Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Background Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the β-hemoglobinopathies to reduce the risk to the fetus. Method Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with β-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. Results Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The β-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. Conclusion This probe capture NGS assay demonstrates the potential of NIPT for β-hemoglobinopathies.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses.

Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings.

To elucidate the little-known bioenergetic pathways of host immune cells in tuberculosis, a granulomatous disease caused by the intracellular pathogen Mycobacterium tuberculosis , we characterized infected murine lung tissue by transcriptomic profiling and confocal imaging. Transcriptomic analysis revealed changes of host energy metabolism during the course of infection that are characterized by upregulation of key glycolytic enzymes and transporters for glucose uptake and downregulation of enzymes participating in the tricarboxylic acid cycle and oxidative phosphorylation. Consistent with elevated glycolysis, we also observed upregulation of a transporter for lactate secretion and a V type H + -ATPase involved in cytosolic pH homeostasis. Transcription profiling results were corroborated by immunofluorescence microscopy showing increased expression of key glycolytic enzymes in macrophages and T cells in granulomatous lesions. Moreover, we found increased mRNA and protein levels in macrophages and T cells of hypoxia inducible factor 1 alpha (HIF-1α), the regulatory subunit of HIF-1, a master transcriptional regulator. Thus, our findings suggest that immune cells predominantly utilize aerobic glycolysis in response to M. tuberculosis infection. This bioenergetic shift is similar to the Warburg effect, the metabolic signature of cancer cells. Finding immunometabolic changes during M. tuberculosis infection opens the way to new strategies for immunotherapy against tuberculosis.

A major hurdle facing tuberculosis (TB) investigators who want to utilize a rapidly growing body of data from both systems biology approaches and omics technologies is the lack of a standard vocabulary for data annotation and reporting. Lacking a means to readily compare samples from different research groups, a significant quantity of potentially informative data is largely ignored by researchers. To facilitate standardizing data across studies, a simple ontology of TB terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address animal models and experimental systems, and existing clinically focused terminology was modified and adapted. This ontology can be used to annotate host TB data in public databases and collaborations, thereby standardizing database searches and allowing researchers to more easily compare results. To demonstrate the utility of a standard TB ontology for host systems biology, a web application was developed to annotate and compare human and animal model gene expression data sets.

Vitamin D has long been linked to resistance to tuberculosis, an infectious respiratory disease that is increasingly hard to treat because of multidrug resistance. Previous work established that vitamin D induces macrophage antimicrobial functions against Mycobacterium tuberculosis. In this article, we report a novel, metabolic role for vitamin D in tuberculosis identified through integrated transcriptome and mechanistic studies. Transcriptome analysis revealed an association between vitamin D receptor (VDR) and lipid metabolism in human tuberculosis and infected macrophages. Vitamin D treatment of infected macrophages abrogated infection-induced accumulation of lipid droplets, which are required for intracellular M. tuberculosis growth. Additional transcriptomics results showed that vitamin D downregulates the proadipogenic peroxisome proliferator–activated receptor γ (PPARγ) in infected macrophages. PPARγ agonists reversed the antiadipogenic and the antimicrobial effects of VDR, indicating a link between VDR and PPARγ signaling in regulating both vitamin D functions. These findings suggest the potential for host-based, adjunct antituberculosis therapy targeting lipid metabolism.

Analyses of highly polymorphic HLA and KIR locus data require specialized tools and methods, but most modern analytical programs are tailored for SNPs. A typical analytical workflow for HLA and KIR data requires trafficking data between several programs, which is time intensive, error prone and limits reproducibility. The IDAWG has been developing an integrated data-management and analysis system tailored to these important genetic systems. We have developed the Toolkit for Immunogenomic Data Exchange and Storage (TIDES) and Bridging ImmunoGenomic Data-Analysis Workflow Gaps (BIGDAWG) systems to address the unmet need for consistent HLA and KIR data management and analysis. TIDES is a free, open-source system that converts HLA and KIR genotypes derived from widely used genotyping platforms into Genotype List (GL) Strings, which are registered with the NMDP’s GL Service (gl.immunogenomics.org). BIGDAWG is an automated pipeline, scripted in R, that performs common data analyses of multi-locus highly polymorphic genetic data characteristic of HLA and KIR genes. TIDES stores GL String-encoded data with project-related data, and exports these to PED and POP data-analysis formats. TIDES can be deployed on an AWS EC2 or equivalent Linux environment, on a local network or on a standalone machine, per user preference. A prototype TIDES implementation is deployed at tides.immunogenomics.org. Starting with unambiguous multi-locus genotype data for case-control groups, BIGDAWG estimates user-specified haplotypes, bins low-frequency haplotypes to enable chi-squared testing, calculates odds-ratios, confidence intervals and p-values for each haplotype, and generates figures and tables for each comparison. The integration of TIDES and BIGDAWG will create a workflow that accepts ambiguous genotype data and returns case-control analysis results. This approach streamlines data analysis and allows the consistent storage and exchange of HLA and KIR genotype data.

