteria have proven to require dietary vitamin B1 because they lack the de novo vitamin B1 synthetic pathway. Moreover, we demonstrated that vitamin B1 is involved in the production of butyrate, along with the amount of acetate in the intestinal environment. We established the causality of possible associations and obtained mechanical insight, through in vivo murine experiments and in silico pathway analyses. These findings serve as a reference to support the development of methods to establish optimal intestinal environment conditions for healthy lifestyles.     

乌鲁木齐地区线粒体12SrRNA基因突变筛查分析

目的　研究乌鲁木齐地区非综合征性聋患者线粒体12S rRNA基因突变情况。方法　收集乌鲁木齐非综合征性聋患者标本609例,对其进行临床和分子遗传学评估。结果　12S rRNA基因突变分析共发现11个突变位点,已知的A1555G、961DelT、C1494T突变分别占2.96%,1.15%,0.16%。另外A1047G突变相关报道较少,A1585G突变未见相关报道。其他突变均为多态性位点。结论　线粒体12S rRNA突变是引起遗传性聋的重要因素,此次乌鲁木齐地区线粒体12S rRNA A1555G突变在聋病人群中的检出率与前期报道相比有所降低,可能与耳毒性药物使用量降低有关。A1047G与新发现的A1585G突变是否与聋相关,还需进一步研究。对筛查中阳性突变携带者及其母系家庭成员需告知氨基糖苷类抗生素使用风险,使其避免使用,逐步降低药物性聋的发生率。     

Gut Microbiota Markers and Dietary Habits Associated with Extreme Longevity in Healthy Sardinian Centenarians

This study was aimed at characterizing the gut microbiota (GM) and its functional profile in two groups of Sardinian subjects with a long healthy life expectancy, overall named Long-Lived Subjects (LLS) [17 centenarians (CENT) and 29 nonagenarians (NON)] by comparing them to 46 healthy younger controls (CTLs). In addition, the contribution of genetics and environmental factors to the GM phenotype was assessed by comparing a subgroup of seven centenarian parents (CPAR) with a paired cohort of centenarians’ offspring (COFF). The analysis was performed through Next Generation Sequencing (NGS) of the V3 and V4 hypervariable region of the 16S rRNA gene on the MiSeq Illumina platform. The Verrucomicrobia phylum was identified as the main biomarker in CENT, together with its members Verrucomicrobiaceae, Akkermansia and Akkermansia muciniphila. In NON, the strongest associations concern Actinobacteria phylum, Bifidobacteriaceae and Bifidobacterium, while in CTLs were related to the Bacteroidetes phylum, Bacteroidaceae, Bacteroides and Bacteroides spp. Intestinal microbiota of CPAR and COFF did not differ significantly from each other. Significant correlations between bacterial taxa and clinical and lifestyle data, especially with Mediterranean diet adherence, were observed. We observed a harmonically balanced intestinal community structure in which the increase in taxa associated with intestinal health would limit and counteract the action of potentially pathogenic bacterial species in centenarians. The GM of long-lived individuals showed an intrinsic ability to adapt to changing environmental conditions, as confirmed by functional analysis. The GM analysis of centenarians’ offspring suggest that genetics and environmental factors act synergistically as a multifactorial cause in the modulation of GM towards a phenotype similar to that of centenarians, although these findings need to be confirmed by larger study cohorts and by prospective studies.     

神经型布鲁菌病临床特点和实验室诊断分析

目的总结神经型布鲁菌病（NB）的临床及实验室诊断过程,为提高临床和实验室对该病的认识和早期诊疗提供经验。 方法回顾性分析2015年1月至2017年1月于首都医科大学宣武医院确诊的12例神经型布鲁菌病患者的临床表现、流行病学资料及脑脊液（CSF）培养等病原学检查、影像学表现和其他实验室检查结果,并与非神经型布鲁菌病患者比较平均发病年龄,CSF白细胞数、蛋白含量、氯化物、葡萄糖水平等。 结果12例神经型布鲁菌病患者中男性9例,女性3例,平均年龄为（43 ± 14）岁;10例患者来自流行地区,9例有明确的流行病学接触史,其中3例既往诊断布鲁菌病;12例患者均出现不同程度发热,6例有头痛伴脑膜刺激征表现;3例患者表现为复视、视力下降;伴有听力减退伴步态不稳,肢体麻木无力,精神状态改变者各4例。血清布鲁杆菌凝集试验阳性（100%）较CSF凝集阳性率（58.3%）和CSF培养阳性率（46.1%）对临床诊断神经型布鲁菌病价值较大,差异具有统计学意义（χ² = 52.68、P = 0.005,χ² = 73.79、P = 0.005）;12例NB患者CSF白细胞数（单核为主）和蛋白均不同程度升高,葡萄糖、氯化物正常或减低,与非NB感染者相比,CSF葡萄糖检测具有重要价值（P均< 0.05）。给予多西环素或米诺环素+利福平+头孢曲松治疗后12例患者均预后良好。 结论神经型布鲁菌病临床表现多样,易误诊或漏诊,非疫区医务人员应重视CSF血瓶培养及布鲁杆菌凝集,加强多学科协作交流以及早确诊及早治疗。     

慢性骨盆疼痛综合征前列腺液16S rRNA基因检测及临床意义

目的 :通过前列腺液 16SrRNA基因的检测 ,探讨慢性骨盆疼痛综合征 (CPPS)的细菌感染性病因及其对抗生素疗效的预测价值。　方法 :用通用引物PCR法检测 5 9例CPPS病人前列腺液 (EPS)和初段尿 (VB1)细菌16SrRNA基因。若只在EPS检测到条带 ,或EPS信号比VB1强 10倍以上 ,则细菌信号判断为阳性。所有病人口服左氧氟沙星及阿奇霉素治疗 4周。与治疗前相比 ,治疗后症状严重性积分指数 (SSI)、症状频度问卷积分 (SFQ)、前列腺炎症状积分指数 (NIH CPSI)中疼痛问题的总积分 (quasi CPSI)减少 5 0 %以上 ,或病人主观总体评价改善5 0 %以上作为治疗有效的标准。　结果 :5 9例CPPS病人中细菌信号阳性 46例、阴性 13例。细菌信号阳性与阴性两组治疗有效率分别为 6 5 %～ 74%、0 ,疗效差异有极显著性 (P <0 .0 1)。　结论 :部分CPPS与细菌感染有关 ,前列腺 16SrRNA基因检测的结果对选择抗生素治疗有指导意义。