基因表达聚类分析技术的现状与发展

随着多个生物基因组测序的完成、DNA芯片技术的广泛应用,基因表达数据分析已成为后基因组时代的研究热点.聚类分析能将功能相关的基因按表达谱的相似程度归纳成类,有助于对未知功能的基因进行研究,是目前基因表达分析研究的主要计算技术之一.已有多种聚类分析算法用于基因表达数据分析,各种算法因其着眼点、原理等方面的差异,而各有其优缺点.如何对各种聚类算法的有效性进行分析、并开发新型的、适合于基因表达数据分析的方法已是当务之急.     

用基因表达谱芯片研究中医经脉-脏腑相关的机理

基因芯片(Genechip),又称DNA芯片、DNA微阵列(DNA microarray),包括寡核苷酸微阵列(Oligo-microarray)及DNAmicroarray,是生物芯片(biochip)中应用最广泛、技术最成熟的分支。一般可分为基因表达谱芯片和DNA测序芯片。现就基因表达谱芯片在中医经脉脏腑相关研究中应用思路及前景作一探讨。     

酿酒酵母中对抗真菌药物克霉唑敏感的突变菌株的筛选

克霉唑是一种临床上用于抗真菌感染的抗真菌药物,同时也具有治疗其他疾病如疟疾、脚气病、足癣和癌症的前景。通过96孔板液体培养及倍比稀释的方法,对模式真菌酿酒酵母双倍体单基因缺失株文库中的4 532个非必需基因缺失株进行初步筛选及表型验证,对筛选出的敏感菌株测定了加克霉唑和不加克霉唑细胞内的ROS水平变化。筛选得到了127个克霉唑敏感的单基因缺失株,127个敏感菌株缺失的基因,其功能分别参与细胞的代谢过程、细胞周期和DNA加工过程、转录过程、蛋白命运(折叠,修饰,靶向运输)、细胞运输、细胞通讯/信号转导机制、细胞命运、细胞组分的发生和起源、不同细胞类型的分化、细胞营救、防御及毒力、细胞与环境交流和调节代谢及蛋白功能的过程,也有33个基因的功能未知。ROS测定结果显示,加入克霉唑之后,和野生型相比,25个菌株细胞内ROS显著升高,13个菌株细胞内ROS水平反而显著降低。克霉唑的抗真菌作用可能是多个细胞过程的集合,研究结果为克霉唑的改进利用及新的抗真菌药物研发提供理论基础。     

外源质粒DNA对小鼠脾脏物质能量代谢的影响

研究外源质粒DNA经胃肠道途径对免疫激活状态下小鼠脾脏代谢的影响。给Balb/c小鼠灌喂质粒pcDNA3 200μg,分别在灌喂后4h和18h分离脾脏,提取脾脏总RNA。利用Affymetrix基因表达谱芯片对灌喂质粒pcDNA3后的Balb/c小鼠脾脏进行基因表达谱研究。采用Genmmapp和MAPPFinder软件进行功能聚类分析。对上调倍数两倍及两倍以上的基因进行功能聚类发现,灌喂后4h,外源质粒刺激小鼠脾脏产生免疫应答的同时,脾脏中嘌呤代谢及蛋白质合成,胆固醇合成、脂肪酸合成、糖酵解、三羧酸循环及线粒体氧化磷酸化途径等大量代谢过程受到诱导。在灌喂后18h也得到类似的结果。结果表明外源质粒DNA可通过胃肠道途径吸收影响小鼠脾脏物质能量代谢过程。结果有助于在分子水平上深入了解外源质粒DNA经胃肠道吸收后的作用机制。     