Background CpG-containing oligodeoxynucleotides (CpG-ODNs) are potent inhibitors of T helper 2 mediated allergic airway disease in sensitised mice challenged with allergen. A single treatment has transient effects but a limited series of treatments has potential to achieve clinically meaningful sustained inhibition of allergic airway disease. Objective To optimise the treatment regimen for sustained efficacy and to determine the mechanisms of action in mice of an inhaled form of CpG-ODN being developed for human asthma treatment. Methods We set up a chronic allergic-asthma model using ragweed-sensitised mice exposed weekly to intranasal ragweed. Using this model, the effects of a limited series of weekly intranasal 1018 ISS (CpG-ODN; B-class) treatments were evaluated during treatment and for several weeks after treatments had stopped but weekly allergen exposures continued. Treatment efficacy was evaluated by measuring effects on lung T helper 2 cytokines and eosinophilia, and lung dendritic cell function and T-cell responses. Results Twelve intranasal 1018 ISS treatments induced significant suppression of bronchoalveolar lavage eosinophilia and interleukin 4, 5 and 13 levels. This suppression of allergic T helper 2 parameters was maintained through 13 weekly ragweed exposures administered after treatment cessation. Subsequent experiments demonstrated that at least five treatments were required for lasting suppression. Although CpG-ODN induced moderate T helper 1 responses, suppression of allergic airway disease did not require interferon γ but was associated with induction of a regulatory T-cell response. Conclusions A short series of CpG-ODN treatments results in sustained suppression of allergic lung inflammation induced by a clinically relevant allergen.

