Decision tree supported substructure prediction of metabolites from GC-MS profiles

Gas chromatography coupled to mass spectrometry (GC-MS) is one of the most widespread routine technologies applied to the large scale screening and discovery of novel metabolic biomarkers. However, currently the majority of mass spectral tags (MSTs) remains unidentified due to the lack of authenticated pure reference substances required for compound identification by GC-MS. Here, we accessed the information on reference compounds stored in the Golm Metabolome Database (GMD) to apply supervised machine learning approaches to the classification and identification of unidentified MSTs without relying on library searches. Non-annotated MSTs with mass spectral and retention index (RI) information together with data of already identified metabolites and reference substances have been archived in the GMD. Structural feature extraction was applied to sub-divide the metabolite space contained in the GMD and to define the prediction target classes. Decision tree (DT)-based prediction of the most frequent substructures based on mass spectral features and RI information is demonstrated to result in highly sensitive and specific detections of sub-structures contained in the compounds. The underlying set of DTs can be inspected by the user and are made available for batch processing via SOAP (Simple Object Access Protocol)-based web services. The GMD mass spectral library with the integrated DTs is freely accessible for non-commercial use at http://gmd.mpimp-golm.mpg.de/. All matching and structure search functionalities are available as SOAP-based web services. A XML + HTTP interface, which follows Representational State Transfer (REST) principles, facilitates read-only access to data base entities

A pivotal role for starch in the reconfiguration of 14C-partitioning and allocation in Arabidopsis thaliana under short-term abiotic stress.

Plant carbon status is optimized for normal growth but is affected by abiotic stress. Here, we used 14C-labeling to provide the first holistic picture of carbon use changes during short-term osmotic, salinity, and cold stress in Arabidopsis thaliana. This could inform on the early mechanisms plants use to survive adverse environment, which is important for efficient agricultural production. We found that carbon allocation from source to sinks, and partitioning into major metabolite pools in the source leaf, sink leaves and roots showed both conserved and divergent responses to the stresses examined. Carbohydrates changed under all abiotic stresses applied; plants re-partitioned 14C to maintain sugar levels under stress, primarily by reducing 14C into the storage compounds in the source leaf, and decreasing 14C into the pools used for growth processes in the roots. Salinity and cold increased 14C-flux into protein, but as the stress progressed, protein degradation increased to produce amino acids, presumably for osmoprotection. Our work also emphasized that stress regulated the carbon channeled into starch, and its metabolic turnover. These stress-induced changes in starch metabolism and sugar export in the source were partly accompanied by transcriptional alteration in the T6P/SnRK1 regulatory pathway that are normally activated by carbon starvation

Phylogeographic Analysis Elucidates the Influence of the Ice Ages on the Disjunct Distribution of Relict Dragonflies in Asia

Unusual biogeographic patterns of closely related groups reflect events in the past, and molecular analyses can help to elucidate these events. While ample research on the origin of disjunct distributions of different organism groups in the Western Paleartic has been conducted, such studies are rare for Eastern Palearctic organisms. In this paper we present a phylogeographic analysis of the disjunct distribution pattern of the extant species of the strongly cool-adapted Epiophlebia dragonflies from Asia. We investigated sequences of the usually more conserved 18 S rDNA and 28 S rDNA genes and the more variable sequences of ITS1, ITS2 and CO2 of all three currently recognised Epiophlebia species and of a sample of other odonatan species. In all genes investigated the degrees of similarity between species of Epiophlebia are very high and resemble those otherwise found between different populations of the same species in Odonata. This indicates that substantial gene transfer between these populations occurred in the comparatively recent past. Our analyses imply a wide distribution of the ancestor of extant Epiophlebia in Southeast Asia during the last ice age, when suitable habitats were more common. During the following warming phase, its range contracted, resulting in the current disjunct distribution. Given the strong sensitivity of these species to climatic parameters, the current trend to increasing global temperatures will further reduce acceptable habitats and seriously threaten the existences of these last representatives of an ancient group of Odonata

The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis

Background:To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer.Methods:Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis.Results:Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer.Conclusion:Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression. © 2011 Cancer Research UK All rights reserved

Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphoma

peer-reviewedThe Epstein–Barr virus (EBV) is linked to various B-cell lymphomas, including Burkitt lymphoma (BL), classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) at frequencies ranging, by routine techniques, from 5 to 10% of cases in DLBCL to >95% in endemic BL. Using higher-sensitivity methods, we recently detected EBV traces in a few EBV-negative BL cases, possibly suggesting a “hit-and-run” mechanism. Here, we used routine and higher-sensitivity methods (qPCR and ddPCR for conserved EBV genomic regions and miRNAs on microdissected tumor cells; EBNA1 mRNA In situ detection by RNAscope) to assess EBV infection in a larger lymphoma cohort [19 BL, 34 DLBCL, 44 cHL, 50 follicular lymphomas (FL), 10 T-lymphoblastic lymphomas (T-LL), 20 hairy cell leukemias (HCL), 10 mantle cell lymphomas (MCL)], as well as in several lymphoma cell lines (9 cHL and 6 BL). qPCR, ddPCR, and RNAscope consistently documented the presence of multiple EBV nucleic acids in rare tumor cells of several cases EBV-negative by conventional methods that all belonged to lymphoma entities clearly related to EBV (BL, 6/9 cases; cHL, 16/32 cases; DLBCL, 11/30 cases), in contrast to fewer cases (3/47 cases) of FL (where the role of EBV is more elusive) and no cases (0/40) of control lymphomas unrelated to EBV (HCL, T-LL, MCL). Similarly, we revealed traces of EBV infection in 4/5 BL and 6/7 HL cell lines otherwise conventionally classified as EBV negative. Interestingly, additional EBV-positive cases (1 DLBCL, 2 cHL) relapsed as EBV-negative by routine methods while showing EBNA1 expression in rare tumor cells by RNAscope. The relapse specimens were clonally identical to their onset biopsies, indicating that the lymphoma clone can largely loose the EBV genome over time but traces of EBV infection are still detectable by high-sensitivity methods. We suggest EBV may contribute to lymphoma pathogenesis more widely than currently acknowledged