酵母SOD1基因缺失突变体应答刚果红胁迫的转录组学分析（英文）

SOD1基因编码的铜锌超氧化物歧化酶是酵母细胞中最重要的抗氧化酶.前期研究发现,SOD1基因缺失(sod1Δ)导致酵母细胞对真菌细胞壁抑制剂刚果红(Congo red,CR)的敏感性增加,提示细胞抗氧化能力与细胞壁稳定性相关.本研究采用酵母全基因组表达谱芯片,比较了CR胁迫条件下,野生型酵母细胞和sod1Δ酵母细胞的转录表达谱.结果表明,与野生型酵母细胞相比,sod1Δ酵母细胞中260个基因发生了显著差异表达(140个基因表达上调、120个基因表达下调).随机选取12个差异表达基因采用定量PCR验证,结果与芯片分析结果一致.差异表达基因功能主要涉及细胞壁(几丁质合成)、细胞代谢、细胞防御(抗氧化和热冲击蛋白)、蛋白质合成以及大量功能未知基因.进一步研究发现,CR处理后,细胞壁几丁质含量和细胞内氧化应激指标丙二醛(MDA)含量在sod1Δ酵母细胞中显著升高,而在野生型酵母细胞中无明显变化,与芯片筛选差异表达基因的生物学功能分析结果一致.本研究提供了在全基因组水平上对SOD1基因与细胞壁应激反应之间关联的新认识.     

酵母SOD1基因缺失突变体应答刚果红胁迫的转录组学分析

SOD1基因编码的铜锌超氧化物歧化酶是酵母细胞中最重要的抗氧化酶. 前期研究发现,SOD1基因缺失(sod1Δ)导致酵母细胞对真菌细胞壁抑制剂刚果红(Congo red, CR)的敏感性增加,提示细胞抗氧化能力与细胞壁稳定性相关. 本研究采用酵母全基因组表达谱芯片,比较了CR胁迫条件下,野生型酵母细胞和sod1Δ酵母细胞的转录表达谱. 结果表明,与野生型酵母细胞相比,sod1Δ酵母细胞中260个基因发生了显著差异表达(140个基因表达上调、120个基因表达下调). 随机选取12个差异表达基因采用定量PCR验证,结果与芯片分析结果一致. 差异表达基因功能主要涉及细胞壁(几丁质合成)、细胞代谢、细胞防御(抗氧化和热冲击蛋白)、蛋白质合成以及大量功能未知基因. 进一步研究发现,CR处理后,细胞壁几丁质含量和细胞内氧化应激指标丙二醛(MDA)含量在sod1Δ酵母细胞中显著升高,而在野生型酵母细胞中无明显变化,与芯片筛选差异表达基因的生物学功能分析结果一致. 本研究提供了在全基因组水平上对SOD1基因与细胞壁应激反应之间关联的新认识.     

基于小波包变换的基因微阵列数据预处理方法

目的:基于阿尔茨海默病微阵列基因表达数据,分析研究微阵列基因表达数据预处理的新的有效方法.方法:首先采用标准差滤波、FSC(特征记分准则)和WPT-SAM(小波包变换-微阵列数据显著性分析)方法对微阵列基因表达数据进行预处理,比较处理后获得的基因数和FDR值;然后采用分类聚类方法对处理后的数据进行分类聚类和分层决策聚类,比较分类聚类结果.结果:标准差滤波和FSC方法获得的初筛基因数据较WPT-SAM方法多,但FDR值也高、后续分类聚类结果较WPT-SAM方法差.结论:WPT-SAM方法在预处理微阵列基因表达数据中,是比较灵活理想的分析方法.     

