Identity tests: determination of cell line cross-contamination

Cell line cross-contamination is a phenomenon that arises as a result of the continuous cell line culture. It has been estimated that around 20% of the cell lines are misidentified, therefore it is necessary to carry out quality control tests for the detection of this issue. Since cell line cross-contamination discovery, different methods have been applied, such as isoenzyme analysis for inter-species cross-contamination; HLA typing, and DNA fingerprinting using short tandem repeat and a variable number of tandem repeat for intra-species cross-contamination. The cell banks in this sense represent the organizations responsible for guaranteeing the authenticity of cell lines for future research and clinical uses.     

油莎豆SRAP指纹图谱构建及遗传多样性分析

利用SRAP分子标记构建了14份不同地理来源、表型具有差异的油莎豆品系的分子指纹图谱并进行遗传多样性分析。结果表明100对引物中共有多态性引物42对,扩增出多态性带328条,平均每对引物7.8条。28对引物在12个品系上具有特征谱带,除品系4和14外,均可用1对引物进行鉴定;采用引物组合法仅用Me2/Em6和Me8/Em11这2对引物就可将14份材料区分开,并利用这2对引物构建了上述品系的数字指纹图谱。UPGMA聚类分析表明,所有参试材料间的遗传距离在0.12～0.75之间,平均为0.42,表明我国不同地理来源的油莎豆品系遗传差异较大,具有较为丰富的遗传多样性。     

DNA marker technology for wildlife conservation

Use of molecular markers for identification of protected species offers a greater promise in the field of conservation biology. The information on genetic diversity of wildlife is necessary to ascertain the genetically deteriorated populations so that better management plans can be established for their conservation. Accurate classification of these threatened species allows understanding of the species biology and identification of distinct populations that should be managed with utmost care. Molecular markers are versatile tools for identification of populations with genetic crisis by comparing genetic diversities that in turn helps to resolve taxonomic uncertainties and to establish management units within species. The genetic marker analysis also provides sensitive and useful tools for prevention of illegal hunting and poaching and for more effective implementation of the laws for protection of the endangered species. This review summarizes various tools of DNA markers technology for application in molecular diversity analysis with special emphasis on wildlife conservation.     

Protein repertoire impact of Ubiquitin-Proteasome System impairment: insight into the protective role of beta-estradiol

The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal.     

MALDI-TOF MS proteomic phenotyping of filamentous and other fungi from clinical origin

Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance.     

Universal fingerprinting chip server

The Virtual Hybridization approach predicts the most probable hybridization sites across a target nucleic acid of known sequence, including both perfect and mismatched pairings. Potential hybridization sites, having a user-defined minimum number of bases that are paired with the oligonucleotide probe, are first identified. Then free energy values are evaluated for each potential hybridization site, and if it has a calculated free energy of equal or higher negative value than a user-defined free energy cut-off value, it is considered as a site of high probability of hybridization. The Universal Fingerprinting Chip Applications Server contains the software for visualizing predicted hybridization patterns, which yields a simulated hybridization fingerprint that can be compared with experimentally derived fingerprints or with a virtual fingerprint arising from a different sample. AVAILABILITY: The database is available for free at http://bioinformatica.homelinux.org/UFCVH/     

Genetic diversity of rhizobia nodulating lentil (Lens culinaris) in Bangladesh

In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1-94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5-10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli-R. leguminosarum group and may correspond to novel species in the genus Rhizobium.     

联合应用ESI MS和1H NMR分析含笑属植物种子磷脂

首次尝试利用电喷雾电离质谱(ESI MS)和氢原子核磁共振(1 H NMR)技术分析了含笑属六种植物种子的磷脂特性,发现在两个指纹图谱区发现明显的差异,即在质荷比(m/z)895-910(ESI MS)和5.30～5.40mg/L(1 H NMR)两个特异的区域存在显著差异。这些源于种子磷脂ESI/MS和1 H NMR的谱带差异可以被用来分析不同植物的种子的磷脂特征。而且,相似地,在更广的层面上,特异性的谱带差异可用于分析其他植物种子的磷脂组成和特性,辅助鉴定种子。     

Biotechnology in forensic medicine: new ways of fingerprinting

The impact of biotechnology on forensic science is reflected in the increasing use of recombinant DNA techniques to identify individuals. Different methods of identification are reviewed and include DNA fingerprinting and antibody fingerprinting.     

Multiplexed,linkage group-specific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

A set of single nucleotide polymorphism (SNP) markers has been developed for each of the nine linkage groups of sugar beet. Each set can monitor the polymorphic state at five to six linked marker loci. In each set, the loci selected for marker development are first amplified in a multiplexed reaction. These amplification products are the basis for sequence-specific elongation of primers adjacent to SNP positions. The extension step revealing SNP loci is based on fluorescently labelled nucleotides. In each set, primers developed to reveal SNP alleles differ in length to allow clear peak resolution in capillary electrophoresis. The nine linkage group (LG) –specific sets provide information on the polymorphism at a total of 52 SNP marker loci. Using the SNP-based tool, groups of concerned loci have been anchored to three different linkage maps of sugar beet. In a second experiment, sugar beet breeding lines have been fingerprinted. The use of the nine sets of LG-specific markers in sugar beet genetics and breeding is discussed. The information necessary to specify the 52 marker loci, as well as their map location, and all details concerning SNP assays, including allele type and nature of mutation, are reported.