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1.  The Bioinformatics Open Source Conference (BOSC) 2013 
Bioinformatics  2014;31(2):299-300.
Contact: nlharris@lbl.gov
doi:10.1093/bioinformatics/btu413
PMCID: PMC4287938  PMID: 25024288
3.  500,000 fish phenotypes: The new informatics landscape for evolutionary and developmental biology of the vertebrate skeleton 
The rich phenotypic diversity that characterizes the vertebrate skeleton results from evolutionary changes in regulation of genes that drive development. Although relatively little is known about the genes that underlie the skeletal variation among fish species, significant knowledge of genetics and development is available for zebrafish. Because developmental processes are highly conserved, this knowledge can be leveraged for understanding the evolution of skeletal diversity. We developed the Phenoscape Knowledgebase (KB; http://kb.phenoscape.org) to yield testable hypotheses of candidate genes involved in skeletal evolution. We developed a community anatomy ontology for fishes and ontology-based methods to represent complex free-text character descriptions of species in a computable format. With these tools, we populated the KB with comparative morphological data from the literature on over 2500 teleost fishes (mainly Ostariophysi) resulting in over 500,000 taxon phenotype annotations. The KB integrates these data with similarly structured phenotype data from zebrafish genes (http://zfin.org). Using ontology-based reasoning, candidate genes can be inferred for the phenotypes that vary across taxa, thereby uniting genetic and phenotypic data to formulate evo-devo hypotheses. The morphological data in the KB can be browsed, sorted, and aggregated in ways that provide unprecedented possibilities for data mining and discovery.
doi:10.1111/j.1439-0426.2012.01985.x
PMCID: PMC3377363  PMID: 22736877
4.  A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody 
PLoS ONE  2015;10(6):e0128381.
The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.
doi:10.1371/journal.pone.0128381
PMCID: PMC4451072  PMID: 26030924
5.  Toward Synthesizing Our Knowledge of Morphology: Using Ontologies and Machine Reasoning to Extract Presence/Absence Evolutionary Phenotypes across Studies 
Systematic Biology  2015;64(6):936-952.
The reality of larger and larger molecular databases and the need to integrate data scalably have presented a major challenge for the use of phenotypic data. Morphology is currently primarily described in discrete publications, entrenched in noncomputer readable text, and requires enormous investments of time and resources to integrate across large numbers of taxa and studies. Here we present a new methodology, using ontology-based reasoning systems working with the Phenoscape Knowledgebase (KB; kb.phenoscape.org), to automatically integrate large amounts of evolutionary character state descriptions into a synthetic character matrix of neomorphic (presence/absence) data. Using the KB, which includes more than 55 studies of sarcopterygian taxa, we generated a synthetic supermatrix of 639 variable characters scored for 1051 taxa, resulting in over 145,000 populated cells. Of these characters, over 76% were made variable through the addition of inferred presence/absence states derived by machine reasoning over the formal semantics of the source ontologies. Inferred data reduced the missing data in the variable character-subset from 98.5% to 78.2%. Machine reasoning also enables the isolation of conflicts in the data, that is, cells where both presence and absence are indicated; reports regarding conflicting data provenance can be generated automatically. Further, reasoning enables quantification and new visualizations of the data, here for example, allowing identification of character space that has been undersampled across the fin-to-limb transition. The approach and methods demonstrated here to compute synthetic presence/absence supermatrices are applicable to any taxonomic and phenotypic slice across the tree of life, providing the data are semantically annotated. Because such data can also be linked to model organism genetics through computational scoring of phenotypic similarity, they open a rich set of future research questions into phenotype-to-genome relationships.
doi:10.1093/sysbio/syv031
PMCID: PMC4604830  PMID: 26018570
character conflict; evolutionary mapping; inference; missing data; morphological character; ontology; phenotype; supermatrix
6.  Plasmodium vivax trophozoite-stage proteomes 
Journal of proteomics  2014;115:157-176.
