Antimycobacterial activity of Pichia pastoris-derived mature bovine neutrophil β-defensins 5

Tuberculosis (TB) is an ongoing threat to global health, and the lack of effective therapies for treating it is also a global problem. Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. To our knowledge, there are no reports on the antimycobacterial effects of bovine neutrophil β-defensins so far. Here, we identified the antimicrobial effect of mature bovine neutrophil β-defensins (mBNBD) 5 against Mycobacterium infection both in vitro and in vivo. The mBNBD5 protein was expressed in Pichia pastoris. To increase the yield of β-defensins, a purification method was employed by adding a 6-His·tag to the C-terminus of the mBNBD5 gene. Our results indicated that recombinant mBNBD5 protein was successfully expressed and purified from Pichia pastoris with intact antimicrobial activity. The recombinant protein exhibited potent bactericidal activity in vitro against M. smegmatis and M. bovis, with a dose-dependent manner and a time-dependent manner. The electron microscope results showed that the bacterial cell wall of M. bovis was disrupted when incubated with mBNBD5 for 72 h. Our data also indicated that the exogenous addition of mBNBD5 could reduce the survival of Mycobacterium spp., especially M. tuberculosis and M. bovis in RAW 264.7 macrophages. These results provide foundations for the development of mBNBD5 as a potential new therapeutic agent for TB treatment.     

胰腺分裂内镜治疗价值

目的探讨内镜下治疗胰腺分裂的疗效和安全性。方法收集2006年6月至2013年6月在南京大学医学院附属鼓楼医院消化科就诊的8例胰腺分裂患者的临床资料,对术中及术后情况进行回顾性分析。结果8例胰腺分裂患者共行经内镜逆行胰胆管造影术28例次,均置入胰管支架,其中7例同时行副乳头括约肌切开术,6例行内镜下扩张术,1例行胰管取石术。术后出现1例次轻度胰腺炎。随访2～47个月,1例死于胰腺癌,3例已拔出支架,3例定期复查,1例失访。患者术后慢性胰腺炎急性发作频率及腹痛程度均较术前明显降低。结论内镜下治疗胰腺分裂安全有效,可减少胰腺炎发作频率,减轻术后腹痛程度,术后并发症发生率低。     

急性心肌梗死患者心室膨胀瘤的影响因素及其对患者预后的预测价值研究

背景心室膨胀瘤是急性心肌梗死(AMI)患者常见且危险的并发症,可增加主要不良心血管事件(MACE)发生风险,影响患者预后,但目前国内外鲜有关于AMI患者心室膨胀瘤影响因素的研究报道。目的探讨AMI患者心室膨胀瘤的影响因素及其对患者预后的影响。方法选取2017年7月—2018年7月空军军医大学第一附属医院收治的AMI患者104例,根据心室膨胀瘤发生情况分为发生组(n=30)和未发生组(n=74)。比较两组患者一般资料〔包括年龄,性别,体质指数(BMI),发病至治疗时间,入院时心率(HR),高血压、高脂血症、糖尿病发生情况,纽约心脏病协会(NYHA)分级,前壁梗死情况,病变血管支数,贫血发生情况,冠状动脉溶栓或介入治疗情况,药物使用史〕、白细胞计数(WBC)及心功能指标〔包括左心室射血分数(LVEF)、左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)〕、住院期间及出院后1年预后;AMI患者心室膨胀瘤的影响因素分析采用多因素Logistic回归分析;绘制受试者工作特征曲线(ROC曲线)以评价AMI患者心室膨胀瘤的影响因素对AMI患者预后的预测价值。结果(1)两组患者男性比例、BMI、入院时HR、高血压发生率、高脂血症发生率、糖尿病发生率、NYHA分级>Ⅱ级及病变血管支数≥3支者所占比例、贫血发生率、行冠状动脉溶栓或介入治疗及有药物使用史者所占比例、LVEDD、LVESD比较,差异无统计学意义(P>0.05);发生组患者年龄大于未发生组,发病至治疗时间长于未发生组,前壁梗死发生率、WBC高于未发生组,LVEF低于未发生组(P<0.05)。(2)多因素Logistic回归分析结果显示,年龄〔OR=2.849,95%CI(2.157,3.365)〕、发病至治疗时间〔OR=3.019,95%CI(2.053,4.162)〕、前壁梗死〔OR=2.408,95%CI(2.214,2.685)〕、WBC〔OR=2.680,95%CI(1.985,2.931)〕、LVEF〔OR=3.047,95%CI(2.013,4.121)〕是AMI患者心室膨胀瘤的影响因素(P<0.05)。(3)发生组患者住院期间预后不良者所占比例高于未发生组(P<0.05)。绘制ROC曲线发现,年龄、发病至治疗时间、前壁梗死、WBC、LVEF预测AMI患者住院期间预后不良的曲线下面积(AUC)分别为0.798、0.745、0.712、0.823、0.861。(4)发生组患者出院后1年预后不良者所占比例高于未发生组(P<0.05)。绘制ROC曲线发现,年龄、发病至治疗时间、前壁梗死、WBC、LVEF预测AMI患者出院后1年预后不良的AUC分别为0.701、0.734、0.796、0.772、0.812。结论高龄、发病至治疗时间延长、前壁梗死、高WBC、低LVEF是AMI患者心室膨胀瘤的危险因素,且上述因素对AMI患者短期预后具有一定预测价值。     

