结核分枝杆菌对宿主细胞凋亡效应的调控研究

由结核分枝杆菌（MTB）引起的结核病是严重危害人类健康的重大传染病,全世界约有1/3的人口潜伏感染MTB。近几年随着耐多药MTB和HIV混合感染的流行,使结核病控制形势更加严峻。细胞凋亡是生物有机体一种古老的免疫防御反应,在帮助机体清除胞内菌过程中发挥关键作用。已有研究结果表明,结核分枝杆菌对宿主细胞凋亡的调控过程非常复杂,即表现为有双重性：有些基因能促进凋亡,而有些则表现为抑制效应,且本研究组初步的研究结果表明细菌的毒力与凋亡有一定的内在联系,但是相关的分子机制还很不清楚。因此,鉴定MTB基因组中与凋亡相关的基因可以为研究细菌和宿主细胞之间的相互作用奠定基础,并且有助于研发新的药物靶标和候选疫苗。     

网络药理学与分子对接研究黄芪治疗阿尔兹海默病的作用机制

为了研究黄芪中活性成分治疗阿尔兹海默病的作用机制,采用网络药理学与分子对接模拟方法,利用相关数据库确定疾病靶点,构建靶点相互作用和药物-成分-靶蛋白-疾病网络,在DAVID数据库进行基因本体富集分析和京都基因与基因组百科全书数据库通路富集分析,并对靶点进行分子对接模拟验证。结果表明：筛选到20种黄芪抗阿尔兹海默病的活性成分,其中槲皮素、山奈酚、异鼠李素、刺芒柄花素、 7-O-甲基-异微凸剑叶莎醇为关键成分;筛选出118个交集靶点,含6个关键靶点,各靶点富集于炎症反应细胞凋亡等生物过程;分析得到180条信号通路,作用机制主要与TNF、 PI3K-Akt、 IL-17等通路相关。     

CSB1-Lipo-DOX-miR101靶向治疗乳腺癌及抗耐药效应的体外研究

通过薄膜超声分散技术、硫酸铵主动载药技术构建了靶向乳腺癌(breast cancer, BC)细胞的具有抑制化疗药物多药耐药性(multidrug resistance, MDR)特征的阿霉素(doxorubicin, DOX)脂质体载药体系CSB1-Lipo-DOX-miR101。应用MTT法、损伤修复法、Transwell、细胞核染色、扫描电镜等技术对药物疗效和细胞行为变化进行研究。利用网络数据库分析miRNA101潜在靶向的MDR相关靶基因及BC恶性表征相关基因,以Western blot验证分析miRNA101靶基因的表达及潜在的抗MDR机制。结果表明,CSB1-Lipo-DOX-miR101对BC细胞MCF-7靶向性良好,特别是对耐药性BC细胞MCF-7/ADR具有明显杀伤力,同时对药物的MDR形成具有显著抑制作用,使药物可以持续发挥杀伤作用。结果提示CSB1-Lipo-DOX-miR101可以提高DOX对BC的临床治疗效应、降低毒副作用及复发率,其对BC的治疗效应及对MDR的抑制机理可能是通过抑制ABCC5、EZH2、APEX1、ITGB1、Snail1、MMP2基因表达实...     

