Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.     

外源基因表达造成的群体感应细菌生存压力的建模分析

外源基因的表达及其对细菌种群的影响对于群体感应系统和合成生物学产业的研究具有重要意义。然而,人们对于表达外源蛋白的细菌本身的行为模式仍然知之甚少。为了研究菌落生长和外源基因表达的过程究竟受到哪些因素的影响,文中测量了受Lux类受体调控的外源基因在N-酰基高丝氨酸内酯 (N-acyl homoserine lactone,N-AHL) 信号分子诱导下的表达,并模拟了其对细菌种群动态的影响。文中建立了一个假设性的数学模型,对信号分子诱导表达下细菌种群生长受影响的现象进行了分析。先前的研究通常将细菌种群生长受群体感应系统影响的现象归咎于合成群体感应信号分子的消耗与N-AHL信号分子的毒性,文中提供了对于这种生存压力的另一种可能的解释。     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.     

MALDI–MS Profiling to Address Honey Bee Health Status under Bacterial Challenge through Computational Modeling

Honey bees play a critical role in the maintenance of plant biodiversity and sustainability of food webs. In the past few decades, bees have been subjected to biotic and abiotic threats causing various colony disorders. Therefore, monitoring solutions to help beekeepers to improve bee health are necessary. Matrix‐assisted laser desorption ionization–mass spectrometry (MALDI–MS) profiling has emerged within this decade as a powerful tool to identify in routine micro‐organisms and is currently used in real‐time clinical diagnosis. MALDI BeeTyping is developed to monitor significant hemolymph molecular changes in honey bees upon infection with a series of entomopathogenic Gram‐positive and ‐negative bacteria. A Serratia marcescens strain isolated from one naturally infected honey bee collected from the field is also considered. A series of hemolymph molecular mass fingerprints is individually recorded and to the authors' knowledge, the first computational model harboring a predictive score of 97.92% and made of nine molecular signatures that discriminate and classify the honey bees’ systemic response to the bacteria is built. Hence, the model is challenged by classifying a training set of hemolymphs and an overall recognition of 91.93% is obtained. Through this work, a novel, time and cost saving high‐throughput strategy that addresses honey bee health on an individual scale is introduced.     

医院感染病原菌分布及药物敏感性分析

【摘 要】 目的 探讨医院感染病原菌的分布及其药物敏感性,以指导临床合理用药。方法 对金华市中心医院2009年至2011年临床分离的1 693株病原菌及药物敏感性进行回顾性分析。结果 医院感染病原菌主要是G-菌,占60.7%,G+菌占23.8%;感染部位以呼吸道、泌尿道为主,易感人群主要是血液系统疾病、肝病肝硬化患者,科室主要分布于重症监护病房、放疗科、血液科。G+菌对常用抗菌药耐药严重,但对利福平、呋喃妥因、万古霉素敏感率相对高;常见G-菌对氨基糖甙类、碳青酶烯类、β-内酰胺酶抑制剂敏感率较高。结论 医院感染监控应从感染科室、感染部位、易感人群等多方面进行,及时动态了解医院感染病原菌分布及其药物敏感性,利于指导合理用药。     

The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model

Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo.     

呼气末二氧化碳分压、C-反应蛋白/白蛋白比值及综合脱机指数对重型颅脑损伤机械通气患者撤机失败的预测价值

摘要 目的：探讨呼气末二氧化碳分压（PetCO₂）、C-反应蛋白/白蛋白比值（CRP/Alb）及综合脱机指数（IWI）对重型颅脑损伤机械通气患者撤机失败的预测价值。方法：选择2020年1月至2022年6月在安徽中医药大学附属六安医院进行机械通气的重型颅脑损伤患者96例作为研究对象,按照撤机结局分为撤机失败组（n=31）和撤机成功组（n=65）。比较两组撤机前PetCO₂、CRP/Alb、IWI及临床参数。应用多因素Logistic回归分析重型颅脑损伤机械通气患者撤机失败的危险因素。应用受试者工作特征（ROC）曲线评价PetCO₂、CRP/Alb、IWI对重型颅脑损伤机械通气患者撤机失败的预测价值。结果：撤机失败组PetCO₂、CRP/Alb、急性生理功能和慢性健康状况评分系统II（APACHE II）评分显著高于撤机成功组,机械通气时间长于撤机成功组,IWI、格拉斯哥昏迷评分（GCS）显著低于撤机成功组（P<0.05）。多因素Logistic回归分析显示,PetCO₂≥37.01 mmHg、CRP/Alb≥0.97、IWI≤78.23、GCS≤5.90分、APACHE II评分≥26.17分、机械通气时间≥4.49 d是重型颅脑损伤机械通气患者撤机失败的危险因素（P<0.05）。ROC曲线分析结果显示PetCO₂、CRP/Alb、IWI、GCS、APACHE II对重型颅脑损伤机械通气患者撤机失败均有较高的敏感度、特异度, PetCO₂、CRP/Alb、IWI三项联合检测对重型颅脑损伤机械通气患者撤机失败预测的曲线下面积（AUC）高于PetCO₂、CRP/Alb、IWI、GCS、APACHE II单独检测。结论：PetCO₂、CRP/Alb及IWI联合评估对重型颅脑损伤机械通气患者撤机失败具有较高的预测价值。     

