Publications by authors named "Regina Z Cer"

30 Publications

  • Page 1 of 1

Immunological and Genetic Investigation of SARS-CoV-2 Reinfection in an Otherwise Healthy, Young Marine Recruit.

Pathogens 2021 Dec 8;10(12). Epub 2021 Dec 8.

Genomics and Bioinformatics Department, Biological Defense Research Directorate, Naval Medical Research Center-Frederick, Fort Detrick, MD 21702, USA.

We used epidemiologic and viral genetic information to identify a case of likely reinfection in an otherwise healthy, young Marine recruit enrolled in the prospective, longitudinal COVID-19 Health Action Response for Marines (CHARM) study, and we paired these findings with serological studies. This participant had a positive RT-PCR to SARS-CoV-2 upon routine sampling on study day 7, although he was asymptomatic at that time. He cleared the infection within seven days. On study day 46, he had developed symptoms consistent with COVID-19 and tested positive by RT-PCR for SARS-CoV-2 again. Viral whole genome sequencing was conducted from nares swabs at multiple time points. The day 7 sample was determined to be lineage B.1.340, whereas both the day 46 and day 49 samples were B.1.1. The first positive result for anti-SARS-CoV-2 IgM serology was collected on day 49 and for IgG on day 91. This case appears most consistent with a reinfection event. Our investigation into this case is unique in that we compared sequence data from more than just paired specimens, and we also assayed for immune response after both the initial infection and the later reinfection. These data demonstrate that individuals who have experienced an infection with SARS-CoV-2 may fail to generate effective or long-lasting immunity, similar to endemic human beta coronaviruses.
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http://dx.doi.org/10.3390/pathogens10121589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8709254PMC
December 2021

Manual Annotation Studio (MAS): a collaborative platform for manual functional annotation of viral and microbial genomes.

BMC Genomics 2021 Oct 9;22(1):733. Epub 2021 Oct 9.

Genomics and Bioinformatics Department, Biological Defense Research Directorate, Naval Medical Research Center, Fort Detrick, MD, USA.

Background: Functional genome annotation is the process of labelling functional genomic regions with descriptive information. Manual curation can produce higher quality genome annotations than fully automated methods. Manual annotation efforts are time-consuming and complex; however, software can help reduce these drawbacks.

Results: We created Manual Annotation Studio (MAS) to improve the efficiency of the process of manual functional annotation prokaryotic and viral genomes. MAS allows users to upload unannotated genomes, provides an interface to edit and upload annotations, tracks annotation history and progress, and saves data to a relational database. MAS provides users with pertinent information through a simple point and click interface to execute and visualize results for multiple homology search tools (blastp, rpsblast, and HHsearch) against multiple databases (Swiss-Prot, nr, CDD, PDB, and an internally generated database). MAS was designed to accept connections over the local area network (LAN) of a lab or organization so multiple users can access it simultaneously. MAS can take advantage of high-performance computing (HPC) clusters by interfacing with SGE or SLURM and data can be exported from MAS in a variety of formats (FASTA, GenBank, GFF, and excel).

Conclusions: MAS streamlines and provides structure to manual functional annotation projects. MAS enhances the ability of users to generate, interpret, and compare results from multiple tools. The structure that MAS provides can improve project organization and reduce annotation errors. MAS is ideal for team-based annotation projects because it facilitates collaboration.
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http://dx.doi.org/10.1186/s12864-021-08029-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8501643PMC
October 2021

The temporal RNA virome patterns of a lesser dawn bat () colony revealed by deep sequencing.

Virus Evol 2020 Jan 18;6(1):veaa017. Epub 2020 Mar 18.

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD 20814, USA.

