ICEPO: the ion channel electrophysiology ontology.

Ion channels are transmembrane proteins that selectively allow ions to flow across the plasma membrane and play key roles in diverse biological processes. A multitude of diseases, called channelopathies, such as epilepsies, muscle paralysis, pain syndromes, cardiac arrhythmias or hypoglycemia are due to ion channel mutations. A wide corpus of literature is available on ion channels, covering both their functions and their roles in disease. The research community needs to access this data in a user-friendly, yet systematic manner. However, extraction and integration of this increasing amount of data have been proven to be difficult because of the lack of a standardized vocabulary that describes the properties of ion channels at the molecular level. To address this, we have developed Ion Channel ElectroPhysiology Ontology (ICEPO), an ontology that allows one to annotate the electrophysiological parameters of the voltage-gated class of ion channels. This ontology is based on a three-state model of ion channel gating describing the three conformations/states that an ion channel can adopt: closed, open and inactivated. This ontology supports the capture of voltage-gated ion channel electrophysiological data from the literature in a structured manner and thus enables other applications such as querying and reasoning tools. Here, we present ICEPO (ICEPO ftp site:ftp://ftp.nextprot.org/pub/current_release/controlled_vocabularies/), as well as examples of its use.

Ultrastructure of trichocysts in Hematodinium spp. infecting Atlantic snow crab, Chionoecetes opilio.

Trichocyst morphology and development were explored using transmission electron microscopy in Hematodinium spp. isolated directly from Atlantic snow crab (Chionoecetes opilio) hemolymph and from in vitro cultures. Appearance of trichocysts defines the initiation of a morphological transition in the parasites life cycle from vegetative stage to the transmission stage. Trichocysts within sporonts were found in distinct clusters near the nucleus in close apposition to the Golgi. As cells transitioned to more mature dinospores however, trichocysts were found randomly distributed throughout the cytoplasm. Clusters contained both primordial and maturing trichocysts at various stages indicating an asynchronous development. The random distribution of mature trichocysts suggests deployment to the cell membrane for future extrusion. Mature trichocysts of Hematodinium spp. appeared structurally similar to trichocysts from photosynthetic dinoflagellates. Hematodinium spp. trichocysts differed by the presence of peripheral tubules associated with novel cuboidal appendages in the apical region rather than a network of central electron dense fibres as found in photosynthetic dinoflagellates. Additionally, the trichocyst membrane of Hematodinium spp. was in close apposition to the square crystalline core. Trichocyst expulsion was not observed during our study which along with features of development and maturation within Hematodinium life stages should provide insight into proposed roles in host attachment or defense that could further our understanding of the mechanisms of pathogenesis and transmission of the parasite.

Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Collaborative annotation of genes and proteins between UniProtKB/Swiss-Prot and dictyBase.

UniProtKB/Swiss-Prot, a curated protein database, and dictyBase, the Model Organism Database for Dictyostelium discoideum, have established a collaboration to improve data sharing. One of the major steps in this effort was the 'Dicty annotation marathon', a week-long exercise with 30 annotators aimed at achieving a major increase in the number of D. discoideum proteins represented in UniProtKB/Swiss-Prot. The marathon led to the annotation of over 1000 D. discoideum proteins in UniProtKB/Swiss-Prot. Concomitantly, there were a large number of updates in dictyBase concerning gene symbols, protein names and gene models. This exercise demonstrates how UniProtKB/Swiss-Prot can work in very close cooperation with model organism databases and how the annotation of proteins can be accelerated through those collaborations.

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

The Gene Ontology (GO) database and informatics resource.

The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.

RasG regulates discoidin gene expression during Dictyostelium growth.

