The Canadian VirusSeq Data Portal & Duotang: open resources for SARS-CoV-2 viral sequences and genomic epidemiology.

The COVID-19 pandemic led to a large global effort to sequence SARS-CoV-2 genomes from patient samples to track viral evolution and inform public health response. Millions of SARS-CoV-2 genome sequences have been deposited in global public repositories. The Canadian COVID-19 Genomics Network (CanCOGeN - VirusSeq), a consortium tasked with coordinating expanded sequencing of SARS-CoV-2 genomes across Canada early in the pandemic, created the Canadian VirusSeq Data Portal, with associated data pipelines and procedures, to support these efforts. The goal of VirusSeq was to allow open access to Canadian SARS-CoV-2 genomic sequences and enhanced, standardized contextual data that were unavailable in other repositories and that meet FAIR standards (Findable, Accessible, Interoperable and Reusable). In addition, the Portal data submission pipeline contains data quality checking procedures and appropriate acknowledgement of data generators that encourages collaboration. From inception to execution, the portal was developed with a conscientious focus on strong data governance principles and practices. Extensive efforts ensured a commitment to Canadian privacy laws, data security standards, and organizational processes. This Portal has been coupled with other resources like Viral AI and was further leveraged by the Coronavirus Variants Rapid Response Network (CoVaRR-Net) to produce a suite of continually updated analytical tools and notebooks. Here we highlight this Portal, including its contextual data not available elsewhere, and the 'Duotang', a web platform that presents key genomic epidemiology and modeling analyses on circulating and emerging SARS-CoV-2 variants in Canada. Duotang presents dynamic changes in variant composition of SARS-CoV-2 in Canada and by province, estimates variant growth, and displays complementary interactive visualizations, with a text overview of the current situation. The VirusSeq Data Portal and Duotang resources, alongside additional analyses and resources computed from the Portal (COVID-MVP, CoVizu), are all open-source and freely available. Together, they provide an updated picture of SARS-CoV-2 evolution to spur scientific discussions, inform public discourse, and support communication with and within public health authorities. They also serve as a framework for other jurisdictions interested in open, collaborative sequence data sharing and analyses.

Canadians' opinions towards COVID-19 data-sharing: a national cross-sectional survey.

OBJECTIVES: COVID-19 research has significantly contributed to pandemic response and the enhancement of public health capacity. COVID-19 data collected by provincial/territorial health authorities in Canada are valuable for research advancement yet not readily available to the public, including researchers. To inform developments in public health data-sharing in Canada, we explored Canadians' opinions of public health authorities sharing deidentified individual-level COVID-19 data publicly. DESIGN/SETTING/INTERVENTIONS/OUTCOMES: A national cross-sectional survey was administered in Canada in March 2022, assessing Canadians' opinions on publicly sharing COVID-19 datatypes. Market research firm Léger was employed for recruitment and data collection. PARTICIPANTS: Anyone greater than or equal to 18 years and currently living in Canada. RESULTS: 4981 participants completed the survey with a 92.3% response rate. 79.7% were supportive of provincial/territorial authorities publicly sharing deidentified COVID-19 data, while 20.3% were hesitant/averse/unsure. Datatypes most supported for being shared publicly were symptoms (83.0% in support), geographical region (82.6%) and COVID-19 vaccination status (81.7%). Datatypes with the most aversion were employment sector (27.4% averse), postal area (26.7%) and international travel history (19.7%). Generally supportive Canadians were characterised as being ≥50 years, with higher education, and being vaccinated against COVID-19 at least once. Vaccination status was the most influential predictor of data-sharing opinion, with respondents who were ever vaccinated being 4.20 times more likely (95% CI 3.21 to 5.48, p=0.000) to be generally supportive of data-sharing than those unvaccinated. CONCLUSIONS: These findings suggest that the Canadian public is generally favourable to deidentified data-sharing. Identifying factors that are likely to improve attitudes towards data-sharing are useful to stakeholders involved in data-sharing initiatives, such as public health agencies, in informing the development of public health communication and data-sharing policies. As Canada progresses through the COVID-19 pandemic, and with limited testing and reporting of COVID-19 data, it is essential to improve deidentified data-sharing given the public's general support for these efforts.

