Systematic reanalysis of copy number losses of uncertain clinical significance.

BACKGROUND: Reanalysis of exome/genome data improves diagnostic yield. However, the value of reanalysis of clinical array comparative genomic hybridisation (aCGH) data has never been investigated. Case-by-case reanalysis can be challenging in busy diagnostic laboratories. METHODS AND RESULTS: We harmonised historical postnatal clinical aCGH results from ~16 000 patients tested via our diagnostic laboratory over ~7 years with current clinical guidance. This led to identification of 37 009 copy number losses (CNLs) including 33 857 benign, 2173 of uncertain significance and 979 pathogenic. We found benign CNLs to be significantly less likely to encompass haploinsufficient genes compared with the pathogenic or CNLs of uncertain significance in our database. Based on this observation, we developed a reanalysis pipeline using up-to-date disease association data and haploinsufficiency scores and shortlisted 207 CNLs of uncertain significance encompassing at least one autosomal dominant disease-gene associated with haploinsufficiency or loss-of-function mechanism. Clinical scientist reviews led to reclassification of 15 CNLs of uncertain significance as pathogenic or likely pathogenic. This was ~0.7% of the starting cohort of 2173 CNLs of uncertain significance and 7.2% of 207 shortlisted CNLs. The reclassified CNLs included first cases of CNV-mediated disease for some genes where all previously described cases involved only point variants. Interestingly, some CNLs could not be reclassified because the phenotypes of patients with CNLs seemed distinct from the known clinical features resulting from point variants, thus raising questions about accepted underlying disease mechanisms. CONCLUSIONS: Reanalysis of clinical aCGH data increases diagnostic yield.

Genome analysis provides insights into microaerobic toluene-degradation pathway of Zoogloea oleivorans BucT.

Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z . oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.

Danger in the vapor? ECMO for adolescents with status asthmaticus after vaping.

Introduction: Electronic nicotine delivery systems (ENDS) use is on the rise in the adolescent and young adult populations, especially in the wake of sweet flavored ENDS solutions and youth-targeted marketing. While the extent of effect of ENDS use and aerosolized flavorings on airway epithelium is not known, there remains significant concern that use of ENDS adversely affects airway epithelial function, particularly in populations with asthma.Case Study: In this case series, we review two cases of adolescents with history of recent and past ENDS use and asthma who required veno-venous extracorporeal membrane oxygenation (VV-ECMO) for status asthmaticus in the year 2018.Results: Both patients experienced hypercarbic respiratory failure requiring VV-ECMO secondary to their status asthmaticus, with slow recovery on extensive bronchodilator and steroid regimens. They both recovered back to respiratory baseline and were counseled extensively on cessation of ENDS use.Conclusion: While direct causation by exposure to ENDS cannot be determined, exposure likely contributed to symptoms. Based on the severity of these cases and their potential relationship with ENDS use, we advocate for increased physician screening of adolescents for ENDS use, patient and parent education on the risks of use, and family cessation counseling.

Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems.

It has recently been suggested that oxygenic dismutation of NO into N2 and O2 may occur in the anaerobic methanotrophic "Candidatus Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to "Ca Methylomirabilis oxyfera" and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from "Ca Methylomirabilis oxyfera" and HdN1, the novel environmental nod sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinol-dependent NO reductase [qNor] and cytochrome c-dependent NO reductase [cNor]) genes. The abundance of nod genes in the investigated samples ranged from 1.6 × 107 to 5.2 × 1010 copies · g-1 (wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems. IMPORTANCE: NO dismutation into N2 and O2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methane-oxidizing bacterium "Ca Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future.

Elucidating carbon sources driving microbial metabolism during oil sands reclamation.

Microbial communities play key roles in remediation and reclamation of contaminated environments via biogeochemical cycling of organic and inorganic components. Understanding the trends in in situ microbial community abundance, metabolism and carbon sources is therefore a crucial component of effective site management. The focus of this study was to use radiocarbon analysis to elucidate the carbon sources driving microbial metabolism within the first pilot wetland reclamation project in the Alberta oil sands region where the observation of H2S had indicated the occurrence of microbial sulphate reduction. The reclamation project involved construction of a three compartment system consisting of a freshwater wetland on top of a sand cap overlying a composite tailings (CT) deposit. Radiocarbon analysis demonstrated that both dissolved and sediment associated organic carbon associated with the deepest compartments (the CT and sand cap) was primarily fossil (Δ14C = -769 to -955‰) while organic carbon in the overlying peat was hundreds to thousands of years old (Δ14C = -250 to -350‰). Radiocarbon contents of sediment associated microbial phospholipid fatty acids (PLFA) were consistent with the sediment bulk organic carbon pools (Peat: Δ14CPLFA = -257‰; Sand cap Δ14CPLFA = -805‰) indicating that these microbes were using sediment associated carbon. In contrast, microbial PLFA grown on biofilm units installed in wells within the deepest compartments contained much more modern carbon that the associated bulk carbon pools. This implied that the transfer of relatively more modern carbon was stimulating the microbial community at depth within the system. Correlation between cellular abundance estimates based on PLFA concentrations and the Δ14CPLFA indicated that the utilization of this more modern carbon was stimulating the microbial community at depth. These results highlight the importance of understanding the occurrence and potential outcomes of the introduction of relatively bioavailable carbon to mine wastes in order to predict and manage the performance of reclamation strategies.