Plasmodium vivax is the causative infectious agent of 80–300 million annual cases of malaria. Many aspects of this parasite’s biology remain unknown. To further elucidate the interaction of P. vivax with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax’s known reticulocyte host–cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host–parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function.
Biological significance
Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host–cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species’ known reticulocyte host–cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell’s parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress.
doi:10.1016/j.jprot.2014.12.010
PMCID: PMC4436379  PMID: 25545414
Plasmodium vivax; Proteomics; Malaria; Trophozoite stage; Infected red blood cell; Protein oxidation/nitration
7.  Moving the mountain: analysis of the effort required to transform comparative anatomy into computable anatomy 
The diverse phenotypes of living organisms have been described for centuries, and though they may be digitized, they are not readily available in a computable form. Using over 100 morphological studies, the Phenoscape project has demonstrated that by annotating characters with community ontology terms, links between novel species anatomy and the genes that may underlie them can be made. But given the enormity of the legacy literature, how can this largely unexploited wealth of descriptive data be rendered amenable to large-scale computation? To identify the bottlenecks, we quantified the time involved in the major aspects of phenotype curation as we annotated characters from the vertebrate phylogenetic systematics literature. This involves attaching fully computable logical expressions consisting of ontology terms to the descriptions in character-by-taxon matrices. The workflow consists of: (i) data preparation, (ii) phenotype annotation, (iii) ontology development and (iv) curation team discussions and software development feedback. Our results showed that the completion of this work required two person-years by a team of two post-docs, a lead data curator, and students. Manual data preparation required close to 13% of the effort. This part in particular could be reduced substantially with better community data practices, such as depositing fully populated matrices in public repositories. Phenotype annotation required ∼40% of the effort. We are working to make this more efficient with Natural Language Processing tools. Ontology development (40%), however, remains a highly manual task requiring domain (anatomical) expertise and use of specialized software. The large overhead required for data preparation and ontology development contributed to a low annotation rate of approximately two characters per hour, compared with 14 characters per hour when activity was restricted to character annotation. Unlocking the potential of the vast stores of morphological descriptions requires better tools for efficiently processing natural language, and better community practices towards a born-digital morphology.
Database URL: http://kb.phenoscape.org
doi:10.1093/database/bav040
PMCID: PMC4429748  PMID: 25972520
8.  Effects of Hfq on the conformation and compaction of DNA 
Nucleic Acids Research  2015;43(8):4332-4341.
Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq's interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here, we focus on DNA-compacting properties of Hfq. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is presumably related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex. Results are discussed in regard to previous results obtained for H-NS, with important implications for protein binding related gene regulation.
doi:10.1093/nar/gkv268
PMCID: PMC4417175  PMID: 25824948
9.  Plasmodium knowlesi gene expression differs in ex vivo compared to in vitro blood-stage cultures 
Malaria Journal  2015;14:110.
Background
Plasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death. While a zoonosis in humans, this simian malaria parasite species infects macaque monkeys and serves as an experimental model for in vivo, ex vivo and in vitro studies. It has underpinned malaria discoveries relating to host-pathogen interactions, the immune response and immune evasion strategies. This study investigated differences in P. knowlesi gene expression in samples from ex vivo and in vitro cultures.
Methods
Gene expression profiles were generated using microarrays to compare the stage-specific transcripts detected for a clone of P. knowlesi propagated in the blood of a rhesus macaque host and then grown in an ex-vivo culture, and the same clone adapted to long-term in vitro culture. Parasite samples covering one blood-stage cycle were analysed at four-hour intervals. cDNA was generated and hybridized to an oligoarray representing the P. knowlesi genome. Two replicate experiments were developed from in vitro cultures. Expression values were filtered, normalized, and analysed using R and Perl language and applied to a sine wave model to determine changes in equilibrium and amplitude. Differentially expressed genes from ex vivo and in vitro time points were detected using limma R/Bioconductor and gene set enrichment analysis (GSEA).