The virosphere is largely unexplored and the majority of viruses are yet to be represented in public sequence databases. Bats are rich reservoirs of viruses, including several zoonoses. In this study, high throughput sequencing (HTS) of viral RNA extracted from swabs of four body sites per bat per timepoint is used to characterize the virome through a longitudinal study of a captive colony of fruit nectar bats, species in Singapore. Through unbiased shotgun and target enrichment sequencing, we identify both known and previously unknown viruses of zoonotic relevance and define the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. To our knowledge, this is the first study that combines probe-based viral enrichment with HTS to create a viral profile from multiple swab sites on individual bats and their cohort. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Given the known risk for bat-human zoonoses, a more complete understanding of the viral dynamics in South-eastern Asian bats has significant implications for disease prevention and control. The findings of this study will be of interest to U.S. Department of Defense personnel stationed in the Asia-Pacific region and regional public health laboratories engaged in emerging infectious disease surveillance efforts.
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http://dx.doi.org/10.1093/ve/veaa017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079719PMC
January 2020

A Department of Defense Laboratory Consortium Approach to Next Generation Sequencing and Bioinformatics Training for Infectious Disease Surveillance in Kenya.

Front Genet 2020 25;11:577563. Epub 2020 Sep 25.

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States.

Epidemics of emerging and re-emerging infectious diseases are a danger to civilian and military populations worldwide. Health security and mitigation of infectious disease threats is a priority of the United States Government and the Department of Defense (DoD). Next generation sequencing (NGS) and Bioinformatics (BI) enhances traditional biosurveillance by providing additional data to understand transmission, identify resistance and virulence factors, make predictions, and update risk assessments. As more and more laboratories adopt NGS and BI technologies they encounter challenges in building local capacity. In addition to choosing the right sequencing platform and approach, considerations must also be made for the complexity of bioinformatics analyses, data storage, as well as personnel and computational requirements. To address these needs, a comprehensive training program was developed covering wet lab and bioinformatics approaches to NGS. The program is meant to be modular and adaptive to meet both common and individualized needs of medical research and public health laboratories across the DoD. The training program was first deployed internationally to the Basic Science Laboratory of the US Army Medical Research Directorate-Africa in Kisumu, Kenya, which is an overseas Lab of the Walter Reed Army Institute of Research (WRAIR). A week-long workshop with intensive focus on targeted sequencing and the bioinformatics of genome assembly ( = 24 participants) was held. Post-workshop self-assessment (completed by 21 participants) noted significant median gains in knowledge domains related to NGS targeted sequencing, bioinformatics for genome assembly, and sequence quality assessment. The participants also reported that the information on study design, sample preparation, sequencing quality control, data quality assessment, reporting, and basic and advanced bioinformatics analysis were the most useful information presented in the training. While longer-term evaluations are planned, the training resulted in significant short-term improvement of a laboratory's self-reported wet lab and bioinformatics capabilities. This framework can be used for future DoD laboratory development in the area of NGS and BI for infectious disease surveillance, ultimately enhancing this global DoD capability.
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http://dx.doi.org/10.3389/fgene.2020.577563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546821PMC
September 2020

An Observational Study of Sepsis in Takeo Province Cambodia: An in-depth examination of pathogens causing severe infections.

PLoS Negl Trop Dis 2020 08 17;14(8):e0008381. Epub 2020 Aug 17.

Austere environments Consortium for Enhanced Sepsis Outcomes (ACESO), Biological Defense Research Directorate, Naval Medical Research Center-Frederick, Ft. Detrick, Maryland, United States of America.

The world's most consequential pathogens occur in regions with the fewest diagnostic resources, leaving the true burden of these diseases largely under-represented. During a prospective observational study of sepsis in Takeo Province Cambodia, we enrolled 200 patients over an 18-month period. By coupling traditional diagnostic methods such as culture, serology, and PCR to Next Generation Sequencing (NGS) and advanced statistical analyses, we successfully identified a pathogenic cause in 46.5% of our cohort. In all, we detected 25 infectious agents in 93 patients, including severe threat pathogens such as Burkholderia pseudomallei and viral pathogens such as Dengue virus. Approximately half of our cohort remained undiagnosed; however, an independent panel of clinical adjudicators determined that 81% of those patients had infectious causes of their hospitalization, further underscoring the difficulty of diagnosing severe infections in resource-limited settings. We garnered greater insight as to the clinical features of severe infection in Cambodia through analysis of a robust set of clinical data.
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http://dx.doi.org/10.1371/journal.pntd.0008381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430706PMC
August 2020

Corrigendum: Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, .

