对喹诺酮类药物耐药的志贺菌gyrA基因突变的研究

目的研究福氏志贺菌对喹诺酮类药物的耐药机制。方法对38株耐药的福氏志贺菌gyrA基因的N末端编码区进行PCR扩增后,用SSCP方法检测突变,然后测序。结果福氏志贺菌gyrA基因PCR-SSCP显示有8株突变,测序结果揭示存在2个导致氨基酸改变的基因点突变:83位TCG(Ser)→TTG(Leu)突变和87位GAC(Asp)→GGC(Gly)突变。结论gyrA基因点突变与福氏志贺菌对喹诺酮类药物的高水平耐药有密切关系。     

A novel SRD5A2 mutation in an Iranian family with sex development disorder

Disorders of sex development (DSD) are different types of conditions that their accurate diagnosis by using conventional phenotypic and biochemical approaches is a challenging issue. Precise determination of DSD is critical due to the detection of possible life-threatening associated disorders. It may also assist parents in choosing the most suitable management for their affected child. In this study, two affected kids born from consanguineous families who were clinically diagnosed for sex development disorder were investigated for the main cause of the disease. Biochemical analysis failed to make an accurate diagnosis. Karyotype analysis showed an abnormal sex chromosome pattern. Whole exome sequencing was sequentially applied to precisely ascertain the genetic cause of the disease. A novel deletion, g.40936_53878del12943insTG (NG_008365.1), and one known mutation, c.586G>A (p.Gly196Ser), were detected in SRD5A2 gene in case I and case II respectively. Further analysis was performed using polymerase chain reaction, primer walking and Sanger sequencing to detect the nucleotides changes accurately. Segregation analysis in the families confirmed 13kb novel homozygous deletion of SRD5A2 in case I and c.586G>A in case II. The present study confirms the diagnostic value of whole exome sequencing in the detection of DSD aetiology, especially when several differential diagnoses are possible.     

Analysis of pharmacological mechanisms of Yinyanghuo as treatment of erectile dysfunction with network pharmacology-based strategy

Erectile dysfunction is considered an important health problem that impacts the quality of life of men. Yinyanghuo, also called Epimedium or Horny Goat Weed, is a frequently used Chinese traditional herbal medicine, commonly used in treating erectile dysfunction in China. A network pharmacology method was performed systematically, at a molecular level, to analyse the pharmacological mechanism of Yinyanghuo as erectile dysfunction therapy. The network pharmacology method used in this study primarily includes prescreening of the active compounds, prediction of targets, network analysis and gene enrichment analysis. This network analysis proved that 4 targets (AR, NR3C2, PDE5A and BMP2) could be the targets of Yinyanghuo therapy on erectile dysfunction. Besides, gene enrichment analysis predicted that Yinyanghuo might have a role in erectile dysfunction by regulating 10 molecular functions, 8 cellular components, 10 biological processes and 36 possible targets related to 10 signalling pathways. Our study demonstrated the molecular and pharmacological mechanisms of Yinyanghuo against erectile dysfunction with a holistic approach and demonstrated a powerful method for analysing pharmacological mechanisms and rational utilisation of Traditional Chinese Medicine clinically.     

Malignant hyperthermia 2020

Malignant hyperthermia is defined in the International Classification of Diseases as a progressive life-threatening hyperthermic reaction occurring during general anaesthesia. Malignant hyperthermia has an underlying genetic basis, and genetically susceptible individuals are at risk of developing malignant hyperthermia if they are exposed to any of the potent inhalational anaesthetics or suxamethonium. It can also be described as a malignant hypermetabolic syndrome. There are no specific clinical features of malignant hyperthermia and the condition may prove fatal unless it is recognised in its early stages and treatment is promptly and aggressively implemented. The Association of Anaesthetists has previously produced crisis management guidelines intended to be displayed in all anaesthetic rooms as an aide memoire should a malignant hyperthermia reaction occur. The last iteration was produced in 2011 and since then there have been some developments requiring an update. In these guidelines we will provide background information that has been used in updating the crisis management recommendations but will also provide more detailed guidance on the clinical diagnosis of malignant hyperthermia. The scope of these guidelines is extended to include practical guidance for anaesthetists dealing with a case of suspected malignant hyperthermia once the acute reaction has been reversed. This includes information on care and monitoring during and after the event; appropriate equipment and resuscitative measures within the operating theatre and ICU; the importance of communication and teamwork; guidance on counselling of the patient and their family; and how to make a referral of the patient for confirmation of the diagnosis. We also review which patients presenting for surgery may be at increased risk of developing malignant hyperthermia under anaesthesia and what precautions should be taken during the peri-operative management of the patients.     

