Christopher Dubay

Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii

ABSTRACT We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2β (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host “addiction” genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.

Using co-occurrence network structure to extract synonymous gene and protein names from MEDLINE abstracts

BackgroundText-mining can assist biomedical researchers in reducing information overload by extracting useful knowledge from large collections of text. We developed a novel text-mining method based on analyzing the network structure created by symbol co-occurrences as a way to extend the capabilities of knowledge extraction. The method was applied to the task of automatic gene and protein name synonym extraction.ResultsPerformance was measured on a test set consisting of about 50,000 abstracts from one year of MEDLINE. Synonyms retrieved from curated genomics databases were used as a gold standard. The system obtained a maximum F-score of 22.21% (23.18% precision and 21.36% recall), with high efficiency in the use of seed pairs.ConclusionThe method performs comparably with other studied methods, does not rely on sophisticated named-entity recognition, and requires little initial seed knowledge.

Applying task analysis to describe and facilitate bioinformatics tasks

OBJECTIVE To document bioinformatics tasks currently per-formed by researchers in genomics and proteomics in an effort to recognize unmet informatics needs and challenges, identify system features that would enhance the performance of those tasks, and inform the development of new bioinformatics tools. DESIGN A cross-sectional study of bioinformatics tasks performed by OHSU investigators involved in genomics and proteomics research was conducted using task analysis techniques. RESULTS Four major categories emerged from 22 bioinformatics tasks reported by 6 research laboratories. These were: 1) gene analysis, 2) protein analysis, 3) biostatistical analysis, and 4) literature searching. Analysis of the data also raised the following challenging issues: 1) lack of procedural documentation, 2) use of home-grown strategies to accomplish goals, 3) individual needs and preferences, and 4) lack of awareness of existing bioinformatics tools. CONCLUSION Task analysis was effective at documenting bioinformatics tasks performed by researchers in the fields of genomics and proteomics, at identifying potentially desirable system features and useful bioinformatics tools, and at providing a better understanding of some of the unmet needs and challenges faced by these researchers.

The Identification of Novel Ovarian Proteases Through the Use of Genomic and Bioinformatic Methodologies

Abstract Proteolytic activities are essential for follicular growth, ovulation, as well as for luteal formation and regression. Using suppression subtractive hybridization (SSH), a novel mouse ovary-selective gene (termed protease serine 35, Prss35) was identified. Analysis of the mouse genome database using the Prss35 sequence led to the identification of a homologous protease (protease serine 23, Prss23). PRSS35 possesses general features that are characteristic of serine (Ser) proteases, but is unique in that the canonical Ser that defines this enzyme family is replaced by a threonine (Thr). In contrast, PRSS23 possesses the standard catalytic Ser typical for this family of proteases. As determined by real-time polymerase chain reaction (PCR), the Prss35 mRNA levels increased around the time of ovulation and remained elevated in the developing corpus luteum. Steroid ablation/replacement studies demonstrated progesterone-dependent regulation of Prss35 gene expression prior to follicle rupture. Prss35 gene expression was localized to the theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory and ovulatory follicles, as well as to the developing corpus luteum. In contrast, Prss23 mRNA levels decreased transiently after ovulation induction and again in the postovulatory period. Prss23 gene expression was noted primarily in the granulosa cells of the secondary/early antral follicles. PRSS35 and PRSS23 orthologs in the rat, human, rhesus macaque, chimpanzee, cattle, dog, and chicken were identified and found to be highly homologous to one another (75–99% homology). Collectively, these results suggest that the PRSS35 and PRSS23 genes have been conserved as critical ovarian proteases throughout the course of vertebrate evolution.

The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980) - a taxonomic opinion

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437(T) and ATCC 68554 (=strain 775) was Vibrio ordalii LMG 13544(T), with more than 99.5% 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544(T), with 95% AAI, 98% MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77% similarity in AAI, 85% in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67% DNA-DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges-Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

BioQuery: an object framework for building queries to biomedical databases

SUMMARY BioQuery is an application that helps scientists automate database searches. Users can build and store queries to public biomedical databases, and receive periodic updates on the results of those queries when new data is available. The application is implemented on a portable object framework that can provide database-searching capability to other applications. This framework is easily extensible, allowing users to develop plug-ins that provide access to new databases. BioQuery thus provides end-users with a complete database searching interface and updating service, and gives developers a toolkit to provide database-searching capability to their applications. AVAILABILITY Free to all users: http://www.bioquery.org.

