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Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort.Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens.Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks.Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Viruses in the family Rhabdoviridae infect a variety of hosts, including vertebrates, invertebrates, plants and fungi, with important consequences for health and agriculture. This study describes two newly discovered viruses of freshwater mussels from the United States.

Background The frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs). Methods A prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline. Results Two hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection. Conclusions These results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.

The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency’s (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence.ImpactThe COVID-19 pandemic is currently a leading cause of death globally and new SARS-CoV-2 variants continue to emerge. Genomic surveillance of variants is necessary to characterize mutations that could affect transmissibility and spread, antigenicity or virulence. Furthermore, wet lab studies are necessary to evaluate how genetic differences may affect viral fitness and transmissibility. The Delta variant is currently the predominant strain globally and determining factors that drive its ability to spread rapidly is important. In this study, we characterized SARS-CoV-2 positive samples from U.S. military personnel and their beneficiaries by next-generation sequencing and isolation of viable virus. Consistent with other studies, we found higher levels of infectious virus from Delta samples when compared to non-Delta infections. Strikingly, we found the difference in titer between Delta and other strains to be so profound as to be unaffected by vaccination status, suggesting that increased transmissibility of the Delta variant is in part due to higher amounts of virus shedding. This helps explain the rapid spread of the Delta variant and provides the impetus to increase control measures such as vaccination, boosters, masking and distancing requirements. It will be necessary to continue genomic and virological characterization of new variants, such as Omicron.

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.

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.

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. 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. 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. 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.

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 Eonycteris spelaea 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.

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., Author summary We enrolled 200 severely ill patients in an observational study of sepsis in Takeo Province Cambodia. In an effort to provide an in-depth characterization of their illness, we combined standard clinical microbiology practices (culture, microscopy, etc) with serological analyses, newer molecular diagnostics, and next-generation sequencing to produce the most detailed characterization to date of sepsis and microbiological cause for southern Cambodia. Specifically, we were able to identify a causative pathogen in 46.5% of cases. A viral source was identified in 26 (13%) patients and bacterial source in 79 (39.5%) patients. In all, we detected 139 infections in 93 (46.5%) patients. Despite our best efforts though, 52.5% of the cohort remained undiagnosed, further underscoring the urgent need for new infectious disease diagnostics that can identify the cause of infections in near real time.

Bats are rich reservoirs of viruses, including several high consequence zoonoses. In this study, high throughput sequencing was used to characterize the virome through a longitudinal study of a captive colony of lesser dawn bats, species Eonycteris spelaea, in Singapore. This study utilized viral RNA extracted from swabs of four body sites per bat per timepoint. Swabs of the exterior of the bat (head and body) were used to evaluate virus populations and demonstrate utility as a sample site for future surveillance to extrapolate population-level infection. Through unbiased shotgun and target-enrichment sequencing, we identified both the known and previously unknown viruses of zoonotic relevance and defined the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. We observed the population persistence of three zoonotic-related viral families that are known to be associated with spillover from bats to humans: Paramyxoviridae, Reoviridae, and Coronaviridae. To our knowledge, this is the first study that combines probe-based viral enrichment with high-throughput sequencing or that creates a viral profile from multiple swab sites on individual bats and their cohorts. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Noninvasive surveillance methods that target the body of bats not only detect viruses shed within the colony but can also represent viral populations dispersed throughout the entire colony. New knowledge of persistent viral families should inform future directions for the biosurveillance of viruses that have the potential to cross the species barrier from bats to humans or other amplifying hosts.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Y&uacute, nnan, 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 Y&uacute, nnan, 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.

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 Eonycteris spelaea 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.

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., 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.

The taxonomic status of the bacterium Wolbachia persica is described, and based on the evidence presented, transfer of this species to the genus Francisella as Francisella persica comb. nov. is proposed. This reclassification is supported by data generated from genomic comparisons of W. persica ATCC VR-331(T) (=FSC845(T)=DSM 101678(T)) to other near neighbours, including Francisella tularensis subsp. novicida. The full-length 16S rRNA gene sequence of strain ATCC VR-331(T) had 98.5 % nucleotide identity to the cognate gene in F. tularensis, with the highest similarity to subspecies novicida. Phylogenetic trees of full-length 16S rRNA gene, gyrA and recA sequences from species of the genera Wolbachia (class Alphaproteobacteria) and Francisella (class Gammaproteobacteria) indicated that W. persica ATCC VR-331(T) was most closely related to members of the genus Francisella and not Wolbachia. Local collinear blocks within the chromosome of strain ATCC VR-331(T) had considerable similarity with F. tularensis subsp. novicida, but not with any Wolbachia strain. The genomes of strain ATCC VR-331(T) and F. tularensis subsp. novicida Utah 112(T) (=ATCC 15482(T)) contained an average nucleotide identity mean of 88.72 % and median of 89.18 %. Importantly, the genome of strain ATCC VR-331(T) contained one Francisella Pathogenicity Island, similar to F. tularensis subsp. novicida, as well as the Francisella-specific gene fopA1 and F. tularensis-specific genes fopA2 and lpnA (also referred to as tul4). In contrast to the obligate intracellular genus Wolbachia, strain ATCC VR-331(T) and facultative intracellular Francisella can replicate in specialized cell-free media. Collectively, these results demonstrate that Wolbachia persica should be reclassified in the genus Francisella as Francisella persica comb. nov. The type strain of Francisella persica comb. nov. is ATCC VR-331(T) (=FSC845(T)=DSM 101678(T)). An emended description of the family Francisellaceae is also provided.

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.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.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.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.

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.

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, 5mCpG 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, 86.9 % of all SAMMs were assigned to the correct tissue of origin. Thus, although tumors from different tissues may have similar mutation patterns, their SAMMs often display signatures that are characteristic of specific tissues. Electronic supplementary material The online version of this article (doi:10.1007/s00439-015-1566-1) contains supplementary material, which is available to authorized users.

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 (Empowering the Development of Genomics Expertise) 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 et al., 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.

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, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. 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 F. tularensis-exposed hPBMCs.

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.

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.

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.

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).