丹参SmRopGEF1基因的克隆、亚细胞定位与表达分析

目的 克隆丹参SmRopGEF1基因,研究其编码蛋白的亚细胞定位及其对逆境胁迫的应激反应和激素诱导下的表达水平。方法 根据前期丹参转录组测序结果设计SmRopGEF1基因特异引物,利用聚合酶链式反应（PCR）扩增SmRopGEF1基因序列;使用生物信息学方法分析SmRopGEF1蛋白序列特征及进化关系;构建pET28a-SmRopGEF1原核表达载体并转化至大肠埃希氏菌中诱导表达,构建35S启动的SmRopGEF1的绿色荧光蛋白原生质体瞬时表达载体,利用激光共聚焦显微镜观察SmRopGEF1的亚细胞定位;实时荧光定量PCR（qRT-PCR）检测逆境胁迫（低温、盐及干旱）和激素诱导（生长素、脱落酸、茉莉酸甲酯）下的基因表达量。结果 丹参SmRopGEF1基因开放阅读框（ORF）全长为1701 bp,编码566个氨基酸,蛋白相对分子质量是62 362.99。生物信息学分析显示SmRopGEF1蛋白含有1个PRONE保守结构域,系统进化树分析表明SmRopGEF1蛋白与芡欧鼠尾草和一串红RopGEF1蛋白为高同源蛋白。成功构建pET28a-SmRopGEF1原核表达载体并在大肠埃希氏菌Rosetta（DE3）菌株中表达、纯化出SmRopGEF1重组蛋白。亚细胞定位的结果显示SmRopGEF1存在于植物细胞的细胞核中。qRT-PCR实验结果表明盐胁迫和冷胁迫能够显著提高SmRopGEF1的表达量;干旱胁迫后丹参愈伤中SmRopGEF1表达量下降。茉莉酸甲酯诱导后SmRopGEF1表达量升高。结论 克隆了丹参SmRopGEF1基因,其在抗逆胁迫中起到重要作用并且可能参与茉莉酸甲酯信号转导。研究结果丰富了丹参防御反应机制的研究,为选育丹参抗逆优良品种、提高丹参的产量与品质提供了参考。     

当归抽薹开花过程中赤霉素代谢水平及其关键酶基因克隆与表达分析

目的 对当归Angelica sinensis 16个赤霉素（gibberellins,GAs）进行含量测定、GAs代谢酶基因筛选并进行生物信息学分析、关键基因克隆及表达验证,以期为调控当归抽薹开花提供理论依据。方法 利用HPLC-MS/MS对不同材料中GAs含量进行测定与分析,基于当归全长转录组筛选直接参与GAs代谢酶基因,利用在线工具进行生物信息学分析,并对关键酶基因GA2ox6和GA20ox1表达水平进行q RT-PCR检测。结果 在所测定的16个GAs中,8个GAs含量在早薹植株中高于非早薹植株,13个GAs含量随植株发育时期延长逐渐增加。当归全长转录组中含有9个直接参与GAs代谢的酶基因,可分为6个亚家族,共含有6个保守基序,亚细胞定位于细胞质。GA2ox6和GA20ox1基因克隆片段由于插入序列与全长转录组测序片段存在长度差异。GA2ox6基因在抽薹植株、随种苗春化作用和植株发育时期延长呈现低表达,而在冷冻规避春化作用种苗中呈现高表达,GA20ox1基因则相反;GA2ox6和GA20ox1基因在叶和茎中相对于根均呈现高表达。结论 当归含有9个直接参与GAs代谢的酶基因,各成...     

竹节参鲨烯环氧酶基因的克隆及生物信息学分析

目的 克隆竹节参Panax japonicus鲨烯环氧酶基因（squalene epoxidase,PjSE）的全长cDNA序列,进行生物信息学分析和原核表达。方法 设计特异性引物,从竹节参中克隆得到PjSE序列;以PjSE基因序列作为输入数据,利用多序列同源比对等生物信息学分析工具进行序列分析;构建重组原核表达载体pCold-PjSE,转化至大肠杆菌BL21(DE3)中进行蛋白诱导表达;采用实时荧光定量PCR技术分析该基因在竹节参不同组织中的相对表达量。结果 PjSE基因开放阅读框长度为1884 bp,编码627个氨基酸,PjSE蛋白相对分子质量为68 639.49,初步预测其具有跨膜结构域,可能定位在叶绿体上;聚丙烯凝胶电泳结果显示诱导表达蛋白的相对分子质量大小与预期结果一致;该基因在竹节参叶中表达量最高,须根次之,根中表达量最低。结论 PjSE基因的克隆、生信分析及原核表达研究,不仅有助于丰富竹节参中该类功能蛋白的种类和数量,完善竹节参皂苷类成分的生物合成途径,也为后期开展竹节参皂苷的异源合成提供了可供选择的关键基因元件。