低毒兴奋效应模拟热量限制调节酵母转运体基因表达及脂质代谢

【目的】已知H2O2介导的线粒体低毒兴奋效应(Mitohormesis)能模拟热量限制延长酵母寿命,但未知两者是否存在共同作用机理。【方法】利用依时菌落计数法测定酿酒酵母时序寿命(CLS),采用微阵列芯片分析ATP结合盒(ABC)转运体基因表达谱及脂质代谢模式的转变,通过酶学测定法比较超氧化物歧化酶(SOD)活性的动态变化。【结果】经热量限制、H2O2、青蒿琥酯处理后,酵母CLS有不同程度延长,细胞解毒相关ABC转运体基因表达均下调或不变,促进长链脂肪酸运输的过氧化物酶体膜ABC转运体基因以及加速固醇摄取的质膜ABC转运体基因表达则显著上调。相应地,脂质分解(如脂肪酸β-氧化)基因表达上调,脂质合成(如脂肪酸延伸及去饱和)基因表达则下调。不同处理组中催化线粒体H2O2生成的Mn-SOD活性提高,导致催化H2O2降解及转变的抗氧化酶基因表达上调。【结论】低毒兴奋效应及热量限制在酵母中发挥延寿作用,既有赖于抗氧化酶催化的活性氧(ROS)清除反应,也取决于ABC转运体介导的脂质转运及后续的脂质分解及再利用。     

两种药物协同效应对酵母细胞转录组的影响

寻找抗衰老活性分子并研究其作用机制是衰老药物学的研究重点和热点。前期研究发现活性分子多球壳菌素和雷帕霉素可通过分子间协同效应延缓芽殖酵母细胞衰老。为了考察这两种小分子协同效应对细胞转录组的影响,利用DNA Microarray技术及生物信息学手段系统分析了这两种小分子药物协同处理对基因表达谱、基因本体聚类及相关信号通路的影响。研究结果显示,药物协同效应对细胞转录组产生了显著影响,共导致2 546个基因的差异表达(FDR0.05,Fold Change10%),其中1 157个基因显著上调,1 389个基因显著下调。进一步对基因本体聚类及信号通路分析显示,线粒体相关的分子功能、细胞组分、生物学过程和信号通路是此协同效应的一个主要靶点,其他可能的作用靶点还包括核糖体的生物发生、细胞骨架及过氧化物酶体代谢。     

Antifungal drug resistance pattern of Candida. spp isolated from vaginitis in Ilam-Iran during 2013-2014

Vaginal Candidiasis is the most common and important opportunistic fungal infection in women. By increasing use of antifungal drugs in recent years, it has caused drug resistance. This study aims to evaluate antifungal drugs susceptibility of Candida. spp isolated of women with vaginitis from Ilam-Iran during 2013-2014. samples were collected and cultured from 385 women with vaginitis, then Candida.spp was diagnosed by standard method. Antifungal drug susceptibility test for nystatin 100 unit/disk, fluconazole 10µg/disk, itraconazole 10µg/disk, ketoconazole 10µg/disk, amphotericinB 20µg/disk, clotrimazole 10µg/disk, posaconazole 5µg/disk, and voriconazole 1µg/disk were carried out by M44-A method(CLSI). From all culture positive samples, 150 isolates were Candida albicans and 89 isolates were non-albicans. The resistance to fluconazole, itraconazole, ketoconazole, clotrimazole, voriconazole, posaconazole, nystatin and amphotericin B was 76%, 62%, 72%, 55%, 6%, 7%, 1% and 0%. The highest resistance was seen for fluconazole , itraconazole, and the highest susceptible was seen for nystatin and amphotericin B. These results indicate nystatin and amphotericin B can be used as the first line for empirical therapy of vaginal candidiasis in the district.     

Effect of antifungal azoles on the heme detoxification system of malarial parasite

The antimalarial activities of some antifungal azole agents (ketoconazole, miconazole, and clotrimazole) have been known for several years, however, their antimalarial mechanism remains equivocal. Our recent study showed that clotrimazole has a relative high affinity for heme, inhibits reduced glutathione-dependent heme catabolism, and enhances heme-induced hemolysis. In the present study, we have found that clotrimazole can remove heme from histidine rich peptide-heme complex, which initiates heme-polymerization in malaria. In addition, we show that two other azoles (ketoconazole and miconazole) behave similarly to clotrimazole in binding to heme: they bind to heme with similar affinities, remove heme from the histidine rich peptide-heme complex and from the reduced glutathione-heme complex to form stable heme-azole complexes with two nitrogenous ligands derived from the imidazole moieties of two azole molecules. We have also revealed that clotrimazole and miconazole have stronger promoting activities for heme-induced hemolysis than ketoconazole, implying that the stronger antimalarial activities of clotrimazole and miconazole might arise from their stronger ability to promote heme-induced hemolysis of clotrimazole and clotrimazole than that of ketoconazole. These results also suggest that ketoconazole and miconazole, like clotrimazole, might possess an antimalarial mechanism relating to their inhibition of heme polymerization and the degradation of reduced glutathione-dependent heme.     