围妊娠期小鼠胰岛细胞增殖及Survivin表达变化的研究

目的 观察围妊娠期小鼠胰岛β/α细胞量、细胞增殖、Survivin表达变化以及大鼠胰岛细胞瘤细胞株INS-1干扰Survivin表达后催乳素刺激细胞增殖的变化,探讨Survivin与妊娠期胰岛细胞扩增的关系.方法 采用免疫组化染色法检测C57 BL/6雌性小鼠非妊娠对照组（NP）和妊娠10.5、14.5、18.5 d（P10.5、P14.5、P18.5）、产后4、8 d（AP4、AP8）（每组各25只）胰岛β/α细胞量的变化.采用胶原酶消化法分离纯化小鼠胰岛,以流式细胞术检测细胞周期,实时定量聚合酶链反应及Western blotting法检测Survivin mRNA和蛋白的表达.采用脂质体转染siRNA方法干扰INS-1细胞Survivin的表达,催乳素刺激后检测细胞周期.多组数据组间比较采用单因素方差分析,两两比较采用最小差异显著性分析.结果 妊娠期胰岛β/α细胞量均显著增加,高峰在P18.5,与NP组相比,β细胞量增长2.1倍（3.84±0.38比1.81 ±0.09,t =4.75,P＜0.05）,α细胞量增长1.6倍（0.92±0.13比0.56 ±0.08,t =2.69,P＜0.05）,至AP8恢复至NP水平.妊娠期小鼠胰岛细胞增殖显著增加,高峰在P14.5,与NP组相比,增殖指数增长2.7倍（23.07±1.88比7.72±1.12,t=10.89,P＜0.05）,产后恢复正常.Survivin mRNA表达水平在妊娠期明显增高,高峰在P14.5,与NP组相比,增长6.7倍（6.69±1.61比1.00 ±0.00,t =4.57,P＜0.05）,产后恢复正常.P10.5时Survivin蛋白开始表达,高峰在P14.5,与P10.5相比增长3.6倍（3.63±0.26比1.00±0.00,t=9.98,P＜0.05）,P18.5时表达逐渐下降,P20.5时表达消失.催乳素可显著刺激INS-1细胞增殖,而siRNA干扰INS-1细胞Survivin表达后,细胞增殖受阻.结论 妊娠期小鼠胰岛细胞出现明显扩增,并伴有Survivin的动态表达.Survivin介导催乳素刺激的胰岛β细胞扩增作用.     