基于网络药理学和分子对接预测黄芪-莪术治疗胃癌的分子机制

基于网络药理学和分子对接探讨黄芪-莪术治疗胃癌的作用机制。利用中药系统药理学数据库与分析平台筛选出黄芪、莪术的活性成分,从Uniprot数据库获取靶点蛋白的基因名。以gastric cancer为关键词,在GeneCards数据库中检索胃癌的相关靶基因,将其与药对的活性成分靶基因相互映射,筛选出共同靶点。利用Cytoscape 3.7.0 软件绘制活性成分-靶点网络。将筛选出来的靶点在STRING数据库中构建蛋白质-蛋白质相互作用网络,同时,选择Omicshare平台进行基因本体功能及京都基因与基因组百科全书通路的富集分析。使用AutoDock Vina和PyMol软件进行分子对接。从黄芪-莪术药对中共筛选出26个活性成分,包括槲皮素、山奈酚、莪术醇、7,2'-二羟基-3',4'-二甲氧基异黄烷、β-榄香烯等,作用于TP53、MYC、CASP3、AKT1、 JUN等74个靶点,这些靶点主要富集在癌症、癌症中的蛋白聚糖、PI3K-Akt、 MAPK、 Rap1、TNF、 FoxO等信号通路上。该研究为黄芪-莪术药对治疗胃癌的基础研究和临床应用提供了参考依据。     

基于GEO数据库和转录因子调控网络的结核病药物作用靶点筛选及其作用机制

目的 基于GEO数据库和转录因子调控网络筛选抗结核病药物及药物作用靶点。方法 通过NCBI的GEO数据库,筛选结核病患者和健康者之间差异表达的基因;通过AnimalTFDB 3.0数据库预测差异表达基因中的转录因子,并构建转录因子调控网络;通过调控网络中的关键基因筛选相关miRNA,并筛选关键节点,初步阐明结核病致病的分子机制。结果 通过GEO数据库检索,筛选出的差异表达基因为784个;通过AnimalTFDB 3.0数据库筛选出23个转录因子和对应的790个靶基因,构建了转录因子-靶基因的调控网络;通过TargetScanHuman 7.2查询到关键节点对应的miRNA,构建“转录因子-靶基因-miRNA”调控网络,筛选出4个结核病药物靶点(EP300,CREBBP,ELAVL1,HSP90AA1),阐明了其与转录因子和miRNA之间的调控机制。结论 通过构建结核“转录因子-靶基因-miRNA”网络,筛选出结核病新的潜在药物作用靶点——EP300、CREBBP、ELAVL1、HSP90AA1;同时发现,EP300、CREBBP、HSP90AA1通过激活转录因子STAT2,导致机体内炎...     

基于GenePANDA算法的精神分裂症药物靶基因预测

精神分裂症是常见的精神障碍类疾病.目前,治疗精神分裂症的药物存在疗效差、副作用大和耐药性的问题,迫切需要开发新的药物,而发现新的药物靶基因是开发新药的重要环节.为了预测精神分裂症药物的靶基因,我们首先基于DrugBank中药物已知的靶基因,使用网络算法GenePANDA预测出候选靶基因;之后,我们对药物已知靶基因进行功能富集分析,使用富集出的生物学通路筛选候选靶基因,最终得到48个候选靶基因.其中,29个基因被报道和精神分裂症直接相关,13个基因被报道为精神分裂症药物的靶基因.此外,在DrugBank中共有54种药物靶向预测出的基因,其中17种药物被研究报道可用于治疗精神分裂症.     

基于网络药理学探讨尪痹方治疗类风湿性关节炎的机制

基于网络药理学方法探讨尪痹方治疗类风湿性关节炎(rheumatoid arthritis, RA)的潜在机制。应用中药系统药理学数据库与分析平台筛选出尪痹方的化学成分及作用靶点,GeneCards数据库检索RA相关靶点,维恩图获取药物与疾病交集靶点,利用STRING数据库得到蛋白质-蛋白质相互作用(protein-protein interaction, PPI)网络信息,使用Cytoscape构建药物-有效成分-靶点-疾病网络与PPI网络图,通过DAVID数据库对共同靶点进行基因本体(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)分析。使用SYBYL-X 2.1.1软件进行分子对接验证。经过筛选得到32个有效成分及99个相关靶点,核心靶点为IL-6、TNF、AKT1、VEGFA、PTGS2等。GO功能富集分析主要涉及RNA聚合酶II启动子转录的正调控等生物过程,KEGG通路富集分析主要涉及TNF信号通路、T细胞受体信号通路、Toll样受体信号通路、破骨细胞分化等信号通路。分子...     

