Puromycin reveals a distinct conformation of neuronal ribosomes.

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

Real-time evaluation of signal accuracy in wastewater surveillance of pathogens with high rates of mutation.

Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.

SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model.

Extracellular vesicles (EVs) are entering the clinical arena as novel biologics for infectious diseases, potentially serving as the immunogenic components of next generation vaccines. However, relevant human assays to evaluate the immunogenicity of EVs carrying viral antigens are lacking, contributing to challenges in translating rodent studies to human clinical trials. Here, we engineered EVs to carry SARS-CoV-2 Spike to evaluate the immunogenicity of antigen-carrying EVs using human peripheral blood mononuclear cells (PBMCs). Delivery of Spike EVs to PBMCs resulted in specific immune cell activation as assessed through T cell activation marker expression. Further, Spike EVs were taken up largely by antigen-presenting cells (monocytes, dendritic cells and B cells). Taken together, this human PBMC-based system models physiologically relevant pathways of antigen delivery, uptake and presentation. In summary, the current study highlights the suitability of using human PBMCs for evaluating the immunogenicity of EVs engineered to carry antigens for infectious disease therapeutics.

Impact of coagulation on SARS-CoV-2 and PMMoV viral signal in wastewater solids.

Wastewater surveillance (WWS) of SARS-CoV-2 has become a crucial tool for monitoring COVID-19 cases and outbreaks. Previous studies have indicated that SARS-CoV-2 RNA measurement from testing solid-rich primary sludge yields better sensitivity compared to testing wastewater influent. Furthermore, measurement of pepper mild mottle virus (PMMoV) signal in wastewater allows for precise normalization of SARS-CoV-2 viral signal based on solid content, enhancing disease prevalence tracking. However, despite the widespread adoption of WWS, a knowledge gap remains regarding the impact of ferric sulfate coagulation, commonly used in enhanced primary clarification, the initial stage of wastewater treatment where solids are sedimented and removed, on SARS-CoV-2 and PMMoV quantification in wastewater-based epidemiology. This study examines the effects of ferric sulfate addition, along with the associated pH reduction, on the measurement of SARS-CoV-2 and PMMoV viral measurements in wastewater primary clarified sludge through jar testing. Results show that the addition of Fe3+ concentrations in the conventional 0 to 60 mg/L range caused no effect on SARS-CoV-2 N1 and N2 gene region measurements in wastewater solids. However, elevated Fe3+ concentrations were shown to be associated with a statistically significant increase in PMMoV viral measurements in wastewater solids, which consequently resulted in the underestimation of PMMoV-normalized SARS-CoV-2 viral signal measurements (N1 and N2 copies/copies of PMMoV). The observed pH reduction from coagulant addition did not contribute to the increased PMMoV measurements, suggesting that this phenomenon arises from the partitioning of PMMoV viral particles into wastewater solids.

Rapidly developed, optimized, and applied wastewater surveillance system for real-time monitoring of low-incidence, high-impact MPOX outbreak.

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Wastewater surveillance monitoring of SARS-CoV-2 variants of concern and dynamics of transmission and community burden of COVID-19.

Wastewater-based surveillance is a valuable approach for monitoring COVID-19 at community level. Monitoring SARS-CoV-2 variants of concern (VOC) in wastewater has become increasingly relevant when clinical testing capacity and case-based surveillance are limited. In this study, we ascertained the turnover of six VOC in Alberta wastewater from May 2020 to May 2022. Wastewater samples from nine wastewater treatment plants across Alberta were analysed using VOC-specific RT-qPCR assays. The performance of the RT-qPCR assays in identifying VOC in wastewater was evaluated against next generation sequencing. The relative abundance of each VOC in wastewater was compared to positivity rate in COVID-19 testing. VOC-specific RT-qPCR assays performed comparatively well against next generation sequencing; concordance rates ranged from 89% to 98% for detection of Alpha, Beta, Gamma, Omicron BA