LGBTQ+ Students' Peer Victimization and Mental Health before and during the COVID-19 Pandemic.

Given the well-established health disparities between lesbian, gay, bisexual, transgender, queer, and gender-expansive (LGBTQ+) and cisgender, straight youth, scholars predicted the COVID-19 pandemic would disproportionately impact LGBTQ+ students. Yet, few studies have described changes in LGBTQ+ students' school experiences and well-being during the pandemic. Using survey data from 40,904 middle and high school students, we examined changes from before (2018) to during (2021) the pandemic in peer victimization, anxiety, and suicide attempts. We conducted bivariate and multivariate regression analyses to assess changes between the two time points among LGBTQ+ and cisgender, straight students. We found LGBTQ+ students experienced more peer victimization, anxiety, and suicide attempts at both points, before and during the pandemic, than cisgender, straight youth. However, LGBTQ+ students reported increased anxiety, decreased peer victimization, and decreased suicide attempts in 2021, during the pandemic, compared to pre-pandemic 2018 reports. These findings suggest pandemic-related school disruptions may have provided LGBTQ+ students some relief from the harmful effects of poor, in-person school climates.

Different Disease Endotypes in Phenotypically Similar Vasculitides Affecting Small-to-Medium Sized Blood Vessels.

Objectives: Chronic primary vasculitis describes a group of complex and rare diseases that are characterized by blood vessel inflammation. Classification of vasculitis subtypes is based predominantly on the size of the involved vessels and clinical phenotype. There is a recognized need to improve classification, especially for small-to-medium sized vessel vasculitides, that, ideally, is based on the underlying biology with a view to informing treatment. Methods: We performed RNA-Seq on blood samples from children (n = 41) and from adults (n = 11) with small-to-medium sized vessel vasculitis, and used unsupervised hierarchical clustering of gene expression patterns in combination with clinical metadata to define disease subtypes. Results: Differential gene expression at the time of diagnosis separated patients into two primary endotypes that differed in the expression of ~3,800 genes in children, and ~1,600 genes in adults. These endotypes were also present during disease flares, and both adult and pediatric endotypes could be discriminated based on the expression of just 20 differentially expressed genes. Endotypes were associated with distinct biological processes, namely neutrophil degranulation and T cell receptor signaling. Conclusions: Phenotypically similar subsets of small-to-medium sized vessel vasculitis may have different mechanistic drivers involving innate vs. adaptive immune processes. Discovery of these differentiating immune features provides a mechanistic-based alternative for subclassification of vasculitis.

Addressing Privacy Concerns in Sharing Viral Sequences and Minimum Contextual Data in a Public Repository During the COVID-19 Pandemic.

COVID-19 was declared to be a pandemic in March 2020 by the World Health Organization. Timely sharing of viral genomic sequencing data accompanied by a minimal set of contextual data is essential for informing regional, national, and international public health responses. Such contextual data is also necessary for developing, and improving clinical therapies and vaccines, and enhancing the scientific community's understanding of the SARS-CoV-2 virus. The Canadian COVID-19 Genomics Network (CanCOGeN) was launched in April 2020 to coordinate and upscale existing genomics-based COVID-19 research and surveillance efforts. CanCOGeN is performing large-scale sequencing of both the genomes of SARS-CoV-2 virus samples (VirusSeq) and affected Canadians (HostSeq). This paper addresses the privacy concerns associated with sharing the viral sequence data with a pre-defined set of contextual data describing the sample source and case attribute of the sequence data in the Canadian context. Currently, the viral genome sequences are shared by provincial public health laboratories and their healthcare and academic partners, with the Canadian National Microbiology Laboratory and with publicly accessible databases. However, data sharing delays and the provision of incomplete contextual data often occur because publicly releasing such data triggers privacy and data governance concerns. The CanCOGeN Ethics and Governance Expert Working Group thus has investigated several privacy issues cited by CanCOGeN data providers/stewards. This paper addresses these privacy concerns and offers insights primarily in the Canadian context, although similar privacy considerations also exist in other jurisdictions. We maintain that sharing viral sequencing data and its limited associated contextual data in the public domain generally does not pose insurmountable privacy challenges. However, privacy risks associated with reidentification should be actively monitored due to advancements in reidentification methods and the evolving pandemic landscape. We also argue that during a global health emergency such as COVID-19, privacy should not be used as a blanket measure to prevent such genomic data sharing due to the significant benefits it provides towards public health responses and ongoing research activities.

