Chemical constituents from Amentotaxus yunnanensis and Torreyayunnanensis

In a chemical study of taxonomically related Taxaceae plants of Yunnan Province, China, seven compounds, including a new amentoflavone biflavonoid, 2,3-dihydro-7,7' '-dimethoxyamentoflavone (1), were isolated from Amentotaxus yunnanensis, and 12 isolates were obtained from Torreya yunnanensis. From the latter plant, a new abietane diterpene, torreyayunnin (7), is reported for the first time. The known isolates from A. yunnanensis have been identified as sequoiaflavone (3), sotetsuflavone (4), 7,7' '-dimethoxyamentoflavone (5), lutein, beta-sitosterol, and sequoyitol. Amentoflavone (2), sotetsuflavone (4), sciadopitysin (6), 12-hydroxydehydroabietinol, meridinol, balanophonin, (+)-pinoresinol monomethyl ether, (+)-pinoresinol monomethyl ether glucoside, erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-[2-formyl-(E)-vinyl]-2- methoxyphenoxy]propane-1,3-diol, threo-1-(4-hydroxy-3-methoxyphenyl)-2- [4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy] propane-1,3-diol, and (E)-2-butenedioic acid were identified as known isolates from T. yunnanensis. The presence of the amentoflavone biflavonoids (1, 3-5) in A. yunnanensis supports its placement in the Taxaceae. The occurrence of the biflavonoid sotetsuflavone (4) in both A. yunnanensis and T. yunnanensis suggests that these two genera are closely related. The identification and structural elucidation of these isolates were based on spectral data analysis including 1D and 2D NMR.     

人NGAL克隆表达及抗体的制备与鉴定

目的：高效表达人中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin,NGAL）重组蛋白并制备其多克隆及单克隆抗体。方法：人工合成经密码子优化的NGAL基因,构建原核表达重组质粒pW28-NGAL,IPTG诱导表达后分离纯化并分析在溶液中的聚集状态;获得的rhNGAL抗原免疫兔子制备多克隆抗体,免疫小鼠筛选高效价的特异性单克隆抗体并鉴定抗体亚型;Protein G亲合柱纯化,等温滴定量热法及非竞争ELISA法检测抗体的亲和力,SDS-PAGE分析纯度;最后运用Western blot、免疫荧光和免疫组化对抗体进行鉴定。结果：高效获得高纯度NGAL重组蛋白,主要以单体形式存在;成功制备兔多抗,同时筛选获得6株高效价单克隆抗体;其中,25号单抗为IgG1亚型,余者均属IgG2a亚型,且19、35号单抗的亲和常数分别为3.06×109、2.14×109。SDS-PAGE分析表明纯度均大于90%。进一步的鉴定结果表明制备的抗体皆具有良好免疫反应性及特异性,且单抗的特异性明显高于多抗。结论：本研究利用密码子优化技术高效表达制备了NGAL重组蛋白,获得了其高效价多克隆抗体和高特异性单克隆抗体,为NGAL的病理生理作用等功能研究提供了良好的实验材料,为NGAL临床免疫检测诊断试剂的国产化奠定了有力的基础。     

SOCS3结构和作用机制研究进展

SOCS3是酪氨酸蛋白激酶/信号传导子和转录激活子(JAK/STAT)途径的负反馈调节因子之一,由SOCS盒、SH2结构域和激酶抑制区三个部分组成.SOCS3参与了体内多种信号分子转导的调控.本文结合近年的研究成果对其结构和作用机制进行了综述,并对其中尚存在的问题进行展望.     