表观遗传调控滇重楼内生真菌活性物质的合成

提出化学表观筛选——分子遗传平台策略,利用表观遗传理论调控滇重楼内生真菌抗菌活性物质的合成。化学表观遗传筛选是对真菌进行高通量化学表观抑制剂的筛选,旨在经济快捷地获得抗菌活性物质合成受到表观调控的菌株;建立分子遗传平台是根据同源基因克隆真菌的相关表观遗传酶类基因,靶向改变表观遗传酶类的表达,旨在获得遗传稳定性高的改良菌株。     

雷公藤红素治疗类风湿关节炎的组学联合分析

用雷公藤红素处理成纤维样滑膜细胞,通过RNA测序、定量蛋白质组学、分子对接、qPCR和Western blot检测和验证,筛选到差异基因和蛋白质,获得7 803个基因和3 372个蛋白质,雷公藤红素作用机制与氧化应激和氧气水平高低相关,也涉及细胞组分和细胞外基质蛋白的稳定和合成通路.结果显示,雷公藤红素可通过这些机制来...     

美国山核桃不定根形成过程中相关蛋白质 的鉴定及功能分析

美国山核桃是扦插后极难生根的树种,为研究其不定根生根机制,利用MALDI-TOF-MS技术对美国山核桃不定根发育关键时期的差异蛋白进行鉴定分析,发现了48个表达相对差异的蛋白点,选择其中18个差异蛋白进行肽质谱指纹分析并进行同源性比较。结果表明,美国山核桃不定根形成的生理过程中产生了差异蛋白质,其中包括了能量代谢相关蛋白、逆境胁迫相关蛋白和信号传递相关蛋白等差异蛋白。分析可知:能量代谢相关蛋白有ATP合成相关酶类、光合作用相关蛋白及烯醇化酶等; 逆境胁迫相关蛋白与热激蛋白同源; 信号传递相关蛋白主要为细胞色素酶类和血红结合蛋白。     

抗结核化合物的设计与合成

针对日益突出的结核病(TB)药物耐药性问题,对抗结核药物进行了合理的修饰,以增强其结核病治疗的效果.以异烟肼(INH)为原料,与不同的异氰酸酯反应,成功制备了9种新型的不对称取代脲类化合物,这类脲桥结构对结核分枝杆菌(MTB)中的半胱氨酸合酶具有抑制作用,合成的产物经质谱(MS)、氢核磁共振谱(~1H NMR)等方法进行了分析表征.     

Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals

Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon-carbon bond formation can be catalysed by enzymes from the thiolase superfamily, including β-ketoacyl-acyl-carrier protein (ACP) synthases, polyketide synthases, 3-hydroxy-3-methylglutaryl-CoA synthases, and biosynthetic thiolases. Pathways involving these enzymes have been exploited for fuel and chemical production, with fatty-acid biosynthesis (β-ketoacyl-ACP synthases) attracting the most attention in recent years. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids, can also operate in the synthetic direction and thus enable carbon-chain elongation. Here we demonstrate that a functional reversal of the β-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities. This pathway operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), characteristics that enable product synthesis at maximum carbon and energy efficiency. The reversal of the β-oxidation cycle was engineered in Escherichia coli and used in combination with endogenous dehydrogenases and thioesterases to synthesize n-alcohols, fatty acids and 3-hydroxy-, 3-keto- and trans-Δ(2)-carboxylic acids. The superior nature of the engineered pathway was demonstrated by producing higher-chain linear n-alcohols (C?≥?4) and extracellular long-chain fatty acids (C?>?10) at higher efficiency than previously reported. The ubiquitous nature of β-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.     