Preparing for Life: Plasma Proteome Changes and Immune System Development During the First Week of Human Life.

Neonates have heightened susceptibility to infections. The biological mechanisms are incompletely understood but thought to be related to age-specific adaptations in immunity due to resource constraints during immune system development and growth. We present here an extended analysis of our proteomics study of peripheral blood-plasma from a study of healthy full-term newborns delivered vaginally, collected at the day of birth and on day of life (DOL) 1, 3, or 7, to cover the first week of life. The plasma proteome was characterized by LC-MS using our established 96-well plate format plasma proteomics platform. We found increasing acute phase proteins and a reduction of respective inhibitors on DOL1. Focusing on the complement system, we found increased plasma concentrations of all major components of the classical complement pathway and the membrane attack complex (MAC) from birth onward, except C7 which seems to have near adult levels at birth. In contrast, components of the lectin and alternative complement pathways mainly decreased. A comparison to whole blood messenger RNA (mRNA) levels enabled characterization of mRNA and protein levels in parallel, and for 23 of the 30 monitored complement proteins, the whole blood transcript information by itself was not reflective of the plasma protein levels or dynamics during the first week of life. Analysis of immunoglobulin (Ig) mRNA and protein levels revealed that IgM levels and synthesis increased, while the plasma concentrations of maternally transferred IgG1-4 decreased in accordance with their in vivo half-lives. The neonatal plasma ratio of IgG1 to IgG2-4 was increased compared to adult values, demonstrating a highly efficient IgG1 transplacental transfer process. Partial compensation for maternal IgG degradation was achieved by endogenous synthesis of the IgG1 subtype which increased with DOL. The findings were validated in a geographically distinct cohort, demonstrating a consistent developmental trajectory of the newborn's immune system over the first week of human life across continents. Our findings indicate that the classical complement pathway is central for newborn immunity and our approach to characterize the plasma proteome in parallel with the transcriptome will provide crucial insight in immune ontogeny and inform new approaches to prevent and treat diseases.

Systems Biology Methods Applied to Blood and Tissue for a Comprehensive Analysis of Immune Response to Hepatitis B Vaccine in Adults.

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized, studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. Sampling included ~350 mL of blood, 12 microbiome samples, and lymph node fine needle aspirates obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, in vitro T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches-NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design.

Rhinovirus Infection Drives Complex Host Airway Molecular Responses in Children With Cystic Fibrosis.

Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro. Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF.

MDA-MB-231 Breast Cancer Cells Resistant to Pleurocidin-Family Lytic Peptides Are Chemosensitive and Exhibit Reduced Tumor-Forming Capacity.

Direct-acting anticancer (DAA) peptides are cytolytic peptides that show promise as novel anticancer agents. DAA peptides bind to anionic molecules that are abundant on cancer cells relative to normal healthy cells, which results in preferential killing of cancer cells. Due to the mechanism by which DAA peptides kill cancer cells, it was thought that resistance would be difficult to achieve. Here, we describe the generation and characterization of two MDA-MB-231 breast cancer cell-line variants with reduced susceptibility to pleurocidin-family and mastoparan DAA peptides. Peptide resistance correlated with deficiencies in peptide binding to cell-surface structures, suggesting that resistance was due to altered composition of the cell membrane. Peptide-resistant MDA-MB-231 cells were phenotypically distinct yet remained susceptible to chemotherapy. Surprisingly, neither of the peptide-resistant breast cancer cell lines was able to establish tumors in immune-deficient mice. Histological analysis and RNA sequencing suggested that tumorigenicity was impacted by alternations in angiogenesis and extracellular matrix composition in the peptide-resistant MDA-MB-231 variants. Collectively, these data further support the therapeutic potential of DAA peptides as adjunctive treatments for cancer.

Whole blood transcriptional responses of very preterm infants during late-onset sepsis.