Sensitivity of some strains of the genus Epidermophyton to different antifungal agents

The inhibitory activity of some antifungal agents against 17 strains of genus Epidermophyton have been studied. The behaviour of Epidermophyton stockdaleae against antifungal agents tested is clearly different from that observed in the strains belonging to Epidermophyton floccosum, since all of the latter were sensitive to the antifungal agents used and the geophilic species showed resistance to griseofulvin, isoconazole, natamycin and nystatin, and intermediate sensitivity to ketoconazole and miconazole. The low sensitivity of E. stockdaleae (and other geophilic dermatophytes) to antifungal agents could be a problem in the current therapy if further studies or case reports demonstrate their pathogenic role.     

Systemic Candida infection in University Hospital 1997–1999: the distribution of Candida biotypes and antifungal susceptibility patterns

A total of 102 Candida species were isolated from blood cultures from January 1997 to October 1999. Using assimilation of carbohydrate test, 52 (51.0%) of the Candida sp. were identified as C. parapsilosis, 25.5% (26) were C. tropicalis. C. albicans made up 11.8% (12), 6.9% (7) were C. rugosa, 3.8% (4) C. glabrata and 1% (1) C. guilliermondii. No C. dubliniensis was found in the study. In vitro antifungal susceptibility tests showed that all Candida species were sensitive to nystatin, amphotericin B and ketoconazole. Although all isolates remained sensitive to fluconazole, intermediate susceptibility was found in 3 C. rugosa isolates. Antifungal agents with high frequency of resistance were econazole, clotrimazole, miconazole and 5-fluorocytosine. Candida species found to have resistance to these antifungal agents were non-C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

The impact of polyene,azole, and DNA analogue antimycotics on the cell surface hydrophobicity of Candida albicans and Candida tropicalis in HIV infection

Oral candidiasis is the most common opportunistic infection in individuals infected with the human immunodeficiency virus. Though Candida albicans is the major aetiological agent, non-albicans species such Candida tropicalis are now emerging as important agents of such infection. The Candida cell surface hydrophobicity (CSH) is considered a critical factor contributing to its colonization potential and virulence. It is also known that brief exposure to sub-cidal concentrations of antifungal agents is a likely scenario in the oral environment where the administered drugs are diluted continuously due to the flushing action of saliva. Hence the objective of the present study was to compare the CSH of 10 isolates each of C. albicans and C. tropicalis from HIV-infected individuals following brief exposure (1hour) of isolates to sub-therapeutic concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine. The CSH was assessed by a previously described biphasic aqueous-hydrocarbon assay. The mean percentage reduction of CSH of C. albicans following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was27.33 (p < 0.001), 21.34 (p < 0.05), 11.74 (p > 0.05), 18.4 (p > 0.05) and 14.64 (p > 0.05) respectively. The mean percentage reduction of CSH of C. tropicalis following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was 33.81 (p < 0.01), 28.88 (p < 0.01), 12.6 (p > 0.05), 21.53(p > 0.05) and 17.68 (p > 0.05) respectively. A significant inter-species variation in CSH was observed for nystatin and amphoterecin B. Overall the results reveal that the CSH of C. albicans is affected to a significantly lesser degree compared with C. tropicalis when exposed to the antifungals. These data further illustrate another mode of action of antifungals on Candida leading to a reduction in the CSH and thereby the yeast adherence to host tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence and the sensitivity to drugs of fungi other than Candida albicans isolated from the vagina