Draft genomes of meropenem-resistant Pseudomonads from the cloacae of turtles in Lake Opinicon, Canada.

We report the draft genome sequences of seven meropenem-resistant bacterial isolates from the cloacae of painted and musk turtles at Lake Opinicon, Canada. This study improves our understanding of the spread of meropenem-resistant bacteria in the wild.

High-throughput transcriptomics toxicity assessment of eleven data-poor bisphenol A alternatives.

Bisphenol A (BPA), a widely used chemical in the production of plastics and epoxy resins, has garnered significant attention due to its association with adverse health effects, particularly its endocrine-disrupting properties. Regulatory measures aimed at reducing human exposure to BPA have led to a proliferation of alternative chemicals used in various consumer and industrial products. While these alternatives serve to reduce BPA exposure, concerns have arisen regarding their safety and potential toxicity as regrettable substitutes. Previous efforts have demonstrated that in vitro high-throughput transcriptomics (HTTr) studies can be used to assess the endocrine-disrupting potential of BPA alternatives, and this strategy produces transcriptomic points-of-departure (tPODs) that are protective of human health when compared to the PODs from traditional rodent studies. In this study, we used in vitro HTTr to assess the potential for toxicity of eleven data-poor legacy chemicals sharing structural similarities to BPA. Human breast cancer MCF-7 cells were exposed to BPA and 11 alternatives at concentrations ranging from 0.1 to 25 µM to assess toxicity. Analysis of global transcriptomic changes and a previously characterized estrogen receptor alpha (ERα) transcriptomic biomarker signature revealed that 9 of 11 chemicals altered gene expression relative to controls. One of the chemicals (2,4'-Bisphenol A) activated the ERα biomarker at the same concentration as BPA (i.e., 4,4'-BPA) but was deemed to be more potent as it induced global transcriptomic changes at lower concentrations. These results address data gaps in support of ongoing screening assessments to identify BPA alternatives with hazard potential and help to identify potential candidates that may serve as safer alternatives.

Interstitial lung disease in children with Rubinstein-Taybi syndrome.

INTRODUCTION: Rubinstein-Taybi syndrome (RSTS) is a rare genetic syndrome caused primarily by a mutation in the CREBBP gene found on chromosome 16. Patients with RSTS are at greater risk for a variety of medical problems, including upper airway obstruction and aspiration. Childhood interstitial lung disease (ILD) thus far has not been definitively linked to RSTS. Here we present three patients with RSTS who developed ILD and discuss possible mechanisms by which a mutation in CREBBP may be involved in the development of ILD. METHODS: Routine hematoxylin and eosin staining was performed on lung biopsy tissue for histological analysis. Immunofluorescent staining was performed on lung biopsy tissue for markers of fibrosis, surfactant deficiency and histone acetylation. Cases 1 and 2 had standard clinical microarray analysis. Case 3 had whole exome sequencing. Bioinformatics analyses were performed to identify possible causative genes using ToppGene. RESULTS: Computed tomography images in all cases showed consolidated densities overlying ground glass opacities. Lung histopathology revealed accumulation of proteinaceous material within alveolar spaces, evidence of fibrosis, and increased alveolar macrophages. Immunofluorescent staining showed increase in surfactant protein C staining, patchy areas of increased anti-smooth muscle antibody staining, and increased staining for acetylated histone 2 and histone 3 lysine 9. DISCUSSION: Clinical characteristics, radiographic imaging, lung histopathology, and immunofluorescent staining results shared by all cases demonstrated findings consistent with ILD. Immunofluorescent staining suggests two possible mechanisms for the development of ILD: abnormal surfactant metabolism and/or persistent activation of myofibroblasts. These two pathways could be related to dysfunctional CREBBP protein.

A Gut Feeling: Abdominal Symptoms as an Initial Presentation of EVALI.

