Remembering Ralph Grassmann (1957 – 2008)

Friends and colleagues of Ralph Grassmann write their remembrances

MicroRNA miR-146a and further oncogenesis-related cellular microRNAs are dysregulated in HTLV-1-transformed T lymphocytes

<p>Abstract</p> <p>Background</p> <p>Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of a severe and fatal lymphoproliferative disease of mainly CD4⁺T cell origin, adult T cell leukemia, which develops after prolonged viral persistence. Transformation of infected cells involves HTLV-1's oncoprotein Tax, which perturbs cell cycle regulation and modulates cellular gene expression. The latter function is also a hallmark of microRNAs, a rather new layer in the regulation of gene expression. Affecting e.g. proliferation, microRNAs constitute a potential target for viral interference on the way to persistence and transformation. Hence, we explored the interconnections between HTLV-1 and cellular microRNAs.</p> <p>Results</p> <p>We report that several microRNAs – miRs 21, 24, 146a, 155 and 223 – are deregulated in HTLV-1-transformed cells. They are all upregulated except for miR-223, which is downregulated. Each of those microRNAs has ties to cancer. Their expression pattern forms a uniform phenotype among HTLV-transformed cells when compared to HTLV-negative control cells. In particular, miR-146a expression was found to be directly stimulated by Tax via NF-<it>κ</it>B-mediated transactivation of its promoter; a single NF-<it>κ</it>B site proximal to the transcription start point was necessary and sufficient for this to happen. An <it>in silico </it>analysis of potential target genes revealed candidates that might be coregulated by two or more of the aforementioned overexpressed microRNAs.</p> <p>Conclusion</p> <p>These data demonstrate that cellular microRNAs are deregulated in HTLV-1-transformed T cells. In the case of miR-146a, this could be directly attributed to HTLV's oncoprotein Tax. Interference with cellular microRNAs may be crucial to maintaining persistence or may facilitate transformation of host cells.</p

Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition

<p>Abstract</p> <p>Background</p> <p>Pathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult.</p> <p>Results</p> <p>A molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of <it>Enterobacteriaceae</it>, non-fermenter and clinical relevant <it>Candida </it>species. The observed detection limits varied from 10 cells (e.g. <it>E. coli</it>) to 10⁵cells (<it>S. aureus</it>) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241).</p> <p>Conclusion</p> <p>The newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios.</p

The UniProt-GO Annotation database in 2011

The GO annotation dataset provided by the UniProt Consortium (GOA: http://www.ebi.ac.uk/GOA) is a comprehensive set of evidenced-based associations between terms from the Gene Ontology resource and UniProtKB proteins. Currently supplying over 100 million annotations to 11 million proteins in more than 360 000 taxa, this resource has increased 2-fold over the last 2 years and has benefited from a wealth of checks to improve annotation correctness and consistency as well as now supplying a greater information content enabled by GO Consortium annotation format developments. Detailed, manual GO annotations obtained from the curation of peer-reviewed papers are directly contributed by all UniProt curators and supplemented with manual and electronic annotations from 36 model organism and domain-focused scientific resources. The inclusion of high-quality, automatic annotation predictions ensures the UniProt GO annotation dataset supplies functional information to a wide range of proteins, including those from poorly characterized, non-model organism species. UniProt GO annotations are freely available in a range of formats accessible by both file downloads and web-based views. In addition, the introduction of a new, normalized file format in 2010 has made for easier handling of the complete UniProt-GOA data se

An expanded evaluation of protein function prediction methods shows an improvement in accuracy

Background: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging. Results: We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2. Conclusions: The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent. Keywords: Protein function prediction, Disease gene prioritizationpublishedVersio

An Expanded Evaluation of Protein Function Prediction Methods Shows an Improvement In Accuracy

Background: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging. Results: We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2. Conclusions: The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent

Überlebensrelevante Funktionen HTLV-1-infizierter T-Zellen während der Leukämogenese