BACKGROUND: Host immune responses during late-onset sepsis (LOS) in very preterm infants are poorly characterised due to a complex and dynamic pathophysiology and challenges in working with small available blood volumes. We present here an unbiased transcriptomic analysis of whole peripheral blood from very preterm infants at the time of LOS. METHODS: RNA-Seq was performed on peripheral blood samples (6-29 days postnatal age) taken at the time of suspected LOS from very preterm infants <30 weeks gestational age. Infants were classified based on blood culture positivity and elevated C-reactive protein concentrations as having confirmed LOS (n = 5), possible LOS (n = 4) or no LOS (n = 9). Bioinformatics and statistical analyses performed included pathway over-representation and protein-protein interaction network analyses. Plasma cytokine immunoassays were performed to validate differentially expressed cytokine pathways. RESULTS: The blood leukocyte transcriptional responses of infants with confirmed LOS differed significantly from infants without LOS (1,317 differentially expressed genes). However, infants with possible LOS could not be distinguished from infants with no LOS or confirmed LOS. Transcriptional alterations associated with LOS included genes involved in pathogen recognition (mainly TLR pathways), cytokine signalling (both pro-inflammatory and inhibitory responses), immune and haematological regulation (including cell death pathways), and metabolism (altered cholesterol biosynthesis). At the transcriptional-level cytokine responses during LOS were characterised by over-representation of IFN-α/ß, IFN-γ, IL-1 and IL-6 signalling pathways and up-regulation of genes for inflammatory responses. Infants with confirmed LOS had significantly higher levels of IL-1α and IL-6 in their plasma. CONCLUSIONS: Blood responses in very preterm infants with LOS are characterised by altered host immune responses that appear to reflect unbalanced immuno-metabolic homeostasis.

Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze.

Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases, including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present a potentially novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school-aged and school-aged wheezing phenotypes, characterized by aberrant migration patterns and reduced integrin α5ß1 expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of integrin α5ß1, where Akt activation enhanced repair and integrin α5ß1 expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5ß1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib - and its non-COX2-inhibiting analogue, dimethyl-celecoxib - stimulated the PI3K/Akt-integrin α5ß1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical data sets, the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K-integrin α5ß1 axis as a potentially novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial, since relevant animal models to test the crucial underlying premise are unavailable.

AB569, a nontoxic chemical tandem that kills major human pathogenic bacteria.

Antibiotic-resistant superbug bacteria represent a global health problem with no imminent solutions. Here we demonstrate that the combination (termed AB569) of acidified nitrite (A-NO2-) and Na2-EDTA (disodium ethylenediaminetetraacetic acid) inhibited all Gram-negative and Gram-positive bacteria tested. AB569 was also efficacious at killing the model organism Pseudomonas aeruginosa in biofilms and in a murine chronic lung infection model. AB569 was not toxic to human cell lines at bactericidal concentrations using a basic viability assay. RNA-Seq analyses upon treatment of P. aeruginosa with AB569 revealed a catastrophic loss of the ability to support core pathways encompassing DNA, RNA, protein, ATP biosynthesis, and iron metabolism. Electrochemical analyses elucidated that AB569 produced more stable SNO proteins, potentially explaining one mechanism of bacterial killing. Our data implicate that AB569 is a safe and effective means to kill pathogenic bacteria, suggesting that simple strategies could be applied with highly advantageous therapeutic/toxicity index ratios to pathogens associated with a myriad of periepithelial infections and related disease scenarios.

A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection.

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Dynamic molecular changes during the first week of human life follow a robust developmental trajectory.

Systems biology can unravel complex biology but has not been extensively applied to human newborns, a group highly vulnerable to a wide range of diseases. We optimized methods to extract transcriptomic, proteomic, metabolomic, cytokine/chemokine, and single cell immune phenotyping data from <1 ml of blood, a volume readily obtained from newborns. Indexing to baseline and applying innovative integrative computational methods reveals dramatic changes along a remarkably stable developmental trajectory over the first week of life. This is most evident in changes of interferon and complement pathways, as well as neutrophil-associated signaling. Validated across two independent cohorts of newborns from West Africa and Australasia, a robust and common trajectory emerges, suggesting a purposeful rather than random developmental path. Systems biology and innovative data integration can provide fresh insights into the molecular ontogeny of the first week of life, a dynamic developmental phase that is key for health and disease.