Electronic cigarette or vaping product use-associated lung injury (EVALI) is a disease process that has become prevalent in the United States.1 The Centers for Disease Control and Prevention reported there have been almost 2700 cases of this condition in the United States as of January 14, 2020, with >50% of these patients aged ≤24.2 We present a 13-year-old boy with a history of functional abdominal pain who presented with recurrent episodes of nausea, emesis, periodic fevers, and severe episodic abdominal pain after a 12-month history of significant electronic cigarette use. On admission, he had severe abdominal pain and appeared anxious. A computed tomography scan of the abdomen was unremarkable, but a computed tomography scan of the chest demonstrated both multifocal ground-glass and crazy-paving pulmonary opacities bilaterally, with scattered septal thickening and dependent bibasilar opacities associated with volume loss. Inflammatory markers were significantly elevated, and cell counts were remarkable for leukocytosis and neutrophilia. The patient was ultimately diagnosed with EVALI and treated with intravenous methylprednisolone, resulting in improvement. This is an example of a case of EVALI in an adolescent, in which the presenting symptoms are largely gastrointestinal. It is important to keep EVALI in the differential diagnosis of patients who exhibit gastrointestinal symptoms, have markers of increased systemic inflammation, and endorse a history of vaping or are in the age range of electronic cigarette users. Although obtaining an accurate history of vaping can be challenging in the pediatric population, it is especially critical to do so.

Nitric Oxide Dismutase (nod) Genes as a Functional Marker for the Diversity and Phylogeny of Methane-Driven Oxygenic Denitrifiers.

Oxygenic denitrification represents a new route in reductive nitrogen turnover which differs from canonical denitrification in how nitric oxide (NO) is transformed into dinitrogen gas. Instead of NO reduction via N2O to N2, NO is proposed to be directly disproportionated into N2 and O2 in oxygenic denitrification, catalyzed by the putative NO dismutase (Nod). Although a high diversity of nod genes has been recovered from various environments, still little is known about the niche partitioning and ecophysiology of oxygenic denitrifiers. One constraint is that nod as a functional marker for oxygenic denitrifiers is not well established. To address this issue, we compared the diversity and phylogeny of nod, 16S rRNA and pmoA gene sequences of four NC10 enrichments that are capable of methane-driven oxygenic denitrification and one environmental sample. The phylogenies of nod, 16S rRNA and pmoA genes of these cultures were generally congruent. The diversity of NC10 bacteria inferred from different genes was also similar in each sample. A new set of NC10-specific nod primers was developed and used in qPCR. The abundance of NC10 bacteria inferred from nod genes was constantly lower than via 16S rRNA genes, but the difference was within one order of magnitude. These results suggest that nod is a suitable molecular marker for studying the diversity and phylogeny of methane-driven oxygenic denitrifiers, the further investigation of which may be of value to develop enhanced strategies for sustainable nitrogen or methane removal.

Transcriptome-Stable Isotope Probing Provides Targeted Functional and Taxonomic Insights Into Microaerobic Pollutant-Degrading Aquifer Microbiota.

While most studies using RNA-stable isotope probing (SIP) to date have focused on ribosomal RNA, the detection of 13C-labeled mRNA has rarely been demonstrated. This approach could alleviate some of the major caveats of current non-target environmental "omics." Here, we demonstrate the feasibility of total RNA-SIP in an experiment where hydrocarbon-degrading microbes from a BTEX-contaminated aquifer were studied in microcosms with 13C-labeled toluene under microoxic conditions. From the total sequencing reads (∼30 mio. reads per density-resolved RNA fraction), an average of 1.2% of reads per sample were identified as non-rRNA, including mRNA. Members of the Rhodocyclaceae (including those related to Quatrionicoccus spp.) were most abundant and enriched in 13C-rRNA, while well-known aerobic degraders such as Pseudomonas spp. remained unlabeled. Transcripts related to cell motility, secondary metabolite formation and xenobiotics degradation were highly labeled with 13C. mRNA of phenol hydroxylase genes were highly labeled and abundant, while other transcripts of toluene-activation were not detected. Clear labeling of catechol 2,3-dioxygenase transcripts supported previous findings that some of these extradiol dioxygenases were adapted to low oxygen concentrations. We introduce a novel combination of total RNA-SIP with calculation of transcript-specific enrichment factors (EFs) in 13C-RNA, enabling a targeted approach to process-relevant gene expression in complex microbiomes.

RNA-stable isotope probing: from carbon flow within key microbiota to targeted transcriptomes.

Stable isotope probing of RNA has enthused researchers right from its first introduction in 2002. The concept of a labelling-based detection of process-targeted microbes independent of cellular replication or growth has allowed for a much more direct handle on functionally relevant microbiota than by labelling of other biomarkers. This has led to a widespread application of the technology, and breakthroughs in our understanding of carbon flow in natural microbiomes, autotrophic and heterotrophic physiologies, microbial food webs, host-microbe interactions and environmental biotechnology. Recent studies detecting labelled mRNA demonstrate that RNA-SIP is not limited to the analysis of rRNA, but is currently developing towards an approach for accessing targeted transcriptomes. In combination with next-generation sequencing and other methodological advances, RNA-SIP will continue to deliver invaluable insights into the functioning of microbial communities.