Activated rasG, rasG(G12T), was expressed in Dictyostelium cells under the control of the folate-repressible discoidin promoter (pVEII-rasG(G12T)) and found to have a unique pattern of expression when cells were transferred to folate-deficient media: an initial increase of RasG(G12T) resulting from the removal of folate, followed by a rapid decline while cells were still in the early exponential phase of growth. Discoidin levels were considerably lower and declined more rapidly in the pVEII-rasG(G12T) transformant than they did in the wild type, suggesting that RasG(G12T) represses discoidin expression. This was independently confirmed by placing the rasG(G12T) gene under the control of the ribonucleotide reductase (rnrB) promoter. Exposure of cells to 10 mM methyl methanesulfonate (MMS) rapidly generated RasG(G12T) and this was accompanied by an equally rapid decrease in discoidin mRNA levels. rasG null cells also contained decreased levels of discoidin under all conditions tested, indicating that RasG is essential for optimum discoidin expression. However, rasG null cells showed normal regulation of discoidin expression in response to PSF, CMF, folate, bacteria, and axenic media, indicating that RasG is not necessary for any of these responses. These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter.

Biphasic expression of rnrB in Dictyostelium discoideum suggests a direct relationship between cell cycle control and cell differentiation.

Cell differentiation in Dictyostelium is strongly affected by the cell cycle. Cell cycle control is well-understood in other systems, but this has had almost no impact on the study of Dictyostelium cell differentiation, in part because the cell cycle in Dictyostelium is unusual, lacking a G1 phase. Here we describe the cell-cycle regulated expression of rnrB, which codes for the small subunit of ribonucleotide reductase and is a marker of late G1 in many systems. There appear to be two expression peaks, one in mid-G2 and the other near the G2/M transition. Using Xgal/anti-BrdU double staining, we show that cells in asynchronously growing cultures express in both phases, with a gap between them during which the gene is transcriptionally silent. Cold-synchronized cells show exclusively G2/M expression, while mid-G2 expression is seen in high-density synchronized cells and can also be inferred in cells undergoing synchronization by either method. rnrB expression occurs in other systems shortly after cells pass a point (the "restriction point" or "start") at which they commit to complete their current cell cycle. We demonstrate a similar commitment point in Dictyostelium and show that this occurs shortly before the mid-G2 rnrB expression peak. The Dictyostelium cell cycle thus appears to include a well-defined though inconspicuous event, between early and mid-G2, with some features which are normally associated with the G1/S transition. Others have described a switch from stalk to spore differentiation preference at about this time. Since Dictyostelium cells switch back from spore to stalk preference approximately at the G2/M rnrB expression maximum, cell differentiation as well as rnrB expression may be regulated directly by fundamental cell cycle control processes.

Inducible expression of exogenous genes in Dictyostelium discoideum using the ribonucleotide reductase promoter.

We report here the development of a regulated gene expression system for Dictyostelium discoideum based on the DNA-damage inducibility of the rnrB gene. rnrB, which codes for the small subunit of the enzyme ribonucleotide reductase, responds to DNA-damaging agents at all stages of the D.discoideum life cycle. Doses that have little effect on development have previously been shown to increase the level of the rnrB transcript by up to 15-fold. Here we show that all elements necessary for DNA-damage induction are contained in a 450 bp promoter fragment. We used a fusion of the rnrB promoter with the gene encoding GFP to demonstrate an up to 10-fold induction at the RNA level, which appears in all aspects similar to induction of the endogenous rnrB transcript. Using a fusion with the lacZ gene we observed an up to 7-fold induction at the protein level. These results indicate that the rnrB promoter can be used to regulate the expression of specific genes in D.discoideum. This controllable gene expression system provides the following new characteristics: the induction is rapid, taking place in the order of minutes, and the promoter is responsive at all stages of the D.discoideum life cycle.

Enhancement of survival of stored dopaminergic cells and promotion of graft survival by exposure of human fetal nigral tissue to glial cell line--derived neurotrophic factor in patients with Parkinson's disease. Report of two cases and technical considerations.