Front Microbiol 2020 14;11:877. Epub 2020 May 14.

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

[This corrects the article DOI: 10.3389/fmicb.2018.02664.].
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http://dx.doi.org/10.3389/fmicb.2020.00877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241116PMC
May 2020

Detection of Recombinant Rousettus Bat Coronavirus GCCDC1 in Lesser Dawn Bats () in Singapore.

Viruses 2020 05 14;12(5). Epub 2020 May 14.

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.
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http://dx.doi.org/10.3390/v12050539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291116PMC
May 2020

Direct-from-blood RNA sequencing identifies the cause of post-bronchoscopy fever.

BMC Infect Dis 2019 Oct 28;19(1):905. Epub 2019 Oct 28.

Austere environments Consortium for Enhanced Sepsis Outcomes (ACESO), Biological Defense Research Directorate, Naval Medical Research Center-Frederick, 8400 Research Plaza, Fort Detrick, MD, 21702, USA.

Background: Antibiotic resistance is rising at disturbing rates and contributes to the deaths of millions of people yearly. Antibiotic resistant infections disproportionately affect those with immunocompromising conditions, chronic colonization, and frequent antibiotic use such as transplant patients or those with cystic fibrosis. However, clinicians lack the diagnostic tools to confidently diagnose and treat infections, leading to widespread use of empiric broad spectrum antimicrobials, often for prolonged duration.

Case Presentation: A 22 year-old Caucasian female with cystic fibrosis received a bilateral orthotopic lung transplantation 5 months prior to the index hospitalization. She underwent routine surveillance bronchoscopy and was admitted for post-procedure fever. A clear cause of infection was not identified by routine methods. Imaging and bronchoscopic lung biopsy did not identify an infectious agent or rejection. She was treated with a prolonged course of antimicrobials targeting known colonizing organisms from prior bronchoalveolar lavage cultures (Pseudomonas, Staphylococcus aureus, and Aspergillus). However, we identified Stenotrophomonas maltophilia in two independent whole blood samples using direct-pathogen sequencing, which was not identified by other methods.

Conclusions: This case represents a common clinical conundrum: identification of infection in a high-risk, complex patient. Here, direct-pathogen sequencing identified a pathogen that would not otherwise have been identified by common techniques. Had results been clinically available, treatment could have been customized, avoiding a prolonged course of broad spectrum antimicrobials that would only exacerbate resistance. Direct-pathogen sequencing is poised to fill a diagnostic gap for pathogen identification, allowing early identification and customization of treatment in a culture-independent, pathogen-agnostic manner.
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http://dx.doi.org/10.1186/s12879-019-4462-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819639PMC
October 2019

Genomic epidemiology of MRSA infection and colonization isolates among military trainees with skin and soft tissue infection.

Infection 2019 Oct 22;47(5):729-737. Epub 2019 Feb 22.

Walter Reed Army Institute of Research, Silver Spring, MD, USA.

Purpose: Individuals with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection (SSTI) can be simultaneously colonized with MRSA on multiple body sites. Using whole genome sequencing (WGS), the intrahost relatedness of MRSA colonization and infection isolates was investigated.