Global and translational science NIAID TB research program Priorities, funding and collaborations in global TB research Christine F. Sizemore, Ph.D.1 1 Chief, TB, Leprosy and Other Mycobacterial Diseases Section. 1472-9792/$ – see front matter doi:10.1016/j.tube.2012.05.007 US Department of Health and Human Services (DHHS) National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Division of Microbiology and Infectious Diseases (DMID) Bethesda, Maryland, USA The National Institute of Allergy and Infectious Diseases (NIAID), a component of the National Institutes of Health (NIH), is a major global funder of biomedical research in tuberculosis, a recognized global health issue. NIAID’s goal in supporting this research is to enhance understanding of TB as an infectious disease and facilitate transition of fundamental knowledge into new tools and approaches to combat both drug-sensitive and drug-resistant TB in all relevant patient populations. NIAID also offers support and provides resources for the discovery, advancement and clinical evaluation of novel vaccines, drugs, and diagnostics. This presentation will provide an overview of the NIAID extramural TB program and its interactionwith domestic and global partners, and will summarize areas of particular emphasis for TB research, funding opportunities and available resources to assist researchers in advancing biomedical and clinical science in this area. For more information: NIAID Research on Tuberculosis: www.niaid.nih.gov/topics/tuberculosis/Pages/Default.aspx NIAID Research in Global Health: http://www.niaid.nih.gov/topics/globalresearch/Pages/default. aspx NIAID Global Research: Improving Health in a Changing World: http://www.niaid.nih.gov/topics/globalResearch/Documents/ niaidglobal508.pdf NIAID Research Agenda for Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis: www.niaid.nih.gov/topics/tuberculosis/research/documents/ mdrxdrresearchagenda.pdf NIAID Funding Opportunities: http://www.niaid.nih.gov/researchfunding/ann/Pages/opps.aspx NIAID Research Resources www.niaid.nih.gov/labsandresources/resources/Pages/default. aspx New drugs for TB treatment C. Robert Horsburgh1 1 Boston University School of Public Health, Boston, MA, USA No new classes of antituberculosis agents were brought to market between 1970 and 2000. However, in 2000 the Declaration of Cape Town began a movement that has resulted in clinical trials Abstracts / Tuberculosis 93 (2013) 108–114 109s / Tuberculosis 93 (2013) 108–114 109 of six new anti-TB drugs, representing four new antibiotic classes. In addition, renewed efforts to shorten treatment and of TB infection and disease using existing agents have resulted in a large number of clinical studies. Phase 2 EBA studies to establish dosing have been or soon will be completed for PA-824, OPC-67683, AZD5847, SQ-109, and PNU-100480. Phase 2b trials of TMC-207 and OPC-67683 in patients with MDR-TB have demonstrated efficacy in this important population, and 3 phase 3 studies in MDR-TB have been initiated (STREAM, TMC-207 and OPC-67683). Three phase 3 treatment-shortening trials of drug-susceptible TB are also nearly complete (OFLOTUB, ReMox and RIFAQUIN). A threemonth, once weekly regimen of INH and Rifapentine for LTBI has been shown to be as effective as 9 months of INH. Regimens including these new agents hold the promise of shorter and more effective regimens for both drug-susceptible and drug-resistant infection and disease. Such regimens would greatly expand the capacity of global health programs to deliver TB treatment. As additional new drugs become available, regimen design will need to be refined to arrive at the simplest and most effective regimens. However, a major challenge will be preventing the emergence of resistance to the new agents. Inoculum-dependent differences in tuberculin skin test (TST) and interferong release assay (IGRA) responses. Edward C. Jones-López1,2,6, Olive Namugga2, Francis Mumbowa3, Martin Ssebidandi2, Olive Mbabazi4, Stephanie Moine1, Gerald Mboowa3, Matthew P. Fox5, Nancy Reilly6, Irene Ayakaka2, Soyeon Kim7, Alphonse Okwera8,2, Moses Joloba3, Kevin P. Fennelly9,2. 1 Section of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts, USA 2 Makerere University – University of Medicine and Dentistry of New Jersey (UMDNJ) Research Collaboration, Kampala, Uganda 3 Department of Microbiology, Makerere University College of Health Sciences, Kampala, Uganda 4 Infectious Diseases Institute, Makerere University College of Health Sciences, Kampala, Uganda 5 Department of Epidemiology, Center for Global Health and Development, Boston University, Boston, Massachusetts, USA 6 Department of Medicine, New Jersey Medical School – UMDNJ, Newark, New Jersey, USA 7 Department of Preventive Medicine and Community Health, New Jersey Medical School – UMDNJ, Newark, New Jersey, USA 8 Mulago Hospital Tuberculosis Clinic, Mulago Hospital, Kampala, Uganda 9 Southeastern National Tuberculosis Center and Emerging Pathogens Institute, Division of Global Medicine, Department of Medicine, University of Florida, Gainesville, Florida, USA Despite established evidence that Mycobacterium tuberculosis is transmitted via fine aerosols, the sputum acid-fast bacilli (AFB) smear has defined tuberculosis (TB) infectiousness for decades. This prolonged reliance on sputum studies to measure TB transmission has distracted from the importance viable M. tuberculosis aerosols may play in modulating human immunopathology following an infectious exposure. We have developed a method to culture and quantify cough-generated aerosols from patients with pulmonary TB. We performed a prospective household contact study in Kampala, Uganda to measure TST and IGRA responses in household contacts exposed to TB patients producing no aerosols, low aerosols (1-9 colony forming units of M. tuberculosis in aerosols) or high aerosols ( 10 CFU). From May 2009 to January 2011, we enrolled 96 sputum culturepositive index TB cases and their 442 contacts. Compared to contacts of negative aerosol and low aerosol patients, contacts of high aerosol TB cases had: 1) a higher rate of both prevalent (baseline) and incident (six weeks) latent TB infection by either TST or IGRA criteria; 2) a larger TST induration size and higher interferon-g levels, and; 3) larger proportion with concordant TST/IGRA results. We propose that, compared to sputum AFB microscopy, cough aerosols are a new and improved quantitative surrogate for inhaled dose. In future studies, cough aerosols may fulfill an important role as an exposure biomarker to predict infection and, possibly, progression to TB disease in contacts of pulmonary TB patients. Post-initiation regulation of mRNA biogenesis in cells infected with M. tuberculosis and in active tuberculosis Hugh Salamon1, Yaming Qiao2, Jeff C. Cheng2,, Ken D. Yamguchi1, Patricia Soteropoulos2, Michael Weiden3, Maria Laura Gennaro2, and Richard Pine2 1 Knowledge Synthesis, Inc., Berkeley, CA, USA 2 Public Health Research Institute, New Jersey Medical School, UMDNJ, Newark, NJ, USA 3 New York University Medical Center, New York, NY, USA M. tuberculosis infection alters macrophage gene expression and macrophage response to interferon gamma (IFNg), a critical host defense cytokine. Understanding the regulation of changes in host gene expression is likely a prerequisite to developing any effective immunomodulatory therapy. Toward this end, we used an in vitro macrophage infection model to investigate the expression of the transcription factors STAT1 and IRF1, which mediate cellular response to IFNg and host defense against M. tuberculosis, and we analyzed transcriptome data for the model and for clinical studies. In differentiated THP-1 cells infected with M. tuberculosis and stimulated with IFNg, we found evidence for negative postinitiation regulation of mRNA biogenesis based on changes in the abundance of nascent transcripts, total nuclear RNA, nuclear polyAþ RNA and total cellular poly-Aþ RNA, together with lack of change in the half-life of STAT1 and IRF1 transcripts in total nuclear RNA and total poly-Aþ RNA. In contrast, no such regulation appeared to occur upon infection with M. bovis BCG. Consistent with the molecular biology of expression for STAT1 and IRF1, analysis of transcriptome data for the in vitro model and for two clinical studies comparing peripheral blood mononuclear cells demonstrated that expression of post-initiation pathway genes differed significantly with infection in vitro and between active tuberculosis and latent infection. Moreover, most significantly regulated genes associated with those annotations were repressed by infection / active disease. Collectively these results demonstrate a novel means by which M. tuberculosis may curb the immune response, not least by limiting the induction of STAT1 and IRF1, and perhaps other genes. Nitric oxide controls inflammatory pathology in tuberculosis Bibhuti B. Mishra1, Vijay A. K. Rathinam3, Gregory W. Martens2, Hardy Kornfeld2, Katherine A. Fitzgerald3, and Christopher M. Sassetti1, 4 1 Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA 2 Division of Pulmonary, Allergy and Critical Care, Department of Medicine, University of Massachusetts Medical School, Worcester,