Results
Major differences were noted between the ex vivo and in vitro time courses in overall gene expression and the length of the cycle (25.5 hours ex vivo; 33.5 hours in vitro). GSEA of genes up-regulated ex vivo showed an enrichment of various genes including SICAvar, ribosomal- associated and histone acetylation pathway genes. In contrast, certain genes involved in metabolism and cell growth, such as porphobilinogen deaminase and tyrosine phosphatase, and one SICAvar gene, were significantly up-regulated in vitro.
Conclusions
This study demonstrates how gene expression in P. knowlesi blood-stage parasites can differ dramatically depending on whether the parasites are grown in vivo, with only one cycle of development ex vivo, or as an adapted isolate in long-term in vitro culture. These data bring emphasis to the importance of studying the parasite, its biology and disease manifestations in the context of the host.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-015-0612-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12936-015-0612-8
PMCID: PMC4369371  PMID: 25880967
Plasmodium knowlesi; Malaria; Non-human primates; Macaca mulatta; Blood-stage infections; Microarrays; Antigenic variation; Gene expression; SICAvar; Haem
10.  Finding Our Way through Phenotypes 
Deans, Andrew R. | Lewis, Suzanna E. | Huala, Eva | Anzaldo, Salvatore S. | Ashburner, Michael | Balhoff, James P. | Blackburn, David C. | Blake, Judith A. | Burleigh, J. Gordon | Chanet, Bruno | Cooper, Laurel D. | Courtot, Mélanie | Csösz, Sándor | Cui, Hong | Dahdul, Wasila | Das, Sandip | Dececchi, T. Alexander | Dettai, Agnes | Diogo, Rui | Druzinsky, Robert E. | Dumontier, Michel | Franz, Nico M. | Friedrich, Frank | Gkoutos, George V. | Haendel, Melissa | Harmon, Luke J. | Hayamizu, Terry F. | He, Yongqun | Hines, Heather M. | Ibrahim, Nizar | Jackson, Laura M. | Jaiswal, Pankaj | James-Zorn, Christina | Köhler, Sebastian | Lecointre, Guillaume | Lapp, Hilmar | Lawrence, Carolyn J. | Le Novère, Nicolas | Lundberg, John G. | Macklin, James | Mast, Austin R. | Midford, Peter E. | Mikó, István | Mungall, Christopher J. | Oellrich, Anika | Osumi-Sutherland, David | Parkinson, Helen | Ramírez, Martín J. | Richter, Stefan | Robinson, Peter N. | Ruttenberg, Alan | Schulz, Katja S. | Segerdell, Erik | Seltmann, Katja C. | Sharkey, Michael J. | Smith, Aaron D. | Smith, Barry | Specht, Chelsea D. | Squires, R. Burke | Thacker, Robert W. | Thessen, Anne | Fernandez-Triana, Jose | Vihinen, Mauno | Vize, Peter D. | Vogt, Lars | Wall, Christine E. | Walls, Ramona L. | Westerfeld, Monte | Wharton, Robert A. | Wirkner, Christian S. | Woolley, James B. | Yoder, Matthew J. | Zorn, Aaron M. | Mabee, Paula
PLoS Biology  2015;13(1):e1002033.
Imagine if we could compute across phenotype data as easily as genomic data; this article calls for efforts to realize this vision and discusses the potential benefits.
Despite a large and multifaceted effort to understand the vast landscape of phenotypic data, their current form inhibits productive data analysis. The lack of a community-wide, consensus-based, human- and machine-interpretable language for describing phenotypes and their genomic and environmental contexts is perhaps the most pressing scientific bottleneck to integration across many key fields in biology, including genomics, systems biology, development, medicine, evolution, ecology, and systematics. Here we survey the current phenomics landscape, including data resources and handling, and the progress that has been made to accurately capture relevant data descriptions for phenotypes. We present an example of the kind of integration across domains that computable phenotypes would enable, and we call upon the broader biology community, publishers, and relevant funding agencies to support efforts to surmount today's data barriers and facilitate analytical reproducibility.
doi:10.1371/journal.pbio.1002033
PMCID: PMC4285398  PMID: 25562316
11.  Interference of Aspergillus fumigatus with the immune response 
Seminars in Immunopathology  2014;37(2):141-152.
