Early virus detection and characterization is key to successful avian influenza virus (AIV) surveillance for the health of humans as well as domestic poultry. We explored a novel sampling approach and molecular strategy using sediment from wetlands and outdoor waterbodies on poultry farms as a population-level proxy of AIV activity in waterfowls. RNA was extracted using the MoBio RNA PowerSoil Total RNA isolation kit with additional chloroform extraction steps to reduce PCR inhibition. AIV matrix protein (MP) gene was detected in 42/345 (12.2%) samples by RT-qPCR; an additional 64 (18.6%) samples showed evidence of amplification below the threshold and were categorized as "suspect positive." Enrichment-based targeted resequencing (TR) identified AIV sequences in 79/345 (22.9%) samples. TR probes were designed for MP, hemagglutinin (HA), and neuraminidase (NA), however PB2 and PA were also identified. Although RT-qPCR and TR only had fair-moderate agreement, RT-qPCR positivity was predictive of TR-positivity both when using only strictly positive RT-qPCR samples (OR = 11.29) and when coding suspect positives as positive (OR = 7.56). This indicates that RT-qPCR could be used as a screening tool to select samples for virus characterization by TR and that future studies should consider RT-qPCR suspect positives to be positive samples for subsequent resequencing when avoiding false negatives is the priority, for instance in a diagnostic test, and to consider suspect positives to be negative samples when cost efficiency over a large number of samples is the priority, for instance in a surveillance program. A total of 13 HA (H1-7, H9-13, H16) and 9 NA (N1-9) subtypes were identified, with a maximum of 8 HA and 8 NA subtypes detected in a single sample. The optimized RNA extraction and targeted resequencing methods provided increased virus detection and subtyping characterization that could be implemented in an AIV surveillance system.
Proteome profiling and global protein-interaction approaches have significantly improved our knowledge of the protein interactomes of autophagy and other cellular stress-response pathways. New discoveries regarding protein complexes, interaction partners, interaction domains, and biological roles of players that are part of these pathways are emerging. The fourth Vancouver Autophagy Symposium showcased research that expands our understanding of the protein interaction networks and molecular mechanisms underlying autophagy and other cellular stress responses in the context of distinct stressors. In the keynote presentation, Dr. Wade Harper described his team's recent discovery of a novel reticulophagy receptor for selective autophagic degradation of the endoplasmic reticulum, and discussed molecular mechanisms involved in ribophagy and non-autophagic ribosomal turnover. In other presentations, both omic and targeted approaches were used to reveal molecular players of other cellular stress responses including amyloid body and stress granule formation, anastasis, and extracellular vesicle biogenesis. Additional topics included the roles of autophagy in disease pathogenesis, autophagy regulatory mechanisms, and crosstalk between autophagy and cellular metabolism in anti-tumor immunity. The relationship between autophagy and other cell stress responses remains a relatively unexplored area in the field, with future investigations required to understand how the various processes are coordinated and connected in cells and tissues.
Next-generation sequencing of solid tumors has revealed variable signatures of immunogenicity across tumors, but underlying molecular characteristics driving such variation are not fully understood. While expression of endogenous retrovirus (ERV)-containing transcripts can provide a source of tumor-specific neoantigen in some cancer models, associations between ERV levels and immunogenicity across different types of metastatic cancer are not well established. We performed bioinformatics analysis of genomic, transcriptomic and clinical data across an integrated cohort of 199 metastatic breast, colorectal and pancreatic ductal adenocarcinoma (PDAC) patient tumors. Within each cancer type, we identified a subgroup of viral mimicry tumors in which increased ERV levels were coupled with transcriptional signatures of autonomous antiviral response and immunogenicity. In addition, viral mimicry colorectal and pancreatic tumors showed increased expression of DNA demethylation gene TET2. Taken together, these data demonstrate the existence of an ERV-associated viral mimicry phenotype across three distinct metastatic cancer types, while indicating links between ERV abundance, epigenetic dysregulation and immunogenicity.
Despite continuing improvements in the management of classical Hodgkin lymphoma (cHL), relapse remains associated with a risk of lymphoma-related mortality. The biological composition of relapse tumour biopsies shows interpatient variability, which can be leveraged to design prognostic biomarkers. Here, we validated the RHL30 assay, a previously reported gene expression model in an independent cohort of 41 patients with relapsed cHL. Patients classified as high-risk by the RHL30 assay had inferior failure-free survival (FFS) after autologous stem cell transplantation (2-year FFS 41% vs. 92%, P = 0·035). The RHL30 model is a robust biomarker that risk-stratifies patients considered for autologous stem cell transplantation.
Background: Compared with second-generation sequencing technologies, third-generation single-molecule RNA sequencing has unprecedented advantages; the long reads it generates facilitate isoform-level transcript characterization. In particular, the Oxford Nanopore Technology sequencing platforms have become more popular in recent years owing to their relatively high affordability and portability compared with other third-generation sequencing technologies. To aid the development of analytical tools that leverage the power of this technology, simulated data provide a cost-effective solution with ground truth. However, a nanopore sequence simulator targeting transcriptomic data is not available yet.
Findings: We introduce Trans-NanoSim, a tool that simulates reads with technical and transcriptome-specific features learnt from nanopore RNA-sequncing data. We comprehensively benchmarked Trans-NanoSim on direct RNA and complementary DNA datasets describing human and mouse transcriptomes. Through comparison against other nanopore read simulators, we show the unique advantage and robustness of Trans-NanoSim in capturing the characteristics of nanopore complementary DNA and direct RNA reads.
Conclusions: As a cost-effective alternative to sequencing real transcriptomes, Trans-NanoSim will facilitate the rapid development of analytical tools for nanopore RNA-sequencing data. Trans-NanoSim and its pre-trained models are freely accessible at https://github.com/bcgsc/NanoSim.
Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-β receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.
Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the complete 5.5 Mbp mitochondrial genome of Sitka spruce (Picea sitchensis), to date, one of the largest mitochondrial genomes of a gymnosperm. We sequenced the whole genome using Oxford Nanopore MinION, and then identified contigs of mitochondrial origin assembled from these long reads based on sequence homology to the white spruce mitochondrial genome. The assembly graph shows a multipartite genome structure, composed of one smaller 168 kbp circular segment of DNA, and a larger 5.4 Mbp single component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination.
Purpose: Many rare ovarian cancer subtypes such as small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) have poor prognosis due to their aggressive nature and resistance to standard platinum and taxane based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.
Experimental design: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype high grade serous ovarian cancer (HGSC) to identify potential therapeutic targets. Immunohistochemistry of tissue microarrays were used as validation of ASS1 deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in-vitro using cell lines and patient derived xenograft mouse models representing SCCOHT.
Results: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared to HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12/31 cases, and expressed in less than 5% of the tumor cells in 9/31 cases. ASS1 deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in-vitro Furthermore, in two cell line mouse xenograft models and one patient derived mouse xenograft model of SCCOHT, once a week treatment of ADI-PEG20 (30mg/kg and 15mg/kg) inhibited tumor growth in-vivo Conclusions: Preclinical in-vitro and in-vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers including SCCOHT.
Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE-MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.