Methods: In the context of a prospective case-control study of SSTI, we analyzed colonization and infection isolates from US Army Infantry trainees with purulent infection due to MRSA. At the time of clinical presentation for SSTI, culture swabs were obtained from the infection site, as well as from the patient's nasal, oral, inguinal, and perianal regions. S. aureus culture and susceptibility was performed by standard methods. DNA from MRSA isolates was extracted and libraries were produced. Sequences were generated on an Illumina MiSeq, sequence reads were assembled, and single nucleotide variant (SNV) data were analyzed.

Results: Of 74 trainees with MRSA SSTI, 19 (25.7%) were colonized with MRSA. Ten (52.6%) were colonized on more than one body site. Colonization frequency by anatomic site was as follows: inguinal region (33%), nasal region (30%), perianal region (22%), and oral region (14%). A total of 36 MRSA colonization isolates were characterized. The intrahost median number of SNVs between infection and colonization isolates was 17. Among trainees with recurrent MRSA SSTI, limited intrahost diversity suggests that persistent colonization is a major contributor to recurrence risk.

Conclusions: Among military trainees with MRSA SSTI, genomic characterization of infection and colonization isolates revealed a high degree of strain relatedness. Single acquisition events may account for MRSA colonization and infection in this population.
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http://dx.doi.org/10.1007/s15010-019-01282-wDOI Listing
October 2019

High-Quality Draft Genome Sequence of Pseudomonas aeruginosa 268 Isolated from a Patient with a Left Ventricular Assist Device.

Microbiol Resour Announc 2019 Jan 3;8(1). Epub 2019 Jan 3.

Genomics & Bioinformatics Department, Biological Defense Research Directorate, Naval Medical Research Center, Fort Detrick, Maryland, USA.

Pseudomonas aeruginosa is known to cause persistent bloodstream infections associated with left ventricular assist devices (LVAD). Here, we present the high-quality draft genome assembly for a clinical isolate, P. aeruginosa 268. The genome sequence is available in GenBank under accession number CP032761.
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http://dx.doi.org/10.1128/MRA.01403-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318362PMC
January 2019

Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, .

Front Microbiol 2018 19;9:2664. Epub 2018 Nov 19.

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

is the leading cause of skin and soft tissue infections (SSTI). Some strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel -positive plasmid, pC02. Prior characterization of -containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous strains and to . We screened additional -positive clinical isolates and pC02 was present in 27% of those strains; other unique -harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that -containing strains are transmitted among military personnel at Ft. Benning and that strains carrying are associated with SSTIs within this population. The identification of a novel mechanism of conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.
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http://dx.doi.org/10.3389/fmicb.2018.02664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252503PMC
November 2018

Characterizing Phage Genomes for Therapeutic Applications.

Viruses 2018 04 10;10(4). Epub 2018 Apr 10.

Biological Defense Research Directorate, Naval Medical Research Center, Fort Detrick, MD 21702, USA.

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.
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http://dx.doi.org/10.3390/v10040188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923482PMC
April 2018

Brief Protocol for EDGE Bioinformatics: Analyzing Microbial and Metagenomic NGS Data.

Bio Protoc 2017 Dec 5;7(23):e2622. Epub 2017 Dec 5.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA.

Next-generation sequencing (NGS) offers unparalleled resolution for untargeted organism detection and characterization. However, the majority of NGS analysis programs require users to be proficient in programming and command-line interfaces. EDGE bioinformatics was developed to offer scientists with little to no bioinformatics expertise a point-and-click platform for analyzing sequencing data in a rapid and reproducible manner. EDGE (mpowering the evelopment of enomics xpertise) v1.0 released in January 2017, is an intuitive web-based bioinformatics platform engineered for the analysis of microbial and metagenomic NGS-based data ( Li , 2017 ). The EDGE bioinformatics suite combines vetted publicly available tools, and tracks settings to ensure reliable and reproducible analysis workflows. To execute the EDGE workflow, only raw sequencing reads and a project ID are necessary. Users can access in-house data, or run analyses on samples deposited in Sequence Read Archive. Default settings offer a robust first-glance and are often sufficient for novice users. All analyses are modular; users can easily turn workflows on/off, and modify parameters to cater to project needs. Results are compiled and available for download in a PDF-formatted report containing publication quality figures. We caution that interpreting results still requires in-depth scientific understanding, however report visuals are often informative, even to novice users.
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http://dx.doi.org/10.21769/BioProtoc.2622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8438472PMC
December 2017

Differential MicroRNA Analyses of - and -Exposed hPBMCs Reveal Potential Biomarkers.