RNA干扰Axin2对大鼠骨髓基质干细胞的成骨分化表达的影响

目的 通过对大鼠Axin2基因的RNA干扰(RNAi),探讨Axin2对大鼠骨髓基质干细胞(BMSCs)向成骨细胞分化过程的影响.方法 取6周龄雌性SD大鼠双侧股、胫骨骨髓细胞,采用全骨髓培养法进行原代和传代培养.细胞传2代后实验组进行Axin2 mRNA干扰质粒转染,对照组进行阴性干扰质粒转染.转染48 h后换用成骨诱导分化培养基.诱导分化28 d后用 von Kossa方法进行细胞外基质矿化染色.采用适时定量聚合酶链反应(RQ-PCR)技术检测诱导分化后第7、14天β-catenin、骨钙素(OCN)、frizzled-2、Lef-1、Wnt5a mRNA的表达水平.结果 对照组大鼠BMSCs经体外诱导后分化为具备矿化细胞外基质能力的成熟成骨细胞,而实验组BMSCs成骨分化不全;RQ-PCR检测显示,诱导分化后第7天实验组β-catenin、frizzled-2 mRNA表达水平显著低于对照组,而OCN、Lef-1、Wnt5a mRNA表达水平显著高于对照组;诱导分化后第14天实验组frizzled-2、Lef-1和Wnt5a mRNA表达水平均显著高于对照组(P<0.05).结论 对大鼠BMSCs Axin2进行RNAi后,BMSCs向成骨细胞分化晚期细胞外基质矿化能力减弱.     

大鼠肺和脑组织中一氧化氮合成酶mRNA表达的研究

以Ｎｏｒｔｈｅｒｎ印迹杂交法观察了３种一氧化氮合成酶（ＮＯＳ）即脑ＮＯＳ（ｂＮＯＳ）、内皮ＮＯＳ（ｅＮＯＳ）和巨噬细胞ＮＯＳ（ｍａｃＮＯＳ）在大鼠肺和脑组织中ｍＲＮＡ的表达情况。结果显示：肺组织存有ｅＮＯＳ和ｍａｃＮＯＳｍＲＮＡ表达，其ｍＲＮＡ杂交带分子大小分别为４．８ｋｂ和４．５ｋｂ；脑组织只有ｂＮＯＳｍＲＮＡ表达，其杂交带分子大小为１０．５ｋｂ。提示３种ＮＯＳ基因结构各异，且其在大鼠肺和脑组织中的分布和表达亦不相同。     

水质BOD5的快速测定方法

采用便携式无汞压差BOD分析仪表测定了水样的BOD5,并与HJ 505-2009进行了比较.实验结果表明:快速无汞压差BOD分析仪表法具有操作简单、效率高、结果准确可靠、能耗低、少污染的特点,在应急检测、大批量样品检测时可得到广泛应用.     

一次性胃管包的研制与临床应用

目的:探讨一次性胃管包用于胃肠破裂患者治疗中的效果.方法:将60例胃肠破裂患/者随机分为观察组与对照组各30例,观察组采用自制一次性胃管包,对照组采用传统的置胃管前准备方法,观察对比两种方法的效果.结果:两组置胃管前准备时间、患者信任度、患者紧张情况、急救时间以及急救成功率的比较均有极显著性差异(P＜0.01);两组患者满意度比较有显著性差异(P＜0.05).结论:使用一次性胃管包为患者置胃管时方便、快捷、安全,可消除患者紧张情绪,提高患者的满意度及信任度,缓解护患矛盾,降低医疗纠纷,同时减少并发症的发生,提高急救成功率.     