利用TRV过表达和沉默系统验证植物油脂合成相关基因的功能

为探究烟草脆裂病毒过表达和沉默系统进行油脂相关基因研究的可能性,该研究克隆了拟南芥的WRI1和FAD2基因,分别构建了TRV过表达和沉默载体. 利用瞬时侵染技术侵染本氏烟草,取材料进行RT-PCR和脂肪酸含量检测. 结果显示在过表达WRI1基因的本氏烟草中,侵染叶和非侵染叶的WRI1基因及其相关参与脂肪酸合成基因ACP1、KAS1和BCCP2等都有明显上调,且脂肪酸含量检测结果显示分别增加16%和28%. 在沉默FDA2基因的本氏烟草植株中,发现多不饱和脂肪酸含量分别减少约25%和24%. 因此,利用TRV系统对油脂相关基因进行研究是可能的.     

A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis

Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M. tuberculosis have reaffirmed tuberculosis as a primary public health threat. However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years. Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M. tuberculosis. Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M. tuberculosis and promising oral activity in animal infection models. We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.     

烟曲霉细胞壁作为药物靶标的鉴定研究

烟曲霉(Aspergillus fumigatus)作为侵袭性真菌感染的三大机会病原真菌之一,因其高致病率和高致死率而成为威胁人类健康的重大隐患。有限的抗真菌类药物以及近年来耐药性菌株的涌现使得寻找新的药物靶标并开发新型抗真菌药物迫在眉睫。真菌细胞壁作为真菌所特有的结构一直以来被认为是理想的药物靶标。本文概述了烟曲霉细胞壁结构及合成过程的相关研究进展,并分析了参与细胞壁各组分合成的酶作为药靶的可行性。     

Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host

The application of molecular cloning to antibiotic-producing microorganisms should lead to enhanced antibiotic productivity and to the biosynthesis of novel antibiotics by in vitro interspecific recombination. To allow such approaches, the genes for antibiotic synthesis must be isolated, analysed and perhaps modified. Certain Streptomyces species produce nearly two-thirds of the known natural antibiotics; the recent development of cloning systems in the genus makes it possible to isolate and analyse Streptomyces genes. However, antibiotics are metabolites which require sets of several enzymes for their synthesis and attempts to isolate the corresponding genes have so far yielded clones carrying either individual genes of the set, or only incomplete gene sets. We describe here the isolation of a large continuous segment of Streptomyces coelicolor DNA which apparently carries the complete genetic information required for synthesis of an antibiotic, actinorhodin , from simple primary metabolites. Not only can the cloned DNA 'complement' all available classes of actinorhodin non-producing mutants of S. coelicolor but, on introduction into a different host, Streptomyces parvulus , it directs the synthesis of the antibiotic. The tendency for the genes for antibiotic synthesis to be clustered together on the chromosomes of Streptomyces species and the availability of plasmid vectors which can carry stable inserts of DNA larger than 30 kilobase pairs (kb) and which can be introduced efficiently into Streptomyces protoplasts, suggest that the experiments described have general significance for this area of biotechnology.     

Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites.

The incidence of human malaria has increased during the past 20 years; 270 million people are now estimated to be infected with the parasite. An important contribution to this increase has been the appearance of malaria organisms resistant to quinoline-containing antimalarials such as chloroquine and quinine. These drugs accumulate in the acid food vacuoles of the intraerythrocytic-stage malaria parasite, although the mechanism of their specific toxicity in this organelle is uncertain. The primary function of the food vacuole is the proteolysis of ingested red cell haemoglobin to provide the growing parasite with essential amino acids. Haemoglobin breakdown in the food vacuole releases haem, which if soluble can damage biological membranes and inhibit a variety of enzymes. Rather than degrading or excreting the haem, the parasite has evolved a novel pathway for its detoxification by incorporating it into an insoluble crystalline material called haemozoin or malaria pigment. These crystals form in the food vacuole of the parasite concomitant with haemoglobin degradation, where they remain until the infected red cell bursts. The structure of haemozoin comprises a polymer of haems linked between the central ferric ion of one haem and a carboxylate side-group oxygen of another. This structure does not form spontaneously from either free haem or haemoglobin under physiological conditions, and the biochemistry of its formation is unclear. Here we report the identification and characterization of a haem polymerase enzyme activity from extracts of Plasmodium falciparum trophozoites, and show that this enzyme is inhibited by quinoline-containing drugs such as chloroquine and quinine. This provides a possible explanation for the highly stage-specific antimalarial properties of these drugs.     