Int J Genomics 2017 16;2017:6489383. Epub 2017 Jul 16.

Genomics and Bioinformatics Department, Biological Defense Research Directorate, Naval Medical Research Center, Frederick, MD, USA.

Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, K96243 and SHU S4, as well as to the nonpathogenic control DH5. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in -exposed hPBMCs.
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http://dx.doi.org/10.1155/2017/6489383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534298PMC
July 2017

Erratum to: The somatic autosomal mutation matrix in cancer genomes.

Hum Genet 2015 Aug;134(8):865-7

In Silico Research Centers of Excellence, Advanced Biomedical Computing Center, Information Systems Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., P.O. Box B, Frederick, MD, 21702, USA.

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http://dx.doi.org/10.1007/s00439-015-1576-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643558PMC
August 2015

The somatic autosomal mutation matrix in cancer genomes.

Hum Genet 2015 Aug 23;134(8):851-64. Epub 2015 May 23.

In Silico Research Centers of Excellence, Advanced Biomedical Computing Center, Information Systems Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., P.O. Box B, Frederick, MD, 21702, USA.

DNA damage in somatic cells originates from both environmental and endogenous sources, giving rise to mutations through multiple mechanisms. When these mutations affect the function of critical genes, cancer may ensue. Although identifying genomic subsets of mutated genes may inform therapeutic options, a systematic survey of tumor mutational spectra is required to improve our understanding of the underlying mechanisms of mutagenesis involved in cancer etiology. Recent studies have presented genome-wide sets of somatic mutations as a 96-element vector, a procedure that only captures the immediate neighbors of the mutated nucleotide. Herein, we present a 32 × 12 mutation matrix that captures the nucleotide pattern two nucleotides upstream and downstream of the mutation. A somatic autosomal mutation matrix (SAMM) was constructed from tumor-specific mutations derived from each of 909 individual cancer genomes harboring a total of 10,681,843 single-base substitutions. In addition, mechanistic template mutation matrices (MTMMs) representing oxidative DNA damage, ultraviolet-induced DNA damage, (5m)CpG deamination, and APOBEC-mediated cytosine mutation, are presented. MTMMs were mapped to the individual tumor SAMMs to determine the maximum contribution of each mutational mechanism to the overall mutation pattern. A Manhattan distance across all SAMM elements between any two tumor genomes was used to determine their relative distance. Employing this metric, 89.5% of all tumor genomes were found to have a nearest neighbor from the same tissue of origin. When a distance-dependent 6-nearest neighbor classifier was used, 10.4% of the SAMMs had an Undetermined tissue of origin, and 92.2% of the remaining SAMMs were assigned to the correct tissue of origin. [corrected]. Thus, although tumors from different tissues may have similar mutation patterns, their SAMMs often display signatures that are characteristic of specific tissues.
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http://dx.doi.org/10.1007/s00439-015-1566-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495249PMC
August 2015

miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation.

Gigascience 2014 13;3:20. Epub 2014 Oct 13.

Biological Defense Research Directorate, Naval Medical Research Center-Frederick, 8400 Research Plaza, Fort Detrick, MD 21702, USA.