Frequency of occurrence of fungal species distinct from C. albicans isolated from vaginal mucosa and their sensitivity to antimycotic chemotherapeutics was determined. Material consisted of 452 fungal strains isolated from vagina from patients suffering from afflictions within genital area. Fungal strains isolated belonged to 13 genera. Fungi distinct from C. albicans constituted 27.1% of all the strains. Fungi the most frequently isolated from vagina belonged to following genera: T. glabrata 35.2% C. krusei 18.4% C. pseudotropicalis 15.2% S. cerevisiae 10.4%. In the majority of cases of vaginal infections caused by fungi distinct from C. albicans, Lactobacillus sp. was present and normal pH values of vaginal content 3/4 with variable number of leucocytes were observed. Evaluation of sensitivity to antimycotic drugs of fungal strains was performed by agar dilution technique. In this study the following chemotherapeutics were assayed: nystatin, pimaricin, amphotericin B, flucytosine, clotrimazole, miconazole and ketoconazole. It is worth to underline resistance of T. glabrata and S. cerevisiae to clotrimazole and ketoconazole. Moreover, resistance of strains belonging to genera C. krusei and C. pseudotropicalis to amphotericin B and C. krusei strains to nystatin and flucytosine was noted.     

Cryptococcus neoformans: Comparisons of in vitro antifungal susceptibilities of serotypes AD and BC

Thirty-nine isolates of Cryptococcus neoformans, nineteen serotype AD and twenty serotype BC, were assayed for susceptibility to eight antifungal agents using an in vitro agar dilution assay. Media employed were Kimmig agar and yeast nitrogen base supplemented with 10% glucose. The antifungal agents used were ketoconazole, amphotericin B, 5-fluorocytosine, nystatin, miconazole, BAY N 7133, ICI 153,066, and itraconazole. No clinically significant differences in in vitro minimum inhibitory concentrations were detected between serotypes AD and BC against any of the compounds tested. An adverse medium effect was observed in two of the assays, but the outcome of the AD/BC comparison was not affected. This is the first report in which the in vitro antifungal susceptibilities of Cryptococcus neoformans serotypes are analyzed.     

Anti-fungal resistance in candida isolated from oral and diaper rash candidiasis in neonates

The purpose of the present study is to evaluate the sensitivity of Candida species isolated from oral candidiasis and diaper dermatitis infections in children. The children referring to private and public clinics in Ilam, Iran were exmined for oral candidiasis and diaper dermatitis. In this study, 248 oral candidiasis and diaper dermatitis samples were collected and cultured.Candida species were identified by using standard methods. Resistance and sensitivity to amphotericin B, nystatin, ketoconazole, fluconazole, itraconazole, clotrimazole, and posaconazole were determined using the CLSI M44-A standard disk diffusion method. From the 248 studied samples, 149 were positive for Candida, among which the Candida albicans was the most prevalent (64.4%). The resistance of different Candida species to nystatin, itraconazole, fluconazole, ketoconazole, clotrimazole, voriconazole, and posaconazole were 4, 43, 34.2, 34.9, 21.5, 6, and 6.7%, respectively. No resistance to amphotericin B was observed. Considering rather low resistance to nystatin, this drug is the best choice for oral candidiasis and diaper dermatitis.     

Interaction between Candida agglutinins and antifungal agents

Antifungal agents alter the function and morphology of Candida cell membranes and cell walls. We observed that brief (30 minute) exposure to either amphotericin B or clotrimazole inhibited the agglutination of Candida blastoconidia by murine bronchoalveolar lavage fluid. This inhibition required continuous drug presence. Neither amphotericin nor clotrimazole inhibited Candida agglutination by concanavalin A or pooled human serum. These results demonstrate that antifungal drugs can produce rapid changes in the surface characteristics of some fungi.