Human T-lymphotropic virus type 1 (HTLV-1), the cause of adult T cell leukemia, stimulates the growth of infected T cells in cultures and in non-leukemic patients. In the latter, HTLV-1 is found in long-term persisting T cell clones. The persistence of normal T cells is controlled by the growth-stimulating and antiapoptotic functions of costimulatory receptors, while the growth-stimulating HTLV-1 functions are mediated by the viral oncoprotein Tax. Among costimulatory receptors of the tumor necrosis factor receptor superfamily, 4-1BB (TNFRSF9/CD137/ILA) was strongly expressed in HTLV-transformed cells. Upregulated 4-1BB expression was a consistent feature of all HTLV-1-infected cell lines, whether patient-derived or in-vitro-transformed. Tax was sufficient to induce the expression of the endogenous 4-1BB gene in uninfected T cells and it strongly activated (45-fold) the 4-1BB promoter via a single NF-kappaB site. The ligand of 4-1BB was also found on transformed T cell lines opening the way for autostimulatory events. Moreover, 4-1BB expression in patients’ lymphocytes ex vivo correlated with Tax expression, strongly suggesting Tax-mediated 4-1BB activation in vivo. Simultaneous determination of 4-1BB expression and cell cycle phase revealed that HTLV-transformed cells exhibiting high 4-1BB expression appeared more often in a premitotic state, i.e., in G2 and M phase. Thus, 4-1BB upregulation by Tax could contribute to growth, survival and clonal expansion of the infected cells during persistence and disease. MicroRNAs are regulators of gene expression and, as such, also impinge upon growth and apoptosis of cells. Since HTLV-1 does not encode any microRNAs of its own, it has to reuse cellular microRNAs to access their regulatory potential. Based on phenotypic parallels to regulatory T cells and links to oncogenicity, seven cellular microRNAs were investigated in HTLV-transformed T cells. Four out of those, miRs 21, 24, 146a and 155, were significantly upregulated and one, miR-223, significantly downregulated. Subsequent testing for sensitivity of microRNA expression to viral proteins returned one – miR-146a – that was affected by Tax. The effect of Tax turned out to be transactivation of the promoter of MIRN146A gene via the NF-kappaB pathway. This constituted the first description of a direct influence of HTLV-1 on cellular microRNA expression. An in silico target gene analysis singled out genes involved in regulation of transcription and cell survival, which might be used by the virus to maintain persistence after infection.Das Humane T-lymphotrope Virus Typ 1 (HTLV-1) kann eine aggressive Neoplasie verursachen, die adulte T-Zell-Leukämie, und stimuliert sowohl in Zellkultur als auch in asymptomatisch Infizierten das Zellwachstum. In letzteren findet sich HTLV-1 in T-Zellklonen, die über lange Zeit persistieren. Das Überleben von T-Zellen wird unter anderem durch kostimulatorische Rezeptoren reguliert, die wachstumsstimulierende und antiapoptotische Funktionen ausüben. In HTLV-infizierten Zellen werden diese Funktionen vom Virus beeinflusst, hauptsächlich vermittelt durch dessen Onkoprotein Tax. Ein Vertreter der kostimulatorischen Rezeptoren aus der Superfamilie der Tumor-Nekrosefaktor- Rezeptoren, 4-1BB (TNFRSF9/CD137/ILA), war in allen betrachteten HTLV-transformierten Zelllinien stark exprimiert, unabhängig von deren Herkunft aus Patienten oder Immortalisierung in vitro. Tax reichte aus, die endogene Expression von 4-1BB in uninfizierten T-Zellen zu steigern, und es aktivierte den 4-1BB Promotor über eine einzelne NF-kappaB Bindestelle. Da auch der passende Ligand zum 4-1BB Rezeptor, 4-1BBL, auf den transformierten Zelllinien vorhanden war, besteht die Möglichkeit einer autostimulatorischen Schleife. Ex vivo Untersuchungen von Zellen, die aus HTLV-1-infizierten Patienten gewonnen worden waren, bestätigten die aus der Zellkultur gewonnenen Ergebnisse: die Tax-Expression korrelierte mit der von 4-1BB, was eine Stimulation des 4-1BB Gens durch Tax auch in vivo nahe legt. Eine parallele Analyse der Expression von 4-1BB in HTLV-transformierten Zellen und ihrer Zellzyklusphase zeigte ein vermehrtes Vorkommen von Zellen mit hohem 4-1BB Niveau in einem prämitotischen Zustand in der G2- bzw. M-Phase. Dies kann als Hinweis auf eine beschleunigte Proliferation der betreffenden Zellen gedeutet werden. Auf diese Weise könnte HTLV-1 Tax zum Wachstum, zum Überleben und zur klonalen Expansion infizierter Zellen während der Persistenz und HTLV-assoziierter Krankheit beitragen. MicroRNAs sind beteiligt an der Regulation von Genexpression und beeinflussen in dieser Funktion auch das Wachstum und die Apoptose von Zellen. Da HTLV-1 selbst keine microRNAs kodiert, muss das Virus das regulatorische Potential zellulärer microRNAs nutzen. Ausgehend von phänotypischen Gemeinsamkeiten zwischen HTLV-transformierten und regulatorischen T-Zellen sowie Überlegungen zur Onkogenität von microRNAs wurden sieben microRNAs in HTLV-transformierten Zellen untersucht. Vier der sieben – miRs 21, 24, 146a und 155 – waren signifikant überexprimiert und eine, miR-223, signifikant reprimiert. In einem HTLV-freien Zellsystem stieg das Niveau endogener microRNA miR-146a in Gegenwart von ektopisch exprimiertem Tax an. Nachfolgende Analysen des betreffenden MIRN146A Promotors ergaben eine Transaktivierung durch Tax über den NF-kappaB Signalweg. Dies stellt die erste Beschreibung eines direkten Einflusses von HTLV-1 auf die Expression zellulärer microRNAs dar und legt somit eine Funktion in der Transformation von T-Zellen durch HTLV-1 nahe. Die Anwendung bioinformatischer Methoden bei der Suche nach Genen, die möglicherweise durch die erwähnten, deregulierten microRNAs beeinflusst werden, erbrachte eine Liste von Zielgenen, die an der Regulierung von Zellproliferation und Apoptose beteiligt sind

Remembering Ralph Grassmann (1957 – 2008)

Abstract Friends and colleagues of Ralph Grassmann write their remembrances.</p