Background: Understanding the biological roles of microRNAs (miRNAs) is a an active area of research that has produced a surge of publications in PubMed, particularly in cancer research. Along with this increasing interest, many open-source bioinformatics tools to identify existing and/or discover novel miRNAs in next-generation sequencing (NGS) reads become available. While miRNA identification and discovery tools are significantly improved, the development of miRNA differential expression analysis tools, especially in temporal studies, remains substantially challenging. Further, the installation of currently available software is non-trivial and steps of testing with example datasets, trying with one's own dataset, and interpreting the results require notable expertise and time. Subsequently, there is a strong need for a tool that allows scientists to normalize raw data, perform statistical analyses, and provide intuitive results without having to invest significant efforts.

Findings: We have developed miRNA Temporal Analyzer (mirnaTA), a bioinformatics package to identify differentially expressed miRNAs in temporal studies. mirnaTA is written in Perl and R (Version 2.13.0 or later) and can be run across multiple platforms, such as Linux, Mac and Windows. In the current version, mirnaTA requires users to provide a simple, tab-delimited, matrix file containing miRNA name and count data from a minimum of two to a maximum of 20 time points and three replicates. To recalibrate data and remove technical variability, raw data is normalized using Normal Quantile Transformation (NQT), and linear regression model is used to locate any miRNAs which are differentially expressed in a linear pattern. Subsequently, remaining miRNAs which do not fit a linear model are further analyzed in two different non-linear methods 1) cumulative distribution function (CDF) or 2) analysis of variances (ANOVA). After both linear and non-linear analyses are completed, statistically significant miRNAs (P < 0.05) are plotted as heat maps using hierarchical cluster analysis and Euclidean distance matrix computation methods.

Conclusions: mirnaTA is an open-source, bioinformatics tool to aid scientists in identifying differentially expressed miRNAs which could be further mined for biological significance. It is expected to provide researchers with a means of interpreting raw data to statistical summaries in a fast and intuitive manner.
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http://dx.doi.org/10.1186/2047-217X-3-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4212236PMC
November 2014

BLASTPLOT: a PERL module to plot next generation sequencing NCBI-BLAST results.

Source Code Biol Med 2014 Mar 31;9(1). Epub 2014 Mar 31.

Naval Medical Research Center-Frederick, 8400 Research Plaza, Frederick, MD, USA.

Background: The development of Next Generation Sequencing (NGS) during the last decade has created an unprecedented amount of sequencing data, as well as the ability to rapidly sequence specimens of interest. Read-based BLAST analysis of NGS data is a common procedure especially in the case of metagenomic samples. However, coverage is usually not enough to allow for de novo assembly. This type of read-based analysis often creates the question of how the reads that align to the same sequence are distributed. The same question applies to preparation of primers or probes for microarray experiments. Although there are several packages that allow the visualization of DNA segments in relation to a reference, in most cases they require the visualization of one reference at a time and the capture of screen shots for each segment. Such a procedure could be tedious and time consuming. The field is in need of a solution that automates the capture of coverage plots for all the segments of interest.

Results: We have created BLASTPLOT, a PERL module to quickly plot the BLAST results from short sequences (primers, probes, reads) against reference targets.

Conclusions: BLASTPLOT is a simple to use PERL module that allows the generation of PNG graphs for all the reference sequences associated with a BLAST result set.
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http://dx.doi.org/10.1186/1751-0473-9-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3974413PMC
March 2014

Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

PLoS Pathog 2013 10;9(10):e1003684. Epub 2013 Oct 10.

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.
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http://dx.doi.org/10.1371/journal.ppat.1003684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795035PMC
May 2014

Guanine holes are prominent targets for mutation in cancer and inherited disease.

PLoS Genet 2013 26;9(9):e1003816. Epub 2013 Sep 26.

Division of Pharmacology and Toxicology, The University of Texas at Austin, Dell Pediatric Research Institute, Austin, Texas, United States of America ; Advanced Biomedical Computing Center, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.

Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.
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http://dx.doi.org/10.1371/journal.pgen.1003816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784513PMC
March 2014

Non-B DB v2.0: a database of predicted non-B DNA-forming motifs and its associated tools.

