Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC₅₀ and MBRC₅₀) of dextranase were 2 U ml^?1 and 5 U ml^?1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.     

A 3D Homochiral MOF [Cd2(d‐cam)3]•2Hdma•4dma for HPLC Chromatographic Enantioseparation

Up to now, some chiral metal‐organic frameworks (MOFs) have been reported for enantioseparation in liquid chromatography. Here we report a homochiral MOF, [Cd2(d‐cam)3]·2Hdma·4dma, used as a new chiral stationary phase for high‐performance liquid chromatographic enantioseparation. Nine racemates of alcohol, naphthol, ketone, and base compounds were used as analytes for evaluating the separation properties of the chiral MOF packed column. Moreover, some effects such as mobile phase composition, column temperature, and analytes mass for separations on this chiral column also were investigated. The relative standard deviations for the resolution values of run‐to‐run and column‐to‐column were less than 2.1% and 3.2%, respectively. The experimental results indicate that the homochiral MOF offered good recognition ability, which promotes the application of chiral MOFs use as stationary phase for enantioseparation. Chirality 28:340–346, 2016. © 2016 Wiley Periodicals, Inc.     

Production of proinsulin in marker-free transgenic tobacco plants using <Emphasis Type="Italic">CRE</Emphasis>/loxP system

The demand for INSULIN is increasing rapidly along with the increased number of diabetic patients. Using the CRE/loxP system, we developed a selective marker-free system without crossing to produce PROINSULIN in transgenic plant. In frame of this approach, the induced promoter pRD29A was isolated from Arabidopsis. The CRE recombinase gene was placed under the control of pRD29A between two loxP recombination sites together with the selective NPTII gene. Furthermore, the binary vector with CRE recombinase and PROINSULIN was constructed and introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Gene excision was used to remove the sequence between the two loxP sites at the presence of 200 mM NaCl. PCR analysis showed that self-excision occurred in several T₀ transgenic plants. Transgenic plants without any marker gene successfully expressed PROINSULIN. This auto-excision strategy provides efficient means of removing the selectable marker gene from transgenic plants. It is an efficient method for producing bio-safe recombinant protein and other valuable substances in plants.     

高血压患者颈动脉粥样硬化与血脂、血压和血尿酸水平的相关性分析

目的:探讨高血压患者颈动脉粥样硬化(CAS)与血脂、血压以及血尿酸水平的相关性。方法:选择2017年2月至2018年8月在我院就诊的高血压患者117例作为研究组,另选择同期在我院进行体检的健康志愿者50例作为对照组。采用彩色多普勒超声诊断仪测定所有受试者的颈动脉内中膜厚度(IMT),并根据研究组患者的颈动脉IMT将其分为斑块组(IMT≥1.3 mm,33例)、IMT增厚组(1.0 mm≤IMT1.3 mm,49例)和IMT正常组(IMT1.0 mm,35例)。比较研究组与对照组受试者IMT,同时分别比较研究组与对照组受试者以及不同IMT高血压患者平均收缩压(SBP)、平均舒张压(DBP)、血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)以及尿酸水平,并采用Pearson相关性分析法分析高血压患者IMT与各指标的相关性。结果:与对照组比较,研究组IMT、SBP、DBP、TC、TG、LDL-C、血尿酸水平升高,HDL-C水平降低,差异均有统计学意义(P0.05)。斑块组患者SBP、DBP、TC、TG、LDL-C、血尿酸水平高于IMT增厚组和IMT正常组,HDL-C水平低于IMT增厚组和IMT正常组,差异均有统计学意义(P0.05);IMT增厚组患者SBP、DBP、TC、TG、LDL-C、血尿酸水平高于IMT正常组,HDL-C水平低于IMT正常组,差异均有统计学意义(P0.05)。Pearson相关性分析显示,高血压患者的IMT与SBP、DBP、TC、TG、LDL-C、血尿酸均呈正相关,与HDL-C呈负相关(P0.05)。结论:高血压患者IMT与血脂、血压和血尿酸水平均有明显相关性,血压、血脂、血尿酸参与了高血压患者CAS的发生与发展。     

马氏珠母贝(Pinctada fucata martensii)细胞粘附分子3(Pm-CAM3)基因的克隆与表达分析

细胞粘附分子3 (cell adhesion molecule 3, CAM3)是免疫球蛋白家族(immunoglobulin family, IgSF)的一员,在细胞黏连、外源病原体的识别等方面具有重要作用。本实验利用末端快速克隆(RACE)技术,克隆获得马氏珠母贝(Pinctada facata martensii)细胞粘附分子3的全长序列(Pm-CAM3),并使用荧光定量PCR(Real-time PCR, q RT-PCR)技术检测了Pm-CAM3在马氏珠母贝不同组织中的表达模式。结果显示,Pm-CAM3基因全长2 245 bp。其中5'UTR 335 bp,3'UTR 166 bp,开放阅读框(ORF)长度为1 744 bp,共编码581个氨基酸;预测其相对分子量为63.21 kD,等电点为5.07,脂溶指数(aliphatic index)为67.21;总平均亲水性(grand averageofhydropathy, GRAVY)为-0.549,属于亲水性蛋白;Pm-CAM3具有跨膜结构域,其胞外区域具有一个Ig SF家族典型的Ig结构域以及一个Ig-like结构域。多序列比对结果显示,Pm-CAM3在物种间的保守性较低,其中Pm-CAM3与太平洋牡蛎的CAM3 (Crassostrea gigas, Cg-CAM3)的氨基酸序列相似性最高,但仅为37%。qRT-PCR分析表明Pm-CAM3在马氏珠母贝的9个组织中均有表达,其中在鳃中的表达量最高(p<0.05)。     

马氏珠母贝(Pinctada fucata martensii)PmHEXL基因的分子特征与表达分析

几丁质作为有机框架主要成分参与贝壳的形成。β-N-乙酰-己糖胺酶(Beta-hexosaminidase/Beta-N-acetylhexosaminidase, HEX)属于糖苷水解酶家族20,在几丁质水解过程发挥重要作用。为了探究Pm HEXL在马氏珠母贝贝壳形成中的作用,本研究利用RACE技术克隆获得Pm HEXL基因c DNA全长序列并检测其在不同组织的表达模式。研究显示,Pm HEXL基因序列全长2 760 bp,其中5'UTR为167 bp,3'UTR为268 bp,开放阅读框(ORF)为2 325 bp,编码774个氨基酸;预测其相对分子量为89.59 kD,理论等电点为5.93;SMART软件分析PmHEXL蛋白质序列,发现它具有典型的GH20、GH20b结构域和CHB-HEX结构域;多序列比对结果显示Pm HEXL与其它物种的HEX具有较高的保守性;qPCR表达分析显示Pm HEXL在外套膜套膜区表达量最高,边缘区次之。综上所述,Pm HEXL可能参与马氏珠母贝贝壳的形成过程。     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.     

Chemical Composition of Bawei Longzuan Granule and Its Anti‐Arthritic Activity on Collagen‐Induced Arthritis in Rats by Inhibiting Inflammatory Responses

Bawei Longzuan granule (BLG) is a representative Zhuang medicine preparation. The present work aims to characterize the chemical constituents of BLG and evaluate its anti‐arthritic activity. The major chemical constituents of BLG were tentatively identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS), which revealed the presence of some alkaloids (e. g., magnoflorine, sinomenine and nitidine) and flavonoids (e. g., hesperidin, diosmin and sinensetin) that may be partly responsible for the anti‐arthritic effect of BLG. In addition, the collagen‐induced arthritis (CIA) model in rats was induced by intradermal injection of bovine collagen‐II in complete Freund's adjuvant at the base of tail. The CIA rats received oral administration of BLG (1.25, 2.5 and 5 g/kg) for 30 days. Then, various indicators were determined to evaluate its anti‐arthritic activity, including paw swelling, arthritic score, body weight, knee joint pathology, thymus index and spleen index. Additionally, the serum levels of tumor necrosis factor (TNF)‐α, interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐6, IL‐4 and IL‐10 were measured to determine the underlying mechanisms. The results showed that BLG efficiently ameliorated the severity of arthritis in CIA rats by decreasing paw swelling and arthritis score and improving the histological lesions of knee joint. Moreover, the serum levels of several pro‐inflammatory cytokines (i. e., IL‐1β, TNF‐α, IL‐6 and IFN‐γ) were downregulated, whereas two anti‐inflammatory factors (i. e., IL‐4 and IL‐10) were upregulated after BLG administration. These results indicated that BLG possessed promising therapeutic effect on collagen‐induced arthritis by inhibiting inflammatory responses. BLG can be used as a complementary or alternative traditional medicine to treat rheumatoid arthritis.     

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

GhL1L1 affects cell fate specification by regulating GhPIN1‐mediated auxin distribution

Auxin is as an efficient initiator and regulator of cell fate during somatic embryogenesis (SE), but the molecular mechanisms and regulating networks of this process are not well understood. In this report, we analysed SE process induced by Leafy cotyledon1‐like 1 (GhL1L1), a NF‐YB subfamily gene specifically expressed in embryonic tissues in cotton. We also identified the target gene of GhL1L1, and its role in auxin distribution and cell fate specification during embryonic development was analysed. Overexpression of GhL1L1 accelerated embryonic cell formation, associated with an increased concentration of IAA in embryogenic calluses (ECs) and in the shoot apical meristem, corresponding to altered expression of the auxin transport gene GhPIN1. By contrast, GhL1L1‐deficient explants showed retarded embryonic cell formation, and the concentration of IAA was decreased in GhL1L1‐deficient ECs. Disruption of auxin distribution accelerated the specification of embryonic cell fate together with regulation of GhPIN1. Furthermore, we showed that PHOSPHATASE 2AA2 (GhPP2AA2) was activated by GhL1L1 through targeting the G‐box of its promoter, hence regulating the activity of GhPIN1 protein. Our results indicate that GhL1L1 functions as a key regulator in auxin distribution to regulate cell fate specification in cotton and contribute to the understanding of the complex process of SE in plant species.