GeneSpeed: protein domain organization of the transcriptome.

The GeneSpeed database (http://genespeed.uchsc.edu/) is an online database and resource tool facilitating the detailed study of protein domain homology in the transcriptomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans. The population schema for the GeneSpeed database takes advantage of HOWARD parallel cluster technology (http://www.massivelyparallel.com/) and performs exhaustive tBLASTn searches covering all pre-assigned PFAM domain classes in all species (currently 7973 domain families) against the respective Unigene EST databases of the selected four transcriptomes. The resulting database provides a complete annotation of presumed protein domain presence for each Unigene cluster. To complement this domain annotation we have also performed a custom transcription factor-family curation of all Pfam domains, incorporated the Gene Ontology classifications for these domains as well as integrated the Novartis SymAtlas2 dataset for both human and mouse which provides rapid and easy access to tissue-based expression analysis. Consequently, the GeneSpeed database provides the user with the capability to browse or search the database by any of these specialized criteria as well as more traditional means (gene identifier, gene symbol, etc.), thereby enabling a supervised analysis of gene families through a top-down hierarchical basis defined by domain content, all directly linked to an optimized gene expression dataset.

Effect of alginate encapsulation on the cellular transcriptome of human islets.

Encapsulation of human islets may prevent their immune rejection when transplanted into diabetic recipients. To assist in understanding why clinical outcomes with encapsulated islets were not ideal, we examined the effect of encapsulation on their global gene (mRNA) and selected miRNAs (non-coding (nc)RNA) expression. For functional studies, encapsulated islets were transplanted into peritoneal cavity of diabetic NOD-SCID mice. Genomics analysis and transplantation studies demonstrate that islet origin and isolation centres are a major source of variation in islet quality. In contrast, tissue culture and the encapsulation process had only a minimal effect, and did not affect islet viability. Microarray analysis showed that as few as 29 genes were up-regulated and 2 genes down-regulated (cut-off threshold 0.1) by encapsulation. Ingenuity analysis showed that up-regulated genes were involved mostly in inflammation, especially chemotaxis, and vascularisation. However, protein expression of these factors was not altered by encapsulation, raising doubts about the biosignificance of the gene changes. Encapsulation had no effect on levels of islet miRNAs. In vivo studies indicate differences among the centres in the quality of the islets isolated. We conclude that microencapsulation of human islets with barium alginate has little effect on their transcriptome.

Sox6 is necessary for efficient erythropoiesis in adult mice under physiological and anemia-induced stress conditions.

BACKGROUND: Definitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions. METHODOLOGY/PRINCIPAL FINDINGS: Sox6fl/flErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6fl/flCaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6fl/flErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1.

Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function.

Our objective was to establish an experimental model of a self-sustained and bistable extracellular signal-regulated kinase 1/2 (ERK1/2) signaling process. A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 4 h. Repeated stimulation leads to sustained activation of ERK1/2 but not Jun N-terminal protein kinase (JNK), p38, or STAT6. The ERK1/2 activation lasts for 3 to 7 days and depends upon a positive-feedback mechanism involving Sprouty 2. Overexpression of Sprouty 2 induces, and its genetic deletion abrogates, ERK1/2 bistability. Sprouty 2 directly activates Fyn kinase, which then induces ERK1/2 activation. A genome-wide microarray analysis shows that the bistable phospho-ERK1/2 (pERK1/2) does not induce a high level of gene transcription. This is due to its nuclear exclusion and compartmentalization to Rab5+ endosomes. Cells with sustained endosomal pERK1/2 manifest resistance against growth factor withdrawal-induced cell death. They are primed for heightened cytokine production. Epithelial cells from cases of human asthma and from a mouse model of chronic asthma manifest increased pERK1/2, which is associated with Rab5+ endosomes. The increase in pERK1/2 was associated with a simultaneous increase in Sprouty 2 expression in these tissues. Thus, we have developed a cellular model of sustained ERK1/2 activation, which may provide a mechanistic understanding of self-sustained biological processes in chronic illnesses such as asthma.

GeneSpeed Beta Cell: an online genomics data repository and analysis resource tailored for the islet cell biologist.

OBJECTIVE: We here describe the development of a freely available online database resource, GeneSpeed Beta Cell, which has been created for the pancreatic islet and pancreatic developmental biology investigator community. RESEARCH DESIGN AND METHODS: We have developed GeneSpeed Beta Cell as a separate component of the GeneSpeed database, providing a genomics-type data repository of pancreas and islet-relevant datasets interlinked with the domain-oriented GeneSpeed database. RESULTS: GeneSpeed Beta Cell allows the query of multiple published and unpublished select genomics datasets in a simultaneous fashion (multiexperiment viewing) and is capable of defining intersection results from precomputed analysis of such datasets (multidimensional querying). Combined with the protein-domain categorization/assembly toolbox provided by the GeneSpeed database, the user is able to define spatial expression constraints of select gene lists in a relatively rigid fashion within the pancreatic expression space. We provide several demonstration case studies of relevance to islet cell biology and development of the pancreas that provide novel insight into islet biology. CONCLUSIONS: The combination of an exhaustive domain-based compilation of the transcriptome with gene array data of interest to the islet biologist affords novel methods for multidimensional querying between individual datasets in a rapid fashion, presently not available elsewhere.