Aspergillus fumigatus is a saprotrophic filamentous fungus and also the most prevalent airborne fungal pathogen of humans. Depending on the host’s immune status, the variety of diseases caused by A. fumigatus ranges from allergies in immunocompetent hosts to life-threatening invasive infections in patients with impaired immunity. In contrast to the majority of other Aspergillus species, which are in most cases nonpathogenic, A. fumigatus features an armory of virulence determinants to establish an infection. For example, A. fumigatus is able to evade the human complement system by binding or degrading complement regulators. Furthermore, the fungus interferes with lung epithelial cells, alveolar macrophages, and neutrophil granulocytes to prevent killing by these immune cells. This chapter summarizes the different strategies of A. fumigatus to manipulate the immune response. We also discuss the potential impact of recent advances in immunoproteomics to improve diagnosis and therapy of an A. fumigatus infection.
doi:10.1007/s00281-014-0465-1
PMCID: PMC4326658  PMID: 25404120
Aspergillus fumigatus; Immune evasion; Phagocytes; Epithelial cells; Neutrophil extracellular traps (NETs); (immuno-) proteomics; Complement
12.  Annotation of phenotypic diversity: decoupling data curation and ontology curation using Phenex 
Background
Phenex (http://phenex.phenoscape.org/) is a desktop application for semantically annotating the phenotypic character matrix datasets common in evolutionary biology. Since its initial publication, we have added new features that address several major bottlenecks in the efficiency of the phenotype curation process: allowing curators during the data curation phase to provisionally request terms that are not yet available from a relevant ontology; supporting quality control against annotation guidelines to reduce later manual review and revision; and enabling the sharing of files for collaboration among curators.
Results
We decoupled data annotation from ontology development by creating an Ontology Request Broker (ORB) within Phenex. Curators can use the ORB to request a provisional term for use in data annotation; the provisional term can be automatically replaced with a permanent identifier once the term is added to an ontology. We added a set of annotation consistency checks to prevent common curation errors, reducing the need for later correction. We facilitated collaborative editing by improving the reliability of Phenex when used with online folder sharing services, via file change monitoring and continual autosave.
Conclusions
With the addition of these new features, and in particular the Ontology Request Broker, Phenex users have been able to focus more effectively on data annotation. Phenoscape curators using Phenex have reported a smoother annotation workflow, with much reduced interruptions from ontology maintenance and file management issues.
doi:10.1186/2041-1480-5-45
PMCID: PMC4236444  PMID: 25411634
Annotation; Phenotypes; Ontology; Curation; Systematics; Character matrix
13.  Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence 
Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of ΔfhpA, ΔfhpB, or ΔgnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.
doi:10.3389/fmicb.2014.00469
PMCID: PMC4160965  PMID: 25309516
Aspergillus fumigatus; reactive nitrogen intermediates; nitric oxide; virulence
14.  TLR-Mediated Inflammatory Responses to Streptococcus pneumoniae Are Highly Dependent on Surface Expression of Bacterial Lipoproteins 
Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host–pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB–regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2–associated responses were preserved. Experiments using leukocytes from IL-1R–associated kinase-4–deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R–associated kinase-4–mediated inflammatory responses to S. pneumoniae.
doi:10.4049/jimmunol.1401413
PMCID: PMC4170674  PMID: 25172490
15.  Secretion of Prohormone of B-Type Natriutretic Peptide, proBNP1–108, is Increased in Heart Failure 
JACC. Heart failure  2013;1(3):207-212.
OBJECTIVES
Using a novel, specific assay for proBNP1–108 we tested the hypotheses that proBNP1–108 is secreted by both non-failing and failing human hearts and that proBNP1–108 secretion is increased in failing hearts.