Nucleic Acids Res 2013 Jan 3;41(Database issue):D94-D100. Epub 2012 Nov 3.

Advanced Biomedical Computing Center, Information Systems Program, SAIC-Frederick, Inc., Frederick, MD 21702, USA.

The non-B DB, available at http://nonb.abcc.ncifcrf.gov, catalogs predicted non-B DNA-forming sequence motifs, including Z-DNA, G-quadruplex, A-phased repeats, inverted repeats, mirror repeats, direct repeats and their corresponding subsets: cruciforms, triplexes and slipped structures, in several genomes. Version 2.0 of the database revises and re-implements the motif discovery algorithms to better align with accepted definitions and thresholds for motifs, expands the non-B DNA-forming motifs coverage by including short tandem repeats and adds key visualization tools to compare motif locations relative to other genomic annotations. Non-B DB v2.0 extends the ability for comparative genomics by including re-annotation of the five organisms reported in non-B DB v1.0, human, chimpanzee, dog, macaque and mouse, and adds seven additional organisms: orangutan, rat, cow, pig, horse, platypus and Arabidopsis thaliana. Additionally, the non-B DB v2.0 provides an overall improved graphical user interface and faster query performance.
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http://dx.doi.org/10.1093/nar/gks955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531222PMC
January 2013

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells.

J Biol Chem 2011 Mar 1;286(12):10017-26. Epub 2011 Feb 1.

Department of Molecular Carcinogenesis, Science Park-Research Division, The University of Texas, M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ∼70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ∼30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ∼40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.
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http://dx.doi.org/10.1074/jbc.M110.176636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060453PMC
March 2011

Non-B DB: a database of predicted non-B DNA-forming motifs in mammalian genomes.

Nucleic Acids Res 2011 Jan 21;39(Database issue):D383-91. Epub 2010 Nov 21.

Advanced Biomedical Computing Center, Information Systems Program, SAIC-Frederick, Inc, NCI-Frederick, Frederick, MD 21702, USA.

Although the capability of DNA to form a variety of non-canonical (non-B) structures has long been recognized, the overall significance of these alternate conformations in biology has only recently become accepted en masse. In order to provide access to genome-wide locations of these classes of predicted structures, we have developed non-B DB, a database integrating annotations and analysis of non-B DNA-forming sequence motifs. The database provides the most complete list of alternative DNA structure predictions available, including Z-DNA motifs, quadruplex-forming motifs, inverted repeats, mirror repeats and direct repeats and their associated subsets of cruciforms, triplex and slipped structures, respectively. The database also contains motifs predicted to form static DNA bends, short tandem repeats and homo(purine•pyrimidine) tracts that have been associated with disease. The database has been built using the latest releases of the human, chimp, dog, macaque and mouse genomes, so that the results can be compared directly with other data sources. In order to make the data interpretable in a genomic context, features such as genes, single-nucleotide polymorphisms and repetitive elements (SINE, LINE, etc.) have also been incorporated. The database is accessed through query pages that produce results with links to the UCSC browser and a GBrowse-based genomic viewer. It is freely accessible at http://nonb.abcc.ncifcrf.gov.
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http://dx.doi.org/10.1093/nar/gkq1170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013731PMC
January 2011

The zinc-dependent protease activity of the botulinum neurotoxins.

Toxins (Basel) 2010 05 7;2(5):978-97. Epub 2010 May 7.

US Army Medical Research and Materiel Command, Ft. Detrick, MD, USA.

The botulinum neurotoxins (BoNT, serotypes A-G) are some of the most toxic proteins known and are the causative agents of botulism. Following exposure, the neurotoxin binds and enters peripheral cholinergic nerve endings and specifically and selectively cleaves one or more SNARE proteins to produce flaccid paralysis. This review centers on the kinetics of the Zn-dependent proteolytic activities of these neurotoxins, and briefly describes inhibitors, activators and factors underlying persistence of toxin action. Some of the structural, enzymatic and inhibitor data that are discussed here are available at the botulinum neurotoxin resource, BotDB (http://botdb.abcc.ncifcrf.gov).
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http://dx.doi.org/10.3390/toxins2050978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153231PMC
May 2010

Temporal characteristics of botulinum neurotoxin therapy.