Mycobacterium tuberculosis infection alters macrophage gene expression and macrophage response to IFN-γ, a critical host defense cytokine. However, regulation of these changes is poorly understood. We report discordance of changes in nascent transcript and total nuclear RNA abundance for the transcription factors STAT1 and IRF1, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN-γ. The results indicate that negative postinitiation regulation of mRNA biogenesis limits the expression of these factors, which mediate host defense against M. tuberculosis through the cellular response to IFN-γ. Consistent with the results for STAT1 and IRF1, transcriptome analysis reveals downregulation of postinitiation mRNA biogenesis processes and pathways by infection, with and without IFN-γ stimulation. Clinical relevance for regulation of postinitiation mRNA biogenesis is demonstrated by studies of donor samples showing that postinitiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared with cured disease and in active tuberculosis compared with ongoing treatment or with latent tuberculosis. For active disease and latent infection donors from two populations (London, U.K., and The Gambia), each analyzed using a different platform, pathway-related gene expression differences were highly correlated, demonstrating substantial specificity in the effect. Collectively, the molecular and bioinformatic analyses point toward downregulation of postinitiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of postinitiation mRNA biogenesis can constrain the macrophage response to infection and overall host defense against tuberculosis.

Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin(+) and thrombomodulin(-) dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin(+) dendritic cells compared to control dendritic cells and thrombomodulin(-) dendritic cells. Thrombomodulin-treated and thrombomodulin(+) dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin(+) dendritic cells produced more lipoxins than thrombomodulin(-) dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin(+) dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin(+) dendritic cells contained more cells actively dividing than those of thrombomodulin(-) dendritic cells. Production of IL-10 is increased in thrombomodulin(+) dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin(+) dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin(+) dendritic cells, which have significantly altered gene expression compared to thrombomodulin(-) dendritic cells in key immune function pathways.