BACKGROUND
The prohormone of B-type natriuretic peptide (proBNP1–108) is a 108-amino acid peptide produced primarily by the heart and cleaved into biologically active BNP1–32 and the biologically inactive NT-proBNP1–76. It is unknown to what extent increased cardiac proBNP1–108 secretion as compared to reduced peripheral processing is responsible for elevated proBNP1–108 levels in patients with heart failure (HF) as compared to subjects without HF.
METHODS
The transcardiac gradient of proBNP1–108 was determined by collecting arterial blood and blood from the coronary sinus (CS). Samples from subjects without overt heart disease (n=9) were collected during cardiac catheterization after coronary artery disease had been excluded. Samples from HF patients (n=21) were collected during implantation of a biventricular pacemaker. ProBNP1–108 was measured with a new assay (BioRad). Values are median (25th/75th percentile).
RESULTS
The gradient of proBNP1–108 across the non-failing hearts was 8 (2/20) ng/L (aorta: 15 (1/25) ng/dL, CS: 24 (8/41) ng/dL; p=0.018). The transcardiac gradient of proBNP1–108 in the failing hearts was 326 (96/482) ng/dL (arterial: 381 (201/586) ng/dL, CS: 709 (408/1087) ng/dL; p<0.001). The transcardiac gradient was greater in failing than non-failing hearts (p=0.001).
CONCLUSION
ProBNP1–108 is secreted by non-failing and failing human hearts, but more so in the latter. It remains to be established where peripheral processing of proBNP1–108 occurs and how this is affected by disease.
doi:10.1016/j.jchf.2013.03.001
PMCID: PMC4120112  PMID: 24621871
proBNP1–108; heart failure; biomarker; natriuretic peptides
16.  No association found between the detection of either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus and chronic fatigue syndrome in a blinded, multi-site, prospective study by the establishment and use of the SolveCFS BioBank 
BMC Research Notes  2014;7:461.
Background
In 2009, a retrospective study reported the detection of xenotropic murine leukemia virus-related virus (XMRV) in clinical isolates derived from individuals with chronic fatigue syndrome or myalgic encephalomyelitis (CFS). While many efforts to confirm this observation failed, one report detected polytropic murine leukemia virus (pMLV), instead of XMRV. In both studies, Polymerase Chain Reaction (PCR)-based methods were employed which could provide the basis for the development of a practical diagnostic tool. To confirm these studies, we hypothesized that the ability to detect these viruses will not only depend upon the technical details of the methods employed but also on the criteria used to diagnose CFS and the availability of well characterized clinical isolates.
Methods
A repository of clinical isolates from geographically distinct sites was generated by the collection of fresh blood samples from well characterized CFS and healthy subjects. Molecular techniques were used to generate assay positive controls and to determine the lower limit of detection (LLOD) for murine retroviral and Intracisternal A particle (Cell 12(4):963-72, 1977) detection methods.
Results
We report the establishment of a repository of well-defined, clinical isolates from five, geographically distinct regions of the US, the comparative determination of the LLODs and validation efforts for the previously reported detection methods and the results of an effort to confirm the association of these retroviral signatures in isolates from individuals with CFS in a blinded, multi-site, prospective study. We detected various, murine retroviral DNA signatures but were unable to resolve a difference in the incidence of their detection between isolates from CFS (5/72; 6.7%) and healthy (2/37; 5.4%) subjects (Fisher’s Exact Test, p-value = 1). The observed sequences appeared to reflect the detection of endogenous murine retroviral DNA, which was not identical to either XMRV or pMLV.
