Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha

In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim^? phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim^? phenotypes were determined by single recessive nuclear mutations. The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.     

花生miRNA与其靶基因的生物信息学预测

本研究利用生物信息学方法预测花生中的miRNA及其靶基因。将miRBase注册的已知植物miRNA与花生EST和GSS数据库进行比对搜索,筛选得到13条miRNA,进一步预测得到20个靶基因。分析结果显示大多数靶基因属于转录因子,调控植物的生长发育等多种生物过程。     

不同时点果蝇七氟醚麻醉相关基因的筛选与分析

目的:在不同时点筛选七氟醚麻酚相关基因,为阐明其作用的分子机制提供有益的线索.方法:以野生品系黑腹果蝇为实验材料,将果蝇分为两组:(1)七氟醚5min组:①空气小组(H5):通入空气5分钟:②七氟醚小组(1H5):七氟醚麻醉5分钟.(1)七氟醚20min组:①空气小组(H20):通入空气20分钟;②七氟醚小组(1H20):七氟醚麻醉20分钟.采用基因表达谱芯片方法,以每组的空气小组为对照分别筛选其七氟醚麻醉小组的差异表达基因.结果:七氟醚5min组筛选出差异表达基因156个,七氟醚20min组13个.对这些差异表达基因进行功能分析发现七氟醚5min组中一些基因的功能为钠:氢反向转运体活性、GTP结合等.七氟醚20min组中一些基因的功能为核酸结合,mRNA结合等,其中功能与钙离了通道、ATP活性、锌离子结合等有关的基因在两组都有改变.结论:许多基因在麻醉初期都活跃表达,还有一些基因表达贯穿麻醉过程,说明麻醉是一个连续的动态过程.     

啤酒有害菌的PCR检测和SYBR Green实时PCR定量

啤酒有害菌是一些能在啤酒中存活并使啤酒的外观和风味发生改变的细菌,对其进行快速检测和定量是啤酒生产急待解决的问题。我们从华润雪花啤酒（中国）有限公司各工厂提供的样品中分离到28株啤酒有害菌,16S rDNA序列的系统进化分析表明,其中26个菌株属于乳杆菌属（Lactobacillus spp．）、1个菌株为明串珠菌属（Leuconostoc spp．）,1个菌株为片球菌属（Pediococcu sp．）。根据酒花（hop）抗性基因horA、horB和horC的保守序列设计了扩增这3个基因的PCR引物,用这些引物对28株啤酒有害菌进行了常规PCR检测,检出率分别为89％、79％和75％,用hor A—horC双引物进行检测,检出率为100％。用SYBR Green实时定量PCR技术,以horA基因为靶序列,建立了对啤酒有害菌的细胞数进行快速定量的新方法,用该方法测定的污染啤酒样品中有害菌的浓度与平板培养法相近。     

食源性单核细胞增生李斯特菌四环素、红霉素耐药基因研究

研究了食源性单核细胞增生李斯特菌四环素、红霉素耐药基因的分布状况及和耐药表型的关系。采用微量肉汤稀释法对2005~2007年河北省疾病预防控制中心分离到的食源性单核细胞增生李斯特菌株进行四环素、红霉素药敏实验;应用PCR方法对实验菌株进行四环素耐药基因tet(M)、tet(S)、tet(L)、tet(K)、tet(B)、及与tet(M)基因关系密切的转座子Tn916、红霉素核糖体甲基化酶基因ermB、ermC、及与ermB基因关系密切的转座子Tn917检测,对阳性样本序列进行鉴定分析;应用血清学分型、脉冲场凝胶电泳(PFGE)、及脂肪酸聚类分析方法分析四环素耐药菌株之间的相关性,确定基因型和多态性。结果表明,91株单核细胞增生李斯特菌四环素敏感77株、耐药14株;红霉素敏感89株、耐药2株,其中包含1株菌同时交叉耐药四环素和红霉素;14株四环素耐药株中含tet(M)基因的13株,在13株tet(M)基因阳性菌中,tet(M)位于Tn916转座子上的9株;1株同时交叉耐药四环素、红霉素菌同时携带tet(S)、ermB基因;ermC基因、转座子Tn917均为阴性;四环素、红霉素敏感株中未检测到上述任何耐药基因。14株四环素耐药菌株血清型分布以1/2a型为主(n=12),部分菌株PFGE、脂肪酸分型完全一致。食源性单核细胞增生李斯特菌获得tet(M)基因是耐四环素的主要机制之一,具有水平传播耐药基因能力的接合型转座子Tn916与该菌四环素耐药播散有直接关系;ermB基因介导的核糖体靶位点改变存在食源性单核细胞增生李斯特菌红霉素耐药株中;PFGE基因型结合脂肪酸聚类分析能够用来分析菌株之间的相关性。     

