利用短短芽孢杆菌进行酮还原酶CgKR2的高效表达与纯化

酮还原酶CgKR2能够一步还原前手性羰基化合物生成高附加值的手性醇,有望解决手性醇传统制备方法的步骤烦琐和高成本问题,具有很高的经济效益。研究表明,CgKR2催化底物2-氧代-4-苯基丁酸乙酯(OPBE)生成普利类降压药的重要中间体(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]具有良好效果。但CgKR2的生产成本高昂、过程烦琐。利用短短芽孢杆菌胞外分泌表达酮还原酶CgKR2,获得该酶的高效表达,并经简便的一步镍亲和层析纯化,即获得高纯度酮还原酶CgKR2,产率高达每升发酵7. 8mg纯酶。以酶标板法测定其比活力、温度稳定性以及动力学参数等基本酶学性质,结果显示,CgKR2的比活力为(78. 32±7. 62) U/mg、Km为(0. 2±0. 02) mmol/L、Vmax为(117. 64±3. 6)μmol/(min·mg)、Kcat为73s-1,与以往报道的数据一致,并且获得的CgKR2纯酶在30℃下孵育72h依然保持80%的活性,酶活的稳定性远好于以往的制备方法。开发出的一套简便高效的酮还原酶CgKR2表达纯化工艺,降低了生产成本、简化了生产工艺,可推进手性醇生物催化制备的普及,对其他生物催化工程酶的制备方法研究也有借鉴作用。     

分泌性中耳炎患者外周血Th1Th2细胞及T淋巴细胞亚群水平的表达及临床意义

目的:探讨分泌性中耳炎(SOM)患者外周血T辅助细胞1(Th1)、T辅助细胞2(Th2)细胞因子及T淋巴细胞亚群水平的表达及其临床意义。方法:选取我院于2015年1月至2018年1月期间收治的SOM患者135例记为SOM组,根据病程将患者分为急性组(病程14d,49例)、亚急性组(病程14-30d,53例)、慢性组(病程30d,33例)。另外选择同期于我院进行体检的100例健康者为对照组。分别对比SOM组和对照组受试者、不同病程SOM患者外周血Th1细胞因子[干扰素(INF-γ)、白细胞介素-2(IL-2)]、Th2细胞因子[白细胞介素-4(IL-4)、白细胞介素-10(IL-10)]以及T淋巴细胞亚群[CD3~+、CD4~+、CD8~+、CD4~+/CD8~+]水平。采用Pearson相关性分析INF-γ、IL-2、IL-4、IL-10与CD3~+、CD4~+、CD8~+、CD4~+/CD8~+的相关性。结果:SOM组患者外周血INF-γ、IL-2、IL-4、IL-10水平高于对照组(P0.05);急性组患者外周血INF-γ、IL-2、IL-4、IL-10水平低于亚急性组、慢性组,亚急性组患者外周血INF-γ、IL-2、IL-4、IL-10水平低于慢性组(P0.05)。SOM组患者外周血CD3~+、CD4~+、CD4~+/CD8~+水平低于对照组,CD8~+水平高于对照组(P0.05);急性组患者外周血CD3~+、CD4~+、CD4~+/CD8~+水平高于亚急性组、慢性组,CD8~+水平低于亚急性组、慢性组,亚急性组患者外周血CD3~+、CD4~+、CD4~+/CD8~+水平高于慢性组,CD8~+水平低于慢性组(P0.05)。Pearson相关性分析结果显示,SOM患者外周血INF-γ、IL-4与CD8~+呈正相关(P0.05),IL-4与CD3~+、CD4~+呈负相关(P0.05)。结论:SOM患者外周血Th1Th2细胞因子、T淋巴细胞亚群水平均表现异常,且其水平与疾病发生和发展存在一定联系,通过监测Th1Th2细胞因子、T淋巴细胞亚群有助于评估SOM患者病情。     

Natural cavity restoration as an alternative to nest box supplementation

Nest box supplementation is widely used to increase nest‐site availability for cavity nesting animals but the analysis of its effects on individuals breeding in natural cavities is often neglected. This study offers a novel restoration technique to revert abandonment of natural breeding sites by a secondary cavity avian bird, the European roller (Coracias garrulus), and other ecologically similar species. We found that, after a program of nest box supplementation with ensuing monitoring, rollers gradually abandon nesting in natural and seminatural cavities in favor of nest boxes because the latter are of higher quality. We examine whether reducing the entrance size of natural and seminatural cavities improves their suitability for rollers. A 6‐year program reduced the diameter of the entrance of sandstone cavities and cavities in bridges. This led to a high occupancy (59%) of manipulated nest‐sites. Manipulated sites were most frequently occupied by rollers and little owls (Athene noctua) (31 and 18% of sites, respectively). Manipulation did not affect clutch size or fledgling success. We suggest that nest‐site diversity and nesting in natural cavities should be preserved to reduce nest box dependence. Our study illustrates the value of nest boxes when used alongside restoration of natural breeding sites and provides insights for the management of natural cavities.     