Expert Rev Neurother 2010 Jan;10(1):93-103

Combat Casualty Care Research Program, US Army Medical Research and Materiel Command, 504 Scott Street, Ft Detrick, MD 21702-5012, USA.

Botulinum neurotoxin is a pharmaceutical treatment used for an increasing number of neurological and non-neurological indications, symptoms and diseases. Despite the wealth of clinical reports that involve the timing of the therapeutic effects of this toxin, few studies have attempted to integrate these data into unified models. Secondary reactions have also been examined including the development of adverse events, resistance to repeated applications, and nerve terminal sprouting. Our primary intent for conducting this review was to gather relevant pharmacodynamic data from suitable biomedical literature regarding botulinum neurotoxins via the use of automated data-mining techniques. We envision that mathematical models will ultimately be of value to those who are healthcare decision makers and providers, as well as clinical and basic researchers. Furthermore, we hypothesize that the combination of this computer-intensive approach with mathematical modeling will predict the percentage of patients who will favorably or adversely respond to this treatment and thus will eventually assist in developing the increasingly important area of personalized medicine.
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http://dx.doi.org/10.1586/ern.09.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842014PMC
January 2010

Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1.

J Bacteriol 2007 Jan 13;189(1):52-64. Epub 2006 Oct 13.

The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA.

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.
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http://dx.doi.org/10.1128/JB.01313-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797222PMC
January 2007

Sequencing Bacillus anthracis typing phages gamma and cherry reveals a common ancestry.

J Bacteriol 2006 May;188(9):3402-8

The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA.

The genetic relatedness of the Bacillus anthracis typing phages Gamma and Cherry was determined by nucleotide sequencing and comparative analysis. The genomes of these two phages were identical except at three variable loci, which showed heterogeneity within individual lysates and among Cherry, Wbeta, Fah, and four Gamma bacteriophage sequences.
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http://dx.doi.org/10.1128/JB.188.9.3402-3408.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1447464PMC
May 2006

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Proc Natl Acad Sci U S A 2004 Jun 21;101(22):8449-54. Epub 2004 May 21.

Epidemiologic Investigations Laboratory, Meningitis and Special Pathogens Branch, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G34, Atlanta, GA 30333, USA.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.
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http://dx.doi.org/10.1073/pnas.0402414101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC420414PMC
June 2004

The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1.

Nucleic Acids Res 2004 11;32(3):977-88. Epub 2004 Feb 11.

The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA.

We sequenced the complete genome of Bacillus cereus ATCC 10987, a non-lethal dairy isolate in the same genetic subgroup as Bacillus anthracis. Comparison of the chromosomes demonstrated that B.cereus ATCC 10987 was more similar to B.anthracis Ames than B.cereus ATCC 14579, while containing a number of unique metabolic capabilities such as urease and xylose utilization and lacking the ability to utilize nitrate and nitrite. Additionally, genetic mechanisms for variation of capsule carbohydrate and flagella surface structures were identified. Bacillus cereus ATCC 10987 contains a single large plasmid (pBc10987), of approximately 208 kb, that is similar in gene content and organization to B.anthracis pXO1 but is lacking the pathogenicity-associated island containing the anthrax lethal and edema toxin complex genes. The chromosomal similarity of B.cereus ATCC 10987 to B.anthracis Ames, as well as the fact that it contains a large pXO1-like plasmid, may make it a possible model for studying B.anthracis plasmid biology and regulatory cross-talk.
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http://dx.doi.org/10.1093/nar/gkh258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373394PMC
May 2004
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