Li P.-E.,Los Alamos National Laboratory |
Lo C.-C.,Los Alamos National Laboratory |
Anderson J.J.,Defense Threat Reduction Agency |
Anderson J.J.,Naval Medical Research Center Frederick |
And 8 more authors.
Nucleic Acids Research | Year: 2017
Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cuttingedge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice nextgeneration sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research. © 2016 The Author(s).
Loraine A.E.,University of North Carolina |
McCormick S.,University of California at Berkeley |
Estrada A.,University of North Carolina |
Patel K.,University of North Carolina |
And 3 more authors.
Plant Physiology | Year: 2013
Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 59 and 39 untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. © 2013 American Society of Plant Biologists. All Rights Reserved.
Bah E.I.,Donka Hospital |
Lamah M.-C.,Donka Hospital |
Fletcher T.,University of Liverpool |
Jacob S.T.,Hospital Mulago |
And 21 more authors.
New England Journal of Medicine | Year: 2015
Background In March 2014, the World Health Organization was notified of an outbreak of Zaire ebolavirus in a remote area of Guinea. The outbreak then spread to the capital, Conakry, and to neighboring countries and has subsequently become the largest epidemic of Ebola virus disease (EVD) to date. Methods From March 25 to April 26, 2014, we performed a study of all patients with laboratoryconfirmed EVD in Conakry. Mortality was the primary outcome. Secondary outcomes included patient characteristics, complications, treatments, and comparisons between survivors and nonsurvivors. Results Of 80 patients who presented with symptoms, 37 had laboratory-confirmed EVD. Among confirmed cases, the median age was 38 years (interquartile range, 28 to 46), 24 patients (65%) were men, and 14 (38%) were health care workers; among the health care workers, nosocomial transmission was implicated in 12 patients (32%). Patients with confirmed EVD presented to the hospital a median of 5 days (interquartile range, 3 to 7) after the onset of symptoms, most commonly with fever (in 84% of the patients; mean temperature, 38.6°C), fatigue (in 65%), diarrhea (in 62%), and tachycardia (mean heart rate, >93 beats per minute). Of these patients, 28 (76%) were treated with intravenous fluids and 37 (100%) with antibiotics. Sixteen patients (43%) died, with a median time from symptom onset to death of 8 days (interquartile range, 7 to 11). Patients who were 40 years of age or older, as compared with those under the age of 40 years, had a relative risk of death of 3.49 (95% confidence interval, 1.42 to 8.59; P = 0.007). Conclusions Patients with EVD presented with evidence of dehydration associated with vomiting and severe diarrhea. Despite attempts at volume repletion, antimicrobial therapy, and limited laboratory services, the rate of death was 43%. © 2015 Massachusetts Medical Society.
Benitez A.J.,Centers for Disease Control and Prevention |
Diaz M.H.,Centers for Disease Control and Prevention |
Wolff B.J.,Centers for Disease Control and Prevention |
Pimentel G.,Naval Medical Research Center Frederick |
And 3 more authors.
Journal of Clinical Microbiology | Year: 2012
In this study, we evaluated a recently developed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited (n = 4) number of M. pneumoniae-positive primary specimens acquired by the CDC was performed using MLVA. Eighteen distinct VNTR types were identified, including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis, which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge, this is the first report showing variation within the Mpn1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations. Copyright © 2012, American Society for Microbiology. All Rights Reserved.
Frey K.G.,Foundation Medicine |
Frey K.G.,Naval Medical Research Center Frederick |
Bishop-Lilly K.A.,Foundation Medicine |
Bishop-Lilly K.A.,Naval Medical Research Center Frederick
Methods in Microbiology | Year: 2015
Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies. © 2015 Elsevier Ltd.
PubMed | OpGen, Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc, Naval Medical Research Center Frederick, Los Alamos National Laboratory and University of Florida
Type: Journal Article | Journal: PloS one | Year: 2015
Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, ordered restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae, we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.
PubMed | Naval Medical Research Center Frederick, Los Alamos National Laboratory and Defense Threat Reduction Agency
Type: Journal Article | Journal: Nucleic acids research | Year: 2016
Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.
Clark D.V.,U.S. National Institutes of Health |
Jahrling P.B.,U.S. National Institutes of Health |
Lawler J.V.,Naval Medical Research Center Frederick
Viruses | Year: 2012
Filovirus infection presents many unique challenges to patient management. Currently no approved treatments are available, and the recommendations for supportive care are not evidence based. The austere clinical settings in which patients often present and the sporadic and at times explosive nature of filovirus outbreaks have effectively limited the information available to evaluate potential management strategies. This review will summarize the management approaches used in filovirus outbreaks and provide recommendations for collecting the information necessary for evaluating and potentially improving patient outcomes in the future. © 2012 by the authors licensee MDPI, Basel, Switzerland.
Schully K.L.,Naval Medical Research Center Frederick |
Bell M.G.,Naval Medical Research Center Frederick |
Ward J.M.,Global Vet Pathology |
Keane-Myers A.M.,Naval Medical Research Center Frederick
PLoS ONE | Year: 2014
Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure. © 2014, Public Library of Science. All rights reserved.
Gebhart D.,Avidbiotics Corporation |
Williams S.R.,Avidbiotics Corporation |
Bishop-Lilly K.A.,Naval Medical Research Center Frederick |
Govoni G.R.,Avidbiotics Corporation |
And 6 more authors.
Journal of Bacteriology | Year: 2012
Clostridium difficile causes one of the leading nosocomial infections in developed countries, and therapeutic choices are limited. Some strains of C. difficile produce phage tail-like particles upon induction of the SOS response. These particles have bactericidal activity against other C. difficile strains and can therefore be classified as bacteriocins, similar to the R-type pyocins of Pseudomonas aeruginosa. These R-type bacteriocin particles, which have been purified from different strains, each have a different C. difficile-killing spectrum, with no one bacteriocin killing all C. difficile isolates tested. We have identified the genetic locus of these "diffocins" (open reading frames 1359 to 1376) and have found them to be common among the species. The entire diffocin genetic locus of more than 20 kb was cloned and expressed in Bacillus subtilis, and this resulted in production of bactericidal particles. One of the interesting features of these particles is a very large structural protein of~200 kDa, the product of gene 1374. This large protein determines the killing spectrum of the particles and is likely the receptor-binding protein. Diffocins may provide an alternate bactericidal agent to prevent or treat infections and to decolonize individuals who are asymptomatic carriers. © 2012, American Society for Microbiology.