心肌梗死二级预防生活方式和膳食指南/共识的质量评价与内容分析

背景　健康的生活方式和膳食作为心肌梗死二级预防的重要组成部分,可有效降低再发心肌梗死的危险,改善心肌梗死患者预后。有必要检索相关高质量证据进行总结,以促进心肌梗死二级预防的临床实践。目的　系统检索并筛选当前国内外心肌梗死二级预防生活方式和膳食指南/共识,对纳入指南/共识的质量进行严格评价,提取、分析并综合相关指南推荐意见,为心肌梗死二级预防的生活方式和膳食指导提供最佳干预方案,为促进证据的临床转化提供理论依据。方法　计算机系统检索各专业指南网站和中英文数据库,包括国际指南协作网（GIN）、英国国家卫生与临床优化研究所（NICE）、苏格兰校际指南协作网（SIGN）、美国国立指南库（NGC）、澳大利亚临床实践指南数据库（ACPG）、新西兰指南工作组（NZGG）、世界卫生组织（WHO）、PubMed、中国知网（CNKI）、万方数据知识服务平台（WanFang Data）、维普网（VIP）,有关心肌梗死的指南、规范及专家共识,或内容涉及心肌梗死预防生活方式或膳食指导的相关指南/共识,并请心血管方面专家进行指南推荐及追溯参考文献补充检索相关指南,检索时间为2013-01-01至2018-12-31,并于2019年6月完成检索。采用临床指南研究与评估系统Ⅱ（AGREEⅡ）对指南/共识进行质量评价,归纳总结指南/共识中生活方式和膳食指导的推荐意见。结果　共纳入来自美国、英国、澳大利亚、新西兰、中国的10篇指南/共识,其中A级1篇,B级7篇,C级2篇。汇总49条推荐意见,其中生活方式涵盖戒烟、控制饮酒、体质量管理、睡眠管理、情绪管理、体育锻炼6个方面;膳食推荐涵盖饮食习惯、营养结构、膳食模式及膳食目标等相关内容。结论　纳入指南/共识的总体质量较高,但还需进一步规范指南/共识的制定和报告流程;心肌梗死指南/共识中有关生活方式和膳食推荐的内容涉及范围较广,为临床实践提供了证据支持。临床医护人员在进行证据转化时应基于最佳证据,将研究证据充分整合到卫生保健提供中,在综合考虑证据应用的促进及阻碍因素、患者的价值观和偏好等后,选择最佳证据进行临床实践;患者指南的制定者应进一步考虑患者等利益相关者的意见,并更具针对性地细化推荐意见,以最终改善患者结局。     

Assessing the practice of data quality evaluation in a national clinical data research network through a systematic scoping review in the era of real-world data

ObjectiveTo synthesize data quality (DQ) dimensions and assessment methods of real-world data, especially electronic health records, through a systematic scoping review and to assess the practice of DQ assessment in the national Patient-centered Clinical Research Network (PCORnet).Materials and MethodsWe started with 3 widely cited DQ literature—2 reviews from Chan et al (2010) and Weiskopf et al (2013a) and 1 DQ framework from Kahn et al (2016)—and expanded our review systematically to cover relevant articles published up to February 2020. We extracted DQ dimensions and assessment methods from these studies, mapped their relationships, and organized a synthesized summarization of existing DQ dimensions and assessment methods. We reviewed the data checks employed by the PCORnet and mapped them to the synthesized DQ dimensions and methods.ResultsWe analyzed a total of 3 reviews, 20 DQ frameworks, and 226 DQ studies and extracted 14 DQ dimensions and 10 assessment methods. We found that completeness, concordance, and correctness/accuracy were commonly assessed. Element presence, validity check, and conformance were commonly used DQ assessment methods and were the main focuses of the PCORnet data checks.DiscussionDefinitions of DQ dimensions and methods were not consistent in the literature, and the DQ assessment practice was not evenly distributed (eg, usability and ease-of-use were rarely discussed). Challenges in DQ assessments, given the complex and heterogeneous nature of real-world data, exist.ConclusionThe practice of DQ assessment is still limited in scope. Future work is warranted to generate understandable, executable, and reusable DQ measures.     

维持性血液透析患者透析初始血尿酸水平与预后的相关性

目的 探讨维持性血液透析患者透析初始血尿酸水平与预后的相关性。方法 纳入2013年7月～2018年7月于我院透析的309例患者为研究对象,按照尿酸是否升高分为尿酸升高组和尿酸正常组。收集患者临床资料,用二元logistic回归法分析尿酸升高的独立危险因素;Kaplan-Meier生存曲线法分析患者生存预后的影响因素;Log-rank法进行显著性检验。结果 ①共309例患者纳入本研究,男性178例（57.6%）,女性131例（42.4%）。其中,158例（51.1%）患者尿酸升高,151例（48.9%）患者尿酸正常。与尿酸正常组比较,尿酸升高组透析龄更长,但年龄、二氧化碳结合力较低,尿素、前白蛋白较高（均P＜0.05）。②相关性分析结果提示,尿酸与透析龄、尿素、肌酐、甘油三酯、血磷和前白蛋白呈正相关,与年龄、直接胆红素、eGFR、高密度脂蛋白、二氧化碳结合力呈负相关均（均P＜0.05）。③二元logistic逐步回归分析结果显示,男性较女性患者尿酸风险降低（OR=0.086）。随着年龄、二氧化碳结合力和高密度脂蛋白的增加,尿酸的风险减少,但随着前白蛋白和尿素的升高,尿酸风险增加（均P＜0.05）。④全因死亡患者的生存分析结果提示,尿酸升高组患者生存时间高于尿酸正常组（P＜0.05）。结论 前白蛋白和尿素是尿酸升高的危险因素,高尿酸可能是维持性血液透析患者死亡的保护因素。     

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).