CAVEMol: an immersive 3D molecule viewer

As the number of solved molecular structures deposited with the Protein Data Bank (PDB) increases, so too does the desire for more advanced ways of using this data. Traditional applications for viewing and manipulating molecular structures create a computer-generated model on a standard desktop computer screen. The display may employ some method of stereography to create the illusion of depth, but generally the user just sees a flat image. The user is able to interact with the molecule by magnifying it to see a particular area of interest, or by rotating it to see all sides of the molecule. The user may also be able to see animated changes in the molecule over time, or they may even be able to make modifications to the structure in real time. Regardless of the amount of control the user has over the molecule, however, one thing remains the same: the user experiences the molecule as though it were an object floating behind the monitor screen which they can indirectly control using a mouse or other pointing device. An immersive environment, on the other hand, provides a new paradigm for molecular visualization, allowing the user a much more realistic interaction with the molecule. The user becomes part of the viewing experience, traversing a molecule as though walking or flying within it. The molecule can completely surround them on all sides, giving them a true sense of the size and shape of the molecule in three dimensions. The user may also interact with the object directly, moving and rotating it with their hands rather than a mouse. This approach should prove particularly valuable for operations such as interactive docking, which allows a user to manipulate the interface between two molecules to identify favorable interaction sites. This thesis presents the design and implementation of CAVEMol, a molecular visualization application for immersive environments. I will also give an overview of molecular visualization and immersive environments, and then discuss future work that can be done in this area as well as applications where molecular visualization in an immersive environment can offer unique advantages

NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats

Whole-genome sequencing (WGS) of bacterial isolates has become standard practice in many laboratories. Applications for WGS analysis include phylogeography and molecular epidemiology, using single nucleotide polymorphisms (SNPs) as the unit of evolution. NASP was developed as a reproducible method that scales well with the hundreds to thousands of WGS data typically used in comparative genomics applications. In this study, we demonstrate how NASP compares with other tools in the analysis of two real bacterial genomics datasets and one simulated dataset. Our results demonstrate that NASP produces similar, and often better, results in comparison with other pipelines, but is much more flexible in terms of data input types, job management systems, diversity of supported tools and output formats. We also demonstrate differences in results based on the choice of the reference genome and choice of inferring phylogenies from concatenated SNPs or alignments including monomorphic positions. NASP represents a source-available, version-controlled, unit-tested method and can be obtained from tgennorth.github.io/NASP

Genome sequence of Staphylococcus aureus strain CA-347, a USA600 methicillin-resistant isolate

The Staphylococcus aureus clonal lineage CC45 is a predominant colonizer of healthy individuals in northern Europe and constitutes a highly basal cluster of the S. aureus population. Here, we report the complete genome sequence of S. aureus strain CA-347 (NRS648), a representative of the methicillin-resistant USA600 clone predominantly found in the United States

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics

Whole-genome sequencing to determine origin of multinational outbreak of Sarocladium kiliense bloodstream infections

We used whole-genome sequence typing (WGST) to investigate an outbreak of Sarocladium kiliense bloodstream infections (BSI) associated with receipt of contaminated antinausea medication among oncology patients in Colombia and Chile during 2013-2014. Twenty-five outbreak isolates (18 from patients and 7 from medication vials) and 11 control isolates unrelated to this outbreak were subjected to WGST to elucidate a source of infection. All outbreak isolates were nearly indistinguishable (≤5 single-nucleotide polymorphisms), and >21,000 single-nucleotide polymorphisms were identified from unrelated control isolates, suggesting a point source for this outbreak. S. kiliense has been previously implicated in healthcare-related infections; however, the lack of available typing methods has precluded the ability to substantiate point sources. WGST for outbreak investigation caused by eukaryotic pathogens without reference genomes or existing genotyping methods enables accurate source identification to guide implementation of appropriate control and prevention measures. © 2016, Centers for Disease Control and Prevention (CDC). All rights reserved