Conclusions
We were unable to confirm a previously reported association between the detection of XMRV or pMLV sequences and CFS in a prospective, multi-site study. Murine retroviral sequences were detected at a low frequency that did not differ between CFS and control subjects. The nature of these sequences appeared to reflect the detection of pre-existing, endogenous, murine retroviral DNA in the PCR reagents employed.
doi:10.1186/1756-0500-7-461
PMCID: PMC4236736  PMID: 25092471
Chronic fatigue syndrome; Xenotropic murine virus-related virus; Polytropic murine leukemia virus; Retrovirus; PCR; Detection
17.  Tyrosine Phosphorylation of the orphan receptor ESDN/DCBLD2 serves as a scaffold for the signaling adaptor CrkL 
FEBS letters  2013;587(15):2313-2318.
A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor ESDN. Mutagenesis analysis of ESDN’s seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkL and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding.
doi:10.1016/j.febslet.2013.05.064
PMCID: PMC3759512  PMID: 23770091
18.  Viral Oncolysis — Can Insights from Measles Be Transferred to Canine Distemper Virus? 
Viruses  2014;6(6):2340-2375.
Neoplastic diseases represent one of the most common causes of death among humans and animals. Currently available and applied therapeutic options often remain insufficient and unsatisfactory, therefore new and innovative strategies and approaches are highly needed. Periodically, oncolytic viruses have been in the center of interest since the first anecdotal description of their potential usefulness as an anti-tumor treatment concept. Though first reports referred to an incidental measles virus infection causing tumor regression in a patient suffering from lymphoma several decades ago, no final treatment concept has been developed since then. However, numerous viruses, such as herpes-, adeno- and paramyxoviruses, have been investigated, characterized, and modified with the aim to generate a new anti-cancer treatment option. Among the different viruses, measles virus still represents a highly interesting candidate for such an approach. Numerous different tumors of humans including malignant lymphoma, lung and colorectal adenocarcinoma, mesothelioma, and ovarian cancer, have been studied in vitro and in vivo as potential targets. Moreover, several concepts using different virus preparations are now in clinical trials in humans and may proceed to a new treatment option. Surprisingly, only few studies have investigated viral oncolysis in veterinary medicine. The close relationship between measles virus (MV) and canine distemper virus (CDV), both are morbilliviruses, and the fact that numerous tumors in dogs exhibit similarities to their human counterpart, indicates that both the virus and species dog represent a highly interesting translational model for future research in viral oncolysis. Several recent studies support such an assumption. It is therefore the aim of the present communication to outline the mechanisms of morbillivirus-mediated oncolysis and to stimulate further research in this potentially expanding field of viral oncolysis in a highly suitable translational animal model for the benefit of humans and dogs.
doi:10.3390/v6062340
PMCID: PMC4074931  PMID: 24921409
canine distemper virus; measles virus; tumor treatment; viral oncolysis
19.  Erectile dysfunction as an initial presentation of diabetes discovered by taking sexual history 
BMJ Case Reports  2012;2012:bcr1220115289.
This case, as an important clinical reminder, will illustrate improvement of a patient’s quality of life and care in chronic diseases through sexual history taking in the primary care setting. The case report also includes recommended investigation for erectile dysfunction (ED). Family physicians need to maintain awareness of sexual dysfunction as part of the history taking during a general medical investigation to avoid leaving sexual issues untreated including ED. If left untreated, ED can lead to psychological trauma, frustration and lower self-esteem. Additionally, ED is associated with major comorbidities such as cardiovascular disease, hypertension, dyslipidaemia, psychological conditions and diabetes mellitus. Thus, appropriately identifying this medical condition may lead prompt diagnoses and treatment of other major diseases.