Nanoparticles for Local Drug Delivery to the Oral Mucosa: Proof of Principle Studies

Purpose To determine if solid lipid nanoparticles represent a viable strategy for local delivery of poorly water soluble and unstable chemopreventive compounds to human oral tissues. Methods Nanoparticle uptake and compound retention evaluations employed monolayer-cultured human oral squamous cell carcinoma (OSCC) cell lines and normal human oral mucosal explants. Feasibility of nanoparticle delivery was also evaluated with respect to the presence of phase-III efflux transporters in normal oral mucosal tissue and OSCC tissues. Results Functional uptake assays confirmed significantly greater internalization of nanoparticle-delivered fluorescent probe relative to free-fluorescent probe delivery, while concurrently demonstrating nanoparticle uptake rate differences among the OSCC cell lines and the phagocytic control human monocyte cell line. Mucosal explants exhibited nanoparticle penetration and internalization in the spinous and basal epithelial layer

Minimization and management of wastes from biomedical research.

Several committees were established by the National Association of Physicians for the Environment to investigate and report on various topics at the National Leadership Conference on Biomedical Research and the Environment held at the 1--2 November 1999 at the National Institutes of Health in Bethesda, Maryland. This is the report of the Committee on Minimization and Management of Wastes from Biomedical Research. Biomedical research facilities contribute a small fraction of the total amount of wastes generated in the United States, and the rate of generation appears to be decreasing. Significant reductions in generation of hazardous, radioactive, and mixed wastes have recently been reported, even at facilities with rapidly expanding research programs. Changes in the focus of research, improvements in laboratory techniques, and greater emphasis on waste minimization (volume and toxicity reduction) explain the declining trend in generation. The potential for uncontrolled releases of wastes from biomedical research facilities and adverse impacts on the general environment from these wastes appears to be low. Wastes are subject to numerous regulatory requirements and are contained and managed in a manner protective of the environment. Most biohazardous agents, chemicals, and radionuclides that find significant use in research are not likely to be persistent, bioaccumulative, or toxic if they are released. Today, the primary motivations for the ongoing efforts by facilities to improve minimization and management of wastes are regulatory compliance and avoidance of the high disposal costs and liabilities associated with generation of regulated wastes. The committee concluded that there was no evidence suggesting that the anticipated increases in biomedical research will significantly increase generation of hazardous wastes or have adverse impacts on the general environment. This conclusion assumes the positive, countervailing trends of enhanced pollution prevention efforts by facilities and reductions in waste generation resulting from improvements in research methods will continue

Functional Analysis: Evaluation of Response Intensities - Tailoring ANOVA for Lists of Expression Subsets

Background: Microarray data is frequently used to characterize the expression profile of a whole genome and to compare the characteristics of that genome under several conditions. Geneset analysis methods have been described previously to analyze the expression values of several genes related by known biological criteria (metabolic pathway, pathology signature, co-regulation by a common factor, etc.) at the same time and the cost of these methods allows for the use of more values to help discover the underlying biological mechanisms. Results: As several methods assume different null hypotheses, we propose to reformulate the main question that biologists seek to answer. To determine which genesets are associated with expression values that differ between two experiments, we focused on three ad hoc criteria: expression levels, the direction of individual gene expression changes (up or down regulation), and correlations between genes. We introduce the FAERI methodology, tailored from a two-way ANOVA to examine these criteria. The significance of the results was evaluated according to the self-contained null hypothesis, using label sampling or by inferring the null distribution from normally distributed random data. Evaluations performed on simulated data revealed that FAERI outperforms currently available methods for each type of set tested. We then applied the FAERI method to analyze three real-world datasets on hypoxia response. FAERI was able to detect more genesets than other methodologies, and the genesets selected were coherent with current knowledge of cellular response to hypoxia. Moreover, the genesets selected by FAERI were confirmed when the analysis was repeated on two additional related datasets. Conclusions: The expression values of genesets are associated with several biological effects. The underlying mathematical structure of the genesets allows for analysis of data from several genes at the same time. Focusing on expression levels, the direction of the expression changes, and correlations, we showed that two-step data reduction allowed us to significantly improve the performance of geneset analysis using a modified two-way ANOVA procedure, and to detect genesets that current methods fail to detect

Neurotransmitter Transporter-Like: A Male Germline-specific SLC6 Transporter Required for Drosophila Spermiogenesis

The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl), is expressed only in the male germline. Mobilization of a transposon inserted near the 3′ end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism

Positional Cues in the Drosophila Nerve Cord: Semaphorins Pattern the Dorso-Ventral Axis

Positional cues target sensory axons to appropriate volumes of the developing nervous system independently of their synaptic partners