高压氧联合高频rTMS对重型颅脑损伤后意识障碍患者促醒作用、神经电生理和脑损伤标志物的影响

摘要 目的：探讨高压氧联合高频重复经颅磁刺激（rTMS）对重型颅脑损伤后意识障碍（DOC）患者促醒作用、神经电生理和脑损伤标志物的影响。方法：选取2021年6月~2023年6月期间安徽中医药大学附属六安医院收治的的80例重型颅脑损伤后DOC患者。根据随机数字表法分为对照组（n=40,高压氧治疗）和观察组（n=40,高压氧联合高频rTMS治疗）。对比两组疗效、促醒作用、神经电生理和脑损伤标志物水平变化情况。结果：观察组的临床总有效率高于对照组（P<0.05）。两组治疗4周后修订的昏迷恢复量表（CRS-R）、格拉斯哥昏迷量表（GCS）评分升高,观察组高于对照组（P<0.05）。两组治疗4周后脑干听觉诱发电位（BAFP）、四肢体感诱发电位（SEP）、脑电图（EEG）分级有所改善,观察组改善效果优于对照组（P<0.05）。两组治疗4周后髓鞘碱蛋白（MBP）、神经元特异性烯醇化酶（NSE）、S100β蛋白下降,观察组低于对照组（P<0.05）。结论：高压氧联合高频rTMS治疗可有效促醒重型颅脑损伤后DOC患者,还可改善神经电生理活动,减轻脑损伤程度。     

小剂量糖皮质激素联合持续性血液净化治疗儿童严重脓毒症的效果及安全性研究

摘要 目的：分析小剂量糖皮质激素联合持续性血液净化治疗儿童严重脓毒症的效果及安全性。方法：选择自2021年1月至2023年1月接诊的102例儿童严重脓毒症患儿作为研究对象,随机分为对照组和观察组,各51例;对照组予以经典治疗方案,观察组在对照组的基础上,予以小剂量糖皮质激素联合持续性血液净化治疗;记录两组治疗后各项信息,比较两组治疗前后外周血乳酸、中心静脉血氧饱和度（ScvO₂）、血清炎症指标、PCIS评分、APACHE Ⅱ评分,观察主要并发症发生情况。结果：与对照组相比,观察组机械通气、低血压持续及入住ICU等时间较短,7 d内停升压药率较高（P＜0.05）;两组28 d病死率比较无差异（P＞0.05）;观察组治疗后乳酸、降钙素原（PCT）、肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）水平均较对照组低,ScvO₂水平较对照组高（P＜0.05）;观察组治疗后PCIS评分较对照组高,APACHE Ⅱ评分较对照组低（P＜0.05）;观察组主要并发症发生率低于对照组（P＜0.05）。结论：小剂量糖皮质激素联合持续性血液净化治疗有利于儿童严重脓毒症患儿病情转归,减少主要并发症发生,可能与阻断炎症反应有关,值得进一步研究应用。     

妊娠合并乙肝病毒感染患者血清DNMT1、TIM-3与HBV-DNA病毒载量和妊娠结局的关系分析

摘要 目的：探讨妊娠合并乙肝病毒（HBV）感染患者血清脱氧核糖核酸甲基转移酶1（DNMT1）、T细胞免疫球蛋白粘蛋白-3（TIM-3）与乙型肝炎病毒-脱氧核糖核酸（HBV-DNA）病毒载量和妊娠结局的关系。方法：选取2021年1月～2022年1月南京医科大学第一附属医院收治的186例妊娠合并HBV感染患者为HBV感染组,根据HBV-DNA病毒载量分为阳性组56例和阴性组130例,根据妊娠结局分为结局不良组和结局良好组,另选取同期于南京医科大学第一附属医院进行孕检的150名健康孕妇为对照组。采用酶联免疫吸附法检测血清DNMT1、TIM-3水平。比较HBV感染组与对照组、阳性组与阴性组血清DNMT1、TIM-3水平。采用单因素及多因素Logistic回归分析妊娠合并HBV感染患者妊娠结局不良的影响因素,受试者工作特征（ROC）曲线分析血清DNMT1、TIM-3水平对妊娠合并HBV感染患者妊娠结局不良的预测价值。结果：与对照组比较,HBV感染组血清DNMT1、TIM-3水平升高（P＜0.05）。与阴性组比较,阳性组血清DNMT1、TIM-3水平升高（P＜0.05）。186例妊娠合并HBV感染患者妊娠结局不良发生率为55.38%（103/186）。单因素分析显示,妊娠结局不良与HBV感染孕周、HBV-DNA病毒载量、谷草转氨酶（AST）、谷丙转氨酶（ALT）、DNMT1、TIM-3有关（P＜0.05）。多因素Logistic回归分析显示,HBV-DNA病毒载量阳性和DNMT1＞34.94 ng/mL、TIM-3＞18.96 pg/mL为妊娠合并HBV感染患者妊娠结局不良的独立危险因素（P＜0.05）。ROC曲线分析显示,血清DNMT1、TIM-3水平单独和联合检测预测妊娠合并HBV感染患者妊娠结局不良的曲线下面积分别为0.798、0.791、0.870。结论：妊娠合并HBV感染患者血清DNMT1、TIM-3水平升高与HBV-DNA病毒载量阳性和妊娠结局不良密切相关,血清DNMT1、TIM-3水平联合对妊娠合并HBV感染患者妊娠结局预测价值良好。