doi:10.1136/bcr.12.2011.5289
PMCID: PMC3351667  PMID: 22605862
20.  BioHackathon series in 2011 and 2012: penetration of ontology and linked data in life science domains 
Katayama, Toshiaki | Wilkinson, Mark D | Aoki-Kinoshita, Kiyoko F | Kawashima, Shuichi | Yamamoto, Yasunori | Yamaguchi, Atsuko | Okamoto, Shinobu | Kawano, Shin | Kim, Jin-Dong | Wang, Yue | Wu, Hongyan | Kano, Yoshinobu | Ono, Hiromasa | Bono, Hidemasa | Kocbek, Simon | Aerts, Jan | Akune, Yukie | Antezana, Erick | Arakawa, Kazuharu | Aranda, Bruno | Baran, Joachim | Bolleman, Jerven | Bonnal, Raoul JP | Buttigieg, Pier Luigi | Campbell, Matthew P | Chen, Yi-an | Chiba, Hirokazu | Cock, Peter JA | Cohen, K Bretonnel | Constantin, Alexandru | Duck, Geraint | Dumontier, Michel | Fujisawa, Takatomo | Fujiwara, Toyofumi | Goto, Naohisa | Hoehndorf, Robert | Igarashi, Yoshinobu | Itaya, Hidetoshi | Ito, Maori | Iwasaki, Wataru | Kalaš, Matúš | Katoda, Takeo | Kim, Taehong | Kokubu, Anna | Komiyama, Yusuke | Kotera, Masaaki | Laibe, Camille | Lapp, Hilmar | Lütteke, Thomas | Marshall, M Scott | Mori, Takaaki | Mori, Hiroshi | Morita, Mizuki | Murakami, Katsuhiko | Nakao, Mitsuteru | Narimatsu, Hisashi | Nishide, Hiroyo | Nishimura, Yosuke | Nystrom-Persson, Johan | Ogishima, Soichi | Okamura, Yasunobu | Okuda, Shujiro | Oshita, Kazuki | Packer, Nicki H | Prins, Pjotr | Ranzinger, Rene | Rocca-Serra, Philippe | Sansone, Susanna | Sawaki, Hiromichi | Shin, Sung-Ho | Splendiani, Andrea | Strozzi, Francesco | Tadaka, Shu | Toukach, Philip | Uchiyama, Ikuo | Umezaki, Masahito | Vos, Rutger | Whetzel, Patricia L | Yamada, Issaku | Yamasaki, Chisato | Yamashita, Riu | York, William S | Zmasek, Christian M | Kawamoto, Shoko | Takagi, Toshihisa
The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed.
doi:10.1186/2041-1480-5-5
PMCID: PMC3978116  PMID: 24495517
BioHackathon; Bioinformatics; Semantic Web; Web services; Ontology; Visualization; Knowledge representation; Databases; Semantic interoperability; Data models; Data sharing; Data integration
21.  Comparative transcriptomics and metabolomics in a rhesus macaque drug administration study 
We describe a multi-omic approach to understanding the effects that the anti-malarial drug pyrimethamine has on immune physiology in rhesus macaques (Macaca mulatta). Whole blood and bone marrow (BM) RNA-Seq and plasma metabolome profiles (each with over 15,000 features) have been generated for five naïve individuals at up to seven timepoints before, during and after three rounds of drug administration. Linear modeling and Bayesian network analyses are both considered, alongside investigations of the impact of statistical modeling strategies on biological inference. Individual macaques were found to be a major source of variance for both omic data types, and factoring individuals into subsequent modeling increases power to detect temporal effects. A major component of the whole blood transcriptome follows the BM with a time-delay, while other components of variation are unique to each compartment. We demonstrate that pyrimethamine administration does impact both compartments throughout the experiment, but very limited perturbation of transcript or metabolite abundance was observed following each round of drug exposure. New insights into the mode of action of the drug are presented in the context of pyrimethamine's predicted effect on suppression of cell division and metabolism in the immune system.
doi:10.3389/fcell.2014.00054
PMCID: PMC4233942  PMID: 25453034
pyrimethamine; bone marrow; peripheral blood; axes of variation; bayesian network inference; principal component analysis (PCA)
22.  The vertebrate taxonomy ontology: a framework for reasoning across model organism and species phenotypes 
Background
A hierarchical taxonomy of organisms is a prerequisite for semantic integration of biodiversity data. Ideally, there would be a single, expansive, authoritative taxonomy that includes extinct and extant taxa, information on synonyms and common names, and monophyletic supraspecific taxa that reflect our current understanding of phylogenetic relationships.