Characterization of Host Responses during Pseudomonas aeruginosa Acute Infection in the Lungs and Blood and after Treatment with the Synthetic Immunomodulatory Peptide IDR-1002.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial pneumonia and infects patients with cystic fibrosis. P. aeruginosa lung infections are difficult to treat due to bacterial resistance to antibiotics, and strains with multidrug resistance are becoming more prevalent. Here, we examined the use of a small host defense peptide, innate defense regulator 1002 (IDR-1002), in an acute P. aeruginosa lung infection in vivo IDR-1002 significantly reduced the bacterial burden in bronchoalveolar lavage fluid (BALF), as well as MCP-1 in BALF and serum, KC in serum, and interleukin 6 (IL-6) in BALF. Transcriptome sequencing (RNA-Seq) was conducted on lungs and whole blood, and the effects of P. aeruginosa, IDR-1002, and the combination of P. aeruginosa and IDR-1002 were evaluated. Differential gene expression analysis showed that P. aeruginosa increased multiple inflammatory and innate immune pathways, as well as affected hemostasis, matrix metalloproteinases, collagen biosynthesis, and various metabolism pathways in the lungs and/or blood. Infected mice treated with IDR-1002 had significant changes in gene expression compared to untreated infected mice, with fewer differentially expressed genes associated with the inflammatory and innate immune responses to microbial infection, and treatment also affected morphogenesis, certain metabolic pathways, and lymphocyte activation. Overall, these results showed that IDR-1002 was effective in treating P. aeruginosa acute lung infections and associated inflammation.

Broad-Spectrum Adaptive Antibiotic Resistance Associated with Pseudomonas aeruginosa Mucin-Dependent Surfing Motility.

Surfing motility is a novel form of surface adaptation exhibited by the nosocomial pathogen Pseudomonas aeruginosa in the presence of the glycoprotein mucin, which is found in high abundance at mucosal surfaces, especially those of the lungs of cystic fibrosis and bronchiectasis patients. Here, we investigated the adaptive antibiotic resistance of P. aeruginosa under conditions in which surfing occurs compared that in to cells undergoing swimming. P. aeruginosa surfing cells were significantly more resistant to several classes of antibiotics, including aminoglycosides, carbapenems, polymyxins, and fluoroquinolones. This was confirmed by incorporation of antibiotics into growth medium, which revealed a concentration-dependent inhibition of surfing motility that occurred at concentrations much higher than those needed to inhibit swimming. To investigate the basis of resistance, transcriptome sequencing (RNA-Seq) was performed and revealed that surfing influenced the expression of numerous genes. Included among genes dysregulated under surfing conditions were multiple genes from the Pseudomonas resistome; these genes are known to affect antibiotic resistance when mutated. Screening transposon mutants in these surfing-dysregulated resistome genes revealed that several of these mutants exhibited changes in susceptibility to one or more antibiotics under surfing conditions, consistent with a contribution to the observed adaptive resistance. In particular, several mutants in resistome genes, including armR, recG, atpB, clpS, nuoB, and certain hypothetical genes, such as PA5130, PA3576, and PA4292, showed contributions to broad-spectrum resistance under surfing conditions and could be complemented by their respective cloned genes. Therefore, we propose that surfing adaption led to extensive multidrug adaptive resistance as a result of the collective dysregulation of diverse genes.

MetaBridge: enabling network-based integrative analysis via direct protein interactors of metabolites.

Summary: Here, we present MetaBridge, a tool that collates protein interactors (curated metabolite-enzyme interactions) that influence the levels of specific metabolites including both biosynthetic and degradative enzymes. This enables network-based integrative analysis of metabolomics data with other omics data types. MetaBridge is designed to complement a systems-biology approach to analysis, pairing well with network analysis tools such as NetworkAnalyst.ca, but can be used in any bioinformatics workflow. Availability and implementation: MetaBridge has been implemented as a web tool at https://www.metabridge.org, and the source code is available at https://github.com/samhinshaw/metabridge_shiny (GNU GPLv3).

High-throughput detection of RNA processing in bacteria.

BACKGROUND: Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. RESULTS: The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5' ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely - 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. CONCLUSIONS: This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com .

Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.