大麦籽粒油脂肪酸成分分析

 测定、分析大麦籽粒油脂的质量分数及其脂肪酸组成。分别采用索氏抽提法和酸水解法提取大麦籽粒中的粗脂肪油,再经过氢氧化钾/甲醇碱催化甲酯化法进行衍生化处理后,通过气相色谱/质谱联用技术分析脂肪酸的组成。结果显示,两种方法大麦籽粒粗脂肪的提取量分别为1.87g/100g和1.98g/100g,分别鉴定出12种和14种脂肪酸成分,主要成分为亚油酸（52.20%,53.27%）、棕榈酸（22.75%,22.15%）、油酸（16.35%,14.63%）和亚麻酸（4.65%,5.48%）,油脂中不饱和脂肪酸的质量分数都超过75%。研究结果可为大麦类食品的相关研究提供基础数据。     

长棘海星的体内脂肪酸成分及其生物学意义分析

 长棘海星是热带和亚热带海域珊瑚的最重要捕食者。首次报道了长棘海星体内总脂肪酸的提取方法及其成分与含量测定。用石油醚提取低极性组分,经硅胶柱层析分离得到游离脂肪酸（酯）;用乙酸乙酯提取酯溶性神经酰胺及其衍生脂类,提取物和残余的水溶性脂类分别经盐酸甲醇解,水解产物再用石油醚提取。各提取及分离组分经GC MS分析,发现游离脂肪酸中有C14～C20的长链饱和与不饱和脂肪酸,其中二十碳的Ω-3和Ω-6多价不饱和脂肪酸含量高,如：5,8,11,14-二十碳四烯酸甲酯（主要）,8,11,14-二十碳三烯酸,5,8,11,14,17 -二十碳五烯酸甲酯。在盐酸甲醇解产物中,主要检测到系列饱和脂肪酸和高含量的甾醇成分。多价不饱和脂肪酸是维持细胞膜结构和功能的必要组分,也是合成其它生理活性物质的前体化合物。据报道不饱和脂肪酸5,8,11,14-二十碳四烯酸也有诱发长棘海星捕食反应的作用。5,8,11,14-二十碳四烯酸是长棘海星体内主要的必需不饱和脂肪酸,长棘海星体内可能无法合成它,所以必须不断地捕食含有该不饱和脂肪酸的珊瑚。本实验脂肪酸（酯）的分极性段提取处理方法,能充分地提取出脂肪酸成分,排除其它成分的干扰,分析结果可靠,且重现性好。研究发现长棘海星体内有高含量的、具有重要应用价值的多价不饱和脂肪酸和甾体激素,这也为长棘海星的合理开发与利用提供了依据。     

红松种子皮诺敛酸形成积累的多基因协同调控

为探讨红松种子皮诺敛酸生物合成的基因调控规律,以3个不同生长发育期(7月8日、8月10日、9月6日)的红松种子为材料,利用气相色谱飞行时间质谱法测定种子油脂脂肪酸组分及其相对百分含量,采用qRT-PCR方法分析脂肪酸代谢途径相关基因ACC、KAR、EAR、HAD、FATB、KASII、SAD、FAE1、KCR、FAD2、FAD8和⊿5D的表达差异,分析基因表达对皮诺敛酸形成积累的影响。红松种子油脂中共检测出10种脂肪酸,其中油酸、亚油酸和皮诺敛酸是主要脂肪酸。同时研究结果首次揭示了红松种子皮诺敛酸形成积累的多基因协同调控机制,对高皮诺敛酸油料植物的培育具有重要意义。