Description
As a step towards development of such a resource, and to enable large-scale integration of phenotypic data across vertebrates, we created the Vertebrate Taxonomy Ontology (VTO), a semantically defined taxonomic resource derived from the integration of existing taxonomic compilations, and freely distributed under a Creative Commons Zero (CC0) public domain waiver. The VTO includes both extant and extinct vertebrates and currently contains 106,947 taxonomic terms, 22 taxonomic ranks, 104,736 synonyms, and 162,400 cross-references to other taxonomic resources. Key challenges in constructing the VTO included (1) extracting and merging names, synonyms, and identifiers from heterogeneous sources; (2) structuring hierarchies of terms based on evolutionary relationships and the principle of monophyly; and (3) automating this process as much as possible to accommodate updates in source taxonomies.
Conclusions
The VTO is the primary source of taxonomic information used by the Phenoscape Knowledgebase (http://phenoscape.org/), which integrates genetic and evolutionary phenotype data across both model and non-model vertebrates. The VTO is useful for inferring phenotypic changes on the vertebrate tree of life, which enables queries for candidate genes for various episodes in vertebrate evolution.
doi:10.1186/2041-1480-4-34
PMCID: PMC4177199  PMID: 24267744
Data integration; Evolutionary biology; Paleontology; Taxonomic rank
23.  Modulation of cGMP in Heart Failure 
Handbook of experimental pharmacology  2009;10.1007/978-3-540-68964-5_21.
Heart failure (HF) is a common disease that continues to be associated with high morbidity and mortality warranting novel therapeutic strategies. Cyclic guanosine monophosphate (cGMP) is the second messenger of several important signaling pathways based on distinct guanylate cyclases (GCs) in the cardiovascular system. Both the nitric oxide/soluble GC (NO/sGC) as well as the natriuretic peptide/GC-A (NP/GC-A) systems are disordered in HF, providing a rationale for their therapeutic augmentation. Soluble GC activation with conventional nitrovasodilators has been used for more than a century but is associated with cGMP-independent actions and the development of tolerance, actions which novel NO-independent sGC activators now in clinical development lack. Activation of GC-A by administration of naturally occurring or designer natriuretic peptides is an emerging field, as is the inhibition of enzymes that degrade endogenous NPs. Finally, inhibition of cGMP-degrading phosphodiesterases, particularly phosphodiesterase 5 provides an additional strategy to augment cGMP-signaling.
doi:10.1007/978-3-540-68964-5_21
PMCID: PMC3835600  PMID: 19089342
24.  Spleen-Dependent Regulation of Antigenic Variation in Malaria Parasites: Plasmodium knowlesi SICAvar Expression Profiles in Splenic and Asplenic Hosts 
PLoS ONE  2013;8(10):e78014.
Background
Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera.
Principal Findings
We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry.
Significance
SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment.
doi:10.1371/journal.pone.0078014
PMCID: PMC3799730  PMID: 24205067
25.  Minor Papilla Adenoma Management in Patients with Pancreas Divisum and Familial Adenomatous Polyposis 
ACG Case Reports Journal  2013;1(1):47-50.
Several case reports on endoscopic resection of minor papilla adenomas exist in the literature. However, there are no reported cases of endoscopic resection in patients with minor papilla adenomas with associated familial adenomatous polyposis (FAP) and pancreas divisum. We report a case of a minor papilla adenoma in a patient with FAP and pancreas divisum. The case demonstrates a new association between these disease processes. Defining pancreatic ductal anatomy prior to endoscopic intervention is essential. In addition, we demonstrate the safety and feasibility of endoscopic management of minor papilla tumors in patients with FAP and associated pancreas divisum.
doi:10.14309/crj.2013.17
PMCID: PMC4435256  PMID: 26157819

Results 1-25 (87)