Utilizing quantitative immunohistochemistry for relationship analysis of tumor microenvironment of head and neck cancer patients

Meeting abstracts Analysis of tumor-infiltrating immune cells using quantitative immunohistochemistry (IHC) has proved to be a powerful prognostic biomarker in colon cancer [[1][1], [2][2]]. Similar observations have been made in patients with oral, head and neck squamous cell carcinoma (OHNSCC),

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of Cyclophosphamide with Allogenic DRibble Vaccine Alone (DPV-001) or with GM-CSF or Imiquimod for adjuvant treatment of Stage IIIA or IIIB NSCLC

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of cyclophosphamide with allogenic dribble vaccine alone (DPV-001) or with GM-CSF or imiquimod for adjuvant treatment of stage IIIa or IIIb NSCLC Rachel Sanborn, Brian Boulmay, Rui Li, Bradley Spieler, Kyle Happel, Christopher Paustian, Tarsem Mougdil, Zipei Feng, Christopher Dubay, Brenda Fisher, Yoshinobu Koguchi, Sandra Aung, Eileen Mederos, Carlo Bifulco, Michael McNamara, Keith S Bahjat, William Redmond, Augusto C Ochoa, Hong Ming Hu, Bernard Fox, Walter Urba, Traci Hilton

Developing an immunotherapy strategy for the effective treatment of patients with squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck (HNSCC) arises in the oral cavity, oropharynx, larynx or hypopharynx, and is the 6th leading cause of cancer by incidence worldwide. Approximately 600,000 cases arise each year, and only 40-50% of these patients will survive for 5 years.7 HNSCC can express PD-L1 and suppressive factors that interfere with the differentiation and activation of dendritic cells and effector function of T cells. Similar to observations in other malignancies, CD8 effector T cell infiltration or transcriptional signature consistent with an ongoing T cell response is associated with prolonged survival. This suggests that HNSCC lacking a T cell infiltrate may benefit from immune interventions that induce and/or augment a tumor-specific immune response. To develop a strategy to address this problem we have created a HNSCC tumor bank that is cryopreserving enzymatically isolated viable cells from resected tumors (39 specimens). We are also attempting to develop primary cell lines (4 lines established) and are isolating tumor-infiltrating lymphocytes from these specimens. We are looking to compare immunohistochemical, flowcytometric and functional analyses of these specimens. Consistent with previous studies, results document that isolated TIL secrete IFN-γ when stimulated with their autologous tumor cells (n=2). Since a central aim of these studies is to develop a strategy to create therapeutic immunity in patients lacking T cell infiltrates, we plan to investigate which antigens are recognized and whether, antigens present in a novel DRibble vaccine, that expresses antigens common to HNSCC, are recognized by these TIL. This may provide a strategy to monitor anti-tumor immunity in these patients and possibly provide an antigen source for combination immunotherapy of patients with HNSCC.

Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination

BackgroundOne of today’s greatest hurdles for cancer immunotherapy is the absence of information regarding which tumor antigens are already recognized by patients receiving immunotherapies, and whether those therapies then boost or generate an immune response against tumor proteins. For CD8+ T cells in particular, patient-specific immune recognition and responses at the level of individual tumor antigens are rarely characterized. Because of this, some immunologists have turned to serum antibodies as an alternative measure of antigen-specific anti-tumor immunity. In this work, we sought to simultaneously interrogate serum IgG and CD8+ T cell recognition of individual tumor antigens to determine whether antigen-specific serum IgG antibodies provide a window into the behavior of antigen-specific CD8+ T cell responses. Using antibody-based assays to evaluate immune response repertoires and focus T cell antigen exploration could afford substantial advantages for discovering and monitoring the anti-cancer immune responses of patients enrolled on clinical trials.MethodsWe vaccinated female BALB/c mice with a novel combination of an autophagosome-enriched vaccine derived from 4T1 mammary carcinoma along with poly-I:C adjuvant, then screened serum for IgG binding to arrays of 15mer peptides containing known mutation sites in 4T1. Simultaneously, we primed CD8+ T cell cultures from these same animals with 8-11mer peptides derived from these antigens. These primed T cells were then stimulated to measure recognition of the peptides or live 4T1 cells by IFNγ release.ResultsVaccinated animals demonstrate increases in antigen-specific CD8+ T cell recognition of 4T1 tumor cells and peptides. For proteins confirmed in 4T1 cells and vaccine by mass spectrometry, there is a correlation between this increased CD8+ T cell IFNγ release and serum IgG binding to individual peptide antigens.ConclusionsThese results suggest it is possible to observe some features of a patient’s antigen-specific T cell repertoire via an antibody surrogate, which has implications for tumor antigen discovery and clinical monitoring of antigen-specific anti-tumor immunity.