The authors have studied the ability of glial cell line-derived neurotrophic factor (GDNF) to promote survival of human fetal dopaminergic tissue after a storage period of 6 days and subsequent implantation into the human putamen. The results indicate that GDNF promotes survival of stored dopaminergic cells. Cells stored without GDNF had a 30.1% decrease in survival time compared with those exposed to GDNF. Two patients with Parkinson's disease received bilateral putaminal implants of fetal dopaminergic cells exposed to GDNF for 6 days and showed enhancement of graft survival as assessed by positron emission tomography scanning. A mean increase of 107% in putaminal fluorodopa uptake from baseline values was observed 12 months postgrafting.

Regulation of the ribonucleotide reductase small subunit gene by DNA-damaging agents in Dictyostelium discoideum.

In Escherichia coli, yeast and mammalian cells, the genes encoding ribonucleotide reductase, an essential enzyme for de novo DNA synthesis, are up-regulated in response to DNA damaging agents. We have examined the response of the rnrB gene, encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum, to DNA damaging agents. We show here that the accumulation of rnrB transcript is increased in response to methyl methane sulfonate, 4-nitroquinoline-1-oxide and irradiation with UV-light, but not to the ribonucleotide reductase inhibitor hydroxyurea. This response is rapid, transient and independent of protein synthesis. Moreover, cells from different developmental stages are able to respond to the drug in a similar fashion, regardless of the basal level of expression of the rnrB gene. We have defined the cis -acting elements of the rnrB promoter required for the response to methyl methane sulfonate and 4-nitroquinoline-1-oxide by deletion analysis. Our results indicate that there is one element, named box C, that can confer response to both drugs. Two other boxes, box A and box D, specifically conferred response to methyl methane sulfonate and 4-nitroquinoline-1-oxide, respectively.

Identification of cis-regulating elements and trans-acting factors regulating the expression of the gene encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum.

We have examined the promoter of rnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, using lacZ as a reporter gene. Deletion analysis showed that expression of this gene in vegetative cells involves an A/T-rich element, whereas its expression in prespore cells during development requires a region encompassing two G/C-rich elements, designated box A and box B. Removal of boxes A and B results in very low level of activity. When either box A or box B is deleted, prestalk cells adjacent to the prespore zone also express beta-galactosidase. The behavior of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression of rnrB is different from that regulating other known prespore genes. We have used electrophoretic mobility shift assays to identify factors that interact with box A and box B. Box A interacts with a factor that is found in the nuclear fraction. While box B interacts with a factor that is present in the cytosolic fraction throughout growth and development, its presence in the nuclear fraction is developmentally regulated. Results from competition assays suggest that both box A and box B interact with transcriptional activators that have not been characterized previously.

Pediatric tracheostomy and associated complications.

A retrospective analysis of 123 pediatric tracheostomies reveals an overall complication rate of 33%. Immediate complications were present in 12% or 15 patients. The most frequent immediate complications were pneumomediastinum and pneumothorax. Delayed complications occurred in 24% or 30 patients. The most frequent delayed complications were subglottic stenosis, fused vocal cords, and tracheal granuloma. Four patients died because of tracheostomy-related complications. Age, underlying disease, and prior endotracheal intubation had a high degree of correlation with complications. The use of a mechanical respirator following tracheostomy did not appear to be significantly related to complications. Fifty percent of the delayed complications in this series were regarded as being unrelated to the tracheostomy or the trachesotomy tube itself.

Tracheostomy decannulation.

Tracheostomy in pediatric patients involves major morbidity that is often reflected in prolonged decannulation difficulty. A review of 123 consecutive pediatric tracheostomies shows that 44 patients experienced such difficulties. Among those patients suffering decannulation delay, subglottic stenosis had developed in 39%, tracheal granuloma in 25%, fused cords in 11%, and temporary laryngeal abductor failure in 25%. Although the overall incidence of decannulation failure is high, more than 60% of those patients affected respond to treatment when diagnosis is prompt and accurate. In this regard, the laryngologist may play a central role in the management of the pediatric decannulation process.