The effector SIX8 is required for virulence of Fusarium oxysporum f.sp. cubense tropical race 4 to Cavendish banana

Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization. Fusarium oxysporum f. sp. cubense is the agent of wilt disease in banana plantlets and four races of the pathogen have been identified based on the cultivar specificity. A total of 9 SIX genes have been detected in the genome of Foc TR4 and 6 genes detected in Foc1. Among these SIX genes, SIX2 and SIX8 are only detected in Foc TR4, not identified in Foc1. Expression profiles analysis revealed that SIX genes of Foc TR4 are highly induced after inoculation to Cavendish banana plantlets. Virulence analysis of the SIX2 and SIX8 knock-out mutants showed that SIX8 is required for the virulence of Foc TR4 while SIX2 has no obvious functions. Over expression of SIX8-FLAG proteins in the SIX8 knock-out mutant partly restored the virulence. Western blot analysis suggested that SIX8 could be secreted into the extracellular space and a signal peptide resided the N-terminal polypeptide sequence. This study provides some clues for further research on mechanism of SIX8 in regulating virulence of Foc TR4.     

Mesocestoides corti (syn. vogae, cestoda): characterization of genes encoding cysteine-rich secreted proteins (CRISP)

With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.     

Taenia crassiceps organic acids detected in cysticerci

Taenia crassiceps cysticerci is used as an experimental model to cysticercosis studies; however there are subcutaneous cases of cysticercosis caused by these cysticerci. It remains unclear in the literature the energetic and fatty acid metabolism in cestodes. Its metabolic study may provide knowledge of pathways that may serve as potential anti-helminthic drugs sites of action. In this work we studied the citric acid cycle organic acids and the fatty acid oxidation in cysticerci removed from mice with 21 and 42 days of infection in two different evolutive stages: growing and final. The organic acids were extracted using perchloric acid and analyzed by HPLC methodology. We found significant statistically differences in oxalate, malate, lactate, and beta-hydroxybutirate concentrations between cysticerci. These results indicate the aerobic metabolism in vivo in spite of the low oxygen concentration of its habitat, and also indicate the presence of fatty acid oxidation as an alternative energetic source.     

骨折电流疗法的改良

目的:针对电流治疗骨折等疾病时存在的问题,提出相应的改进方法。方法:分析以往使用电流对骨折等疾病进行治疗时存在的电化学副作用、创口易于感染、重复施术不便等问题,提出相应的诸如使用溶液导电、隔离电解物、使电极与创口闭合生长等改进方法;并提出将电流治疗与药物治疗相结合的骨科疾病电流疗法改良方案。结果:将电流与药物相结合治疗骨折等疾病是很有可行性的治疗方法,本法不仅克服了已往疗法存在的一些问题,还为骨伤药物直接进入病患部位提供了有效的平台,使骨折、骨不连等疾病能够快速愈合。今后应加强研究骨折愈合各阶段所需的直接注射药物及其配比。完善电流—药物复合电极使用方法,以便与本法尽快进入临床实践。结论:将电流与药物相结合治疗骨折等疾病是很有可行性的治疗方法。     

Bats relocate maternity colony after the natural loss of roost trees

Understanding the ephemerality of trees used as roosts by wildlife, and the number of roost trees needed to sustain their populations, is important for forest management and wildlife conservation. Several studies indicate that roosts are limiting to bats, but few studies have monitored longevity of roost trees used by bats over several years. From 2004–2007 in Cypress Hills Interprovincial Park, Saskatchewan, Canada, several big brown bats (Eptesicus fuscus) from a maternity group roosted in cavities in trembling aspen (Populus tremuloides) trees approximately 7 km southeast away from their original known roosting area (RA1). Using a long-term data set of the roost trees used by bats in this area from 2000–2007, we evaluated whether the movement of bats to the new roosting area (RA4) corresponded with annual and cumulative losses of roost trees. We also determined whether longevity of the roosts from the time we discovered bats first using them differed between the 2 roosting areas based on Kaplan-Meier estimates. Bats began using RA4 in addition to RA1 in 2004, when the cumulative loss of roost trees in RA1 over 3 consecutive years reached 18%. Most bats exclusively roosted in RA4 in 2007, when the cumulative loss of roost trees over 6 consecutive years had reached 46% in RA1. Annual survival for roost trees, from when we first discovered bats using them, was generally lower in RA1 than in RA4. Our results suggest that the movement of bats to the new roosting area corresponded with high losses of roost trees in RA1. This provides additional evidence that to maintain high densities of suitable roost trees for bats in northern temperature forests over several decades, management plans need to recruit live and dead trees in multiple age classes and stages of decay that will be suitable for the formation of new cavities. © 2019 The Wildlife Society.