The acquisition of cold sensitivity during TRPM8 ion channel evolution

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

Given that temperature influences all biological operations, the evolution of thermosensory adaptation is crucial in shaping the specialized temperature-dependent inhabitation of an organism. At the cellular level, thermosensory neurons in the dorsal root ganglia or trigeminal ganglia innervate the skin and transmit temperature information to the spinal cord and the brain. To bestow such neurons with thermal sensitivity, animals have a toolkit of temperature-sensitive ion channels located on the cell membrane at the molecular level. Accordingly, several members of the transient receptor potential (TRP) superfamily with steep thermosensitivity (referred to as thermoTRP) have attracted the general interest in the field of thermal biology, as they sufficiently cause steep changes in depolarizing currents upon either heating or cooling and thus are considered as the primary molecular sensors of temperature (1–4). Therefore, the evolutionary strategy for directly tuning the thermal activation in thermoTRPs can be employed by animals for their specialized thermosensory adaptation, as seen in vampire bats, pit-bearing snakes, platypus, penguins, squirrels, and camels (5–9).As heat sensation (warmth and extreme heat) provides the precondition of a fundamental and conserved biological survival process, the genes that encode heat sensors are considered ancient in many metazoan organisms. The annotation of trpv1 is consistently available in the genomes of fishes, insects, amphibians, reptiles, birds, and mammals. Despite the species-specific temperature-sensitive ranges, a growing number of studies have reported the functional convergence of these heat-sensitive thermoTRP orthologs at the protein level (10), suggesting the essential role of these channels in heat perception across species. Compared to heat sensors, the cold-sensitive thermoTRP likely evolved late. As the most established cold sensor responsive to low temperatures and cooling compounds, transient receptor potential melastatin 8 (TRPM8) was found in somatosensory neurons, and genetic ablation of trpm8 either in the neurons or mice led to a largely decreased cold sensitivity (4, 11–13). Interestingly, cold activation of amphibian TRPM8 has been tested (14), while sequencing efforts indicated the absence of the trpm8 gene in 12 fish species from 10 different orders (15). Several specific domains that may alter TRPM8 cold activation have been reported, including the pore domain, voltage sensing apparatus, and C terminus (8, 16–19). Notably, although the efficacy of cold activation is largely altered by residue substitutions in the pore domain, the channel mutants are still cold sensitive (8). Therefore, these findings based on domain/residue swapping among cold-sensitive TRPM8 orthologs may not draw an overall picture in functionally important domains responsible for cold sensitivity. How did the trpm8 gene originate? How did TRPM8 integrate and modulate cold sensitivity throughout evolution? The answers to such questions probably lead us to understand the evolution of temperature perception and identify the essential structural elements that shape TRPM8 cold activation.In this study, we show the presence of the young trpm8 gene in lobe-finned fishes, believed to be the ancestors that gave rise to all land vertebrates (20). Such a young type of trpm8 derived from the trpm2 exon shuffling was originated and formed during the expansion of lobe-finned fish genomes. By detecting the positive selection-rich domains, we described the formation of the thermosensitive MHR1-3 domain in amphibian and reptile species that enables TRPM8 to undergo conformational changes at low temperatures. Furthermore, we found that the TRPM8 pore domain of terrestrial vertebrates evolved to tune the efficacy of cold activation, in which a cold-sensitive MHR1-3 domain is indispensable to achieve such a modulatory mechanism. Together, our findings suggest that the trpm8 gene origination and formation of the TRPM8 MHR1-3 domain contributed to the transition of vertebrate life from water to land and that the efficacy of cold activation tuned by the TRPM8 pore domain diversified the setting of temperature-adaptive phenotypes in terrestrial vertebrates.     

基于TCGA数据库应用生物信息学方法分析和挖掘肺腺癌预后和诊断miRNA研究

目的基于TCGA数据库,应用生物信息学方法分析和挖掘肺腺癌预后和诊断miRNA生物学标志物。方法数据下载:从TCGA下载获取肺腺癌miRNA表达谱数据,包括miRNAseq和临床数据。筛选差异表达miRNAs:应用R-version 3.6.2软件中的edgeR包筛选肺腺癌组织和正常肺组织的差异基因,以│logFC│>2,P<0.05为筛选条件。筛选与预后相关miRNAs:应用R-version 3.6.2软件中的survival包绘制KM生存曲线,筛选与预后相关的miRNAs。筛选可作为肺腺癌诊断的miRNAs:应用R-version 3.6.2软件中的pROC包制作受试者工作特征曲线(ROC)评价与预后相关miRNAs诊断肺腺癌的特异性和敏感性。结果共识别到肺腺癌与正常肺组织差异表达的miRNA 144个,其中上调表达119个,下调表达25个。通过K-M生存曲线筛选出与预后显著相关(P<0.05)的miRNA共13个,分别为hsa-miR-139-3p、hsa-miR-328-3p、hsa-let-7g-3p、hsa-miR-142-3p、hsa-miR-147b...