CAVEMol: an immersive 3D molecule viewer

"As the number of solved molecular structures deposited with the Protein Data Bank (PDB) increases, so too does the desire for more advanced ways of using this data. Traditional applications for viewing and manipulating molecular structures create a computer-generated model on a standard desktop computer screen. The display may employ some method of stereography to create the illusion of depth, but generally the user just sees a flat image. The user is able to interact with the molecule by magnifying it to see a particular area of interest, or by rotating it to see all sides of the molecule. The user may also be able to see animated changes in the molecule over time, or they may even be able to make modifications to the structure in real time. Regardless of the amount of control the user has over the molecule, however, one thing remains the same: the user experiences the molecule as though it were an object floating behind the monitor screen which they can indirectly control using a mouse or other pointing device. An immersive environment, on the other hand, provides a new paradigm for molecular visualization, allowing the user a much more realistic interaction with the molecule. The user becomes part of the viewing experience, traversing a molecule as though walking or flying within it. The molecule can completely surround them on all sides, giving them a true sense of the size and shape of the molecule in three dimensions. The user may also interact with the object directly, moving and rotating it with their hands rather than a mouse. This approach should prove particularly valuable for operations such as ""interactive docking,"" which allows a user to manipulate the interface between two molecules to identify favorable interaction sites. This thesis presents the design and implementation of CAVEMol, a molecular visualization application for immersive environments. I will also give an overview of molecular visualization and immersive environments, and then discuss future work that can be done in this area as well as applications where molecular visualization in an immersive environment can offer unique advantages."</p

Novel Odoribacter splanchnicus Strain and Its Outer Membrane Vesicles Exert Immunoregulatory Effects in vitro

Odoribacter splanchnicus, belonging to the order Bacteroidales, is a common, short-chain fatty acid producing member of the human intestinal microbiota. A decreased abundance of Odoribacter has been linked to different microbiota-associated diseases, such as non-alcoholic fatty liver disease, cystic fibrosis and inflammatory bowel disease (IBD). The type strain of O. splanchnicus has been genome-sequenced, but otherwise very little is known about this anaerobic bacterium. The species surfaces in many microbiota studies and, consequently, comprehension on its interactions with the host is needed. In this study, we isolated a novel strain of O. splanchnicus from a healthy fecal donor, identified it by genome sequencing and addressed its adhesive, epithelium reinforcing and immunoregulatory properties. Our results show that O. splanchnicus strain 57 is non-adherent to enterocytes or mucus, does not reinforce nor compromise Caco-2 monolayer integrity and most likely harbors penta-acylated, less endotoxic lipid A as part of its lipopolysaccharide (LPS) structure based on the lack of gene lpxM and in vitro results on low-level NF-κB activity. The studies by transmission electron microscopy revealed that O. splanchnicus produces outer membrane vesicles (OMV). O. splanchnicus cells, culture supernatant i.e., spent medium or OMVs did not induce interleukin-8 (IL-8) response in HT-29 enterocyte cells suggesting a very low proinflammatory capacity. On the contrary, the treatment of HT-29 cells with O. splanchnicus cells, spent medium or OMVs prior to exposure to Escherichia coli LPS elicited a significant decrease in IL-8 production as compared to E. coli LPS treatment alone. Moreover, O. splanchnicus spent supernatant induced IL-10 production by immune cells, suggesting anti-inflammatory activity. Our in vitro findings indicate that O. splanchnicus and its effector molecules transported in OMVs could potentially exert anti-inflammatory action in the gut epithelium. Taken together, O. splanchnicus seems to be a commensal with a primarily beneficial interaction with the host.Peer reviewe

Cryptic Microheteroresistance Explains Mycobacterium tuberculosis Phenotypic Resistance

RationaleMinority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays.ObjectivesTo determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations.MethodsWe used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group).Measurements and main resultsAmong 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n = 20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n = 18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n = 3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n = 53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (&lt;5%) subpopulations (n = 3/66) across both groups.ConclusionsCryptic minor variant mycobacterial subpopulations exist below the resolving capability of current drug susceptibility testing methodologies, and may explain an important proportion of false-negative resistance determinations

Mycobacterium tuberculosis Subculture Results in Loss of Potentially Clinically Relevant Heteroresistance.

Multidrug-resistant tuberculosis (TB) presents a major public health dilemma. Heteroresistance, the coexistence of drug-resistant and drug-susceptible strains or of multiple drug-resistant strains with discrete haplotypes, may affect accurate diagnosis and the institution of effective treatment. Subculture, or passage of cells onto fresh growth medium, is utilized to preserve Mycobacterium tuberculosis cell lines and is universally employed in TB diagnostics. The impact of such passages, typically performed in the absence of drug, on drug-resistant subpopulations is hypothesized to vary according to the competitive costs of genotypic resistance-associated variants. We applied ultradeep next-generation sequencing to 61 phenotypically rifampin-monoresistant (n = 17) and preextensively (n = 41) and extensively (n = 3) drug-resistant isolates with presumptive heteroresistance at two time points in serial subculture. We found significant dynamic loss of minor-variant resistant subpopulations across all analyzed resistance-determining regions, including eight isolates (13%) whose antibiogram data would have transitioned from resistant to susceptible for at least one drug through subculture. Surprisingly, some resistance-associated variants appeared to be selected for in subculture