Microbial community dynamics in replicate membrane bioreactors - Natural reproducible fluctuations

We operated 4 replicate membrane bioreactors (MBRs) in parallel to test if an acclimated seed inoculum would evolve similarly following even distribution into replicates. A cloning and sequencing library of 16S rRNA genes was obtained from the seed inoculum complemented with terminal restriction fragment length polymorphism (T-RFLP; n = 18 per reactor) analysis over the study period (n = 113 d) that targeted the 16S rRNA gene. The amoA functional gene was also monitored by T-RFLP. The T-RFLP results were analyzed by means of diversity indices, an adaptation of a moving window of similarity approach within each MBR, and non-metric multi-dimensional scaling (NMS) accompanied with multi-response permutation procedures (MRPP) to assess community interrelationships amongst MBRs. Based on the 16S rRNA microbial communities, the 4 MBRs initially diverged away from one another, followed by a convergence on Day 4. From thereon, the 16S rRNA-based communities evolved similarly throughout (average p-value = 0.49 from pair-wise MRPP). In contrast, the nitrifying communities did not undergo any discernable shift over time amongst MBRs according to T-RFLP analysis of amoA and revealed one cluster by NMS (average p-value = 0.83 from pair-wise MRPP). The study demonstrates that acclimated microbial communities evolve similarly over time in engineered systems when operational parameters are left unchanged.     

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.     

Characteristics of Staphylococcus hyicus strains isolated from pig carcasses in two different slaughterhouses

In a previous study, we showed that coagulase positive staphylococci, which are often used as indicators for Staphylococcus aureus, are frequently found on pig carcasses. Further characterization of the strains identified only a minor part as S. aureus. Selected non-S. aureus strains were all identified as Staphylococcus hyicus, However, two studies described in this species strains that produce staphylococcal enterotoxins. The aim of the present study was to further characterize such coagulase positive S. hyicus strains isolated from pig carcasses and to assess the results for their food safety relevance. A total of 189 strains from two abattoirs were characterized. Phenotypically, 98.9% showed non-pigmented colonies, 99.5% no haemolysis and 67.7% were egg yolk-positive. DNase activity was found in all but one isolate. Only five of the 189 strains were resistant to the antimicrobials tested. One strain harboured the mecA gene. Exfoliative toxin genes were detected in 31 (16.4%), S. aureus enterotoxin genes in none of the strains.The PFGE genotyping results show only a limited number of clusters. Cluster I included more than 50% of the strains. The fact that similar or closely related PFGE patterns of S. hyicus can be found on carcasses after bleeding in both abattoirs indicates the occurrence of widespread strains in the Swiss pig population. Moreover, the genotyping results revealed a remarkable homogeneity in S. hyicus strains isolated from different process stages in abattoir B, which could indicate a recontamination problem with persisting strains.     

高致病性猪繁殖与呼吸系统综合症病毒荧光定量PCR检测方法的建立

为了对当前爆发流行的高致病性猪繁殖与呼吸系统综合症病毒建立快速准确的检测方法,根据该类病毒在Nsp2基因1594-1680处缺失87个碱基的特点,设计了一对特异性引物和一个Taqman探针,通过对反应条件的优化,建立了荧光定量PCR检测方法.该方法特异性强,灵敏度高,能很好地区分高致病性猪繁殖与呼吸系统综合症病毒和其它病毒,没有发现假阳性和假阴性现象,检测病毒滴度达到1TCID50.用该法对38份疑似样本进行检测,阳性率60.5%,与常规PCR法符合率100%.