5074 Background: Large-scale genomic analyses of high-grade, advanced-stage serous ovarian cancers by The Cancer Genome Atlas (TCGA) project revealed aberrations in genes comprising key proliferation and survival pathways (RB-E2F, RAS, PI3K) in the majority of tumors. Patients with germline BRCA1/2-mutations have more favorable prognoses than non-BRCA carriers, and recent work suggests that BRCA2 carriers do better than BRCA1. We hypothesized that concurrent proliferation pathway aberrations and BRCA1/2 mutations in tumors might play a role in patient outcome. Methods: Mutation, copy number, and clinical data for 309 TCGA-profiled serous ovarian tumors were downloaded from the MSKCC cBIO web portal. Each tumor was scored as aberrant for a pathway if any gene (RB: RB1, CDKN2A, CCND1, CCND2, E2F3, CCNE1; PI3K: PIK3CA, PTEN, AKT1, AKT2; RAS: KRAS, BRAF, NF1) in that pathway was mutated, amplified, or deleted. Results: 205 of 309 tumors had an aberration in at least one of these pathways. The frequency of pathway alteration differed significantly in BRCA1 (82%, 28/34), BRCA2 (52%, 17/33) and BRCA1/2 WT (66%, 160/242) tumors (BRCA1 vs. BRCA2: Fisher’s p= 0.0096). BRCA1 tumors more frequently contained alterations in multiple pathways than BRCA2 or WT tumors (41% vs. 24% or 25%, respectively). RB-E2F pathway alteration frequency was significantly different (BRCA1: 56%, BRCA2: 18%, WT: 43%, p=0.0043), but no significant differences in PI3K and RAS pathway aberration frequencies (BRCA1%: 41, 38; BRCA2%: 36, 27; WT% 28, 27), respectively, were observed. In agreement with the previous report, BRCA2 patients had significantly better overall survival (OS) than either BRCA1 or WT patients (median OS months for BRCA1: 35.9, BRCA2 45.4, BRCA1/2 WT 27.8; p=0.001). Presence of pathway alterations was not significantly associated with OS in BRCA1, BRCA2, or WT patients in this cohort. Conclusions: These results show a negative association between BRCA2 mutations and aberrations in key proliferation and survival pathways. Beyond BRCA1 and BRCA2 genetic mutations, the elevated frequency of pathway alteration in BRCA1 vs. BRCA2 tumors highlights differences that may be important for patient prognosis as well as therapy responses.

Interferon beta (IFNβ) reduces disease burden in relapsing-remitting multiple sclerosis (MS) patients. In this study, IFNβ-1b-treated MS patient gene expression profiles and biological knowledgebases were integrated to study IFNβ's pleiotropic mechanisms of action. Genes involved in immune regulation, mitochondrial fatty acid metabolism and antioxidant activity were discovered. Plausible mediators of neuronal preservation included NRF2, downregulation of OLA1, an antioxidant suppressor, and the antioxidant gene ND6, implicated in optic neuropathy and MS-like lesions. Network analysis highlighted IKBKE, which likely has a role in both viral response and energy metabolism. A comparative analysis of therapy-naive MS- and IFNβ-associated gene expression suggests an IFNβ insufficiency in MS. We observed more gene expression changes in long-term treatment than during acute dosing. These distinct short- and long-term effects were driven by different transcription factors. Multi-gene biomarker signatures of IFNβ treatment effects were developed and subsequently confirmed in independent IFNβ-1b-treated MS studies, but not in glatiramer acetate-treated patients.

Alignment to a genomic sequence is a common task in modern bioinformatics. By improving the methods used, significant amount of time and resources can be saved. We have developed a new genomic alignment search tool, called GAST, for sequences of at least 160 nt. GAST is many times faster than commonly used alignment tools BLAT and Mega BLAST. As the sizes of query sequences and the database increase, the advantage grows. This paper describes the principles of GAST and reports a comparison of GAST with BLAT and Mega BLAST. The effects the query sequence length and the number of queries have on run times were studied using the full human genome and the chromosome 1 of human genome separately. Additionally, the error tolerance and behaviour of GAST when handling sequences with lower similarity to a database was studied. Lastly, we compared the quality of exon mappings produced by the three tools and the genomic