中华绒螯蟹蜕壳生长及其与相关基因表达的关联分析

蜕壳是甲壳动物常见的生长发育现象,但对调控蜕壳与生长的内在机制尚缺乏足够了解。本研究在室内条件下,对中华绒螯蟹(Eriocheir sinensis)一个蜕壳周期内的个体蜕壳与生长现象进行了连续观察,分析了2个蜕壳相关基因,即蜕皮激素受体基因(Ec R)和维甲类X受体基因(RXR),以及1个生长相关基因肌肉生长抑制素基因(MSTN)的表达及其与生长性状的相关性。结果发现,中华绒螯蟹在蜕壳后会出现一个跳跃式生长期,之后进入了一个缓慢持续上升过程,当营养物质积累到一定程度(肥满度达60%左右时)时启动下一次蜕壳;MSTN基因的表达与壳长(r=﹣0.450,P0.05)、壳宽(r=﹣0.410,P0.05)增长率呈显著负相关,而与肥满度呈显著正相关(r=0.450,P0.05),Ec R和RXR基因的表达与体重、壳长和壳宽的增长率均没有显著相关性;相对来说,MSTN在蜕壳后的表达量越高,则增重率越小;而Ec R和RXR在蜕壳后表达量越高,其增重率越大。本研究结果表明,中华绒螯蟹在蜕壳后其生长具有一定的规律性,肥满度可以作为衡量中华绒螯蟹体内营养积累启动蜕壳的指标,Ec R、RXR及MSTN基因表达与生长表型具有一定的相关性。     

AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD+ elevation

AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress‐induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide‐induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP‐RFP‐LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD⁺ levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD⁺ synthesis. In addition, the mechanistic relationship of autophagic flux and NAD⁺ synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress‐induced senescence by improving autophagic flux and NAD⁺ homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD⁺ homeostasis, and it is also valuable in the development of innovative strategies to combat aging.     

Green synthesis of fluorescence carbon nanoparticles from yum and application in sensitive and selective detection of ATP

Fluorescent carbon nanoparticles (CPs), a fascinating class of recently discovered nanocarbons, have been widely known as some of the most promising sensing probes in biological or chemical analysis. In this study, we demonstrate a green synthetic methodology for generating water‐soluble CPs with a quantum yield of approximately 24% via a simple heating process using yum mucilage as a carbon source. The prepared carbon nanoparticles with an ~10 nm size possessed excellent fluorescence properties, and the fluorescence of the CPs was strongly quenched by Fe³⁺, and recovered by adenosine triphosphate (ATP), thus, an ‘off’ and ‘on’ system can be easily established. This ‘CPs‐Fe³⁺‐ATP’ strategy was sensitive and selective at detecting ATP with the linear range of 0.5 µmol L^?1 to 50 µmol L^?1 and with a detection limit of 0.48 µmol L^?1. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

无创正压通气联合纤维支气管镜肺泡灌洗对老年AECOPD合并Ⅱ型呼吸衰竭患者肺功能及血气指标的影响

目的:观察无创正压通气(NIPPV)联合纤维支气管镜(FB)肺泡灌洗对老年急性加重期慢性阻塞性肺疾病(AECOPD)合并Ⅱ型呼吸衰竭患者肺功能及血气指标的影响,为临床治疗方案的选择提供依据。方法:选取82例于2017年1月～2019年1月间在我院住院治疗的老年AECOPD合并II型呼吸衰竭患者。根据治疗方法将患者分为观察组(NIPPV联合FB肺泡灌洗治疗,n=42)与对照组(单独NIPPV治疗,n=40)。观察两组患者的住院时间及抗菌药静脉滴注时间,并比较治疗前及治疗后两组患者的血气指标[pH值(pH)、氧分压(PaO_2)、二氧化碳分压(PaCO_2)、血氧饱和度(SaO_2)]、肺功能指标[一秒钟用力呼气容积(FEV1)、肺活量(FVC)、呼气峰值流速(PEF)]的变化情况。记录两组患者治疗过程中的并发症发生情况。结果:观察组住院时间及抗菌药物静脉滴注时间均明显短于对照组(P0.05)。治疗后,两组pH、PaO_2、SaO_2明显上升,而PaCO_2明显下降(P0.05),且与对照组比较,观察组的pH、PaO_2、SaO_2明显较高,而PaCO_2明显较低(P0.05)。治疗后,对照组FEV1、FVC、PEF无明显变化(P0.05),观察组FEV1、FVC、PEF均明显升高且高于对照组(P0.05)。两组患者不良反应发生率比较差异无统计学意义(P0.05)。结论:NIPPV联合FB肺泡灌洗治疗对老年AECOPD合并II型呼吸衰竭患者血气指标及肺功能均有较好的改善效果,能明显缩短患者的住院时间及抗菌药静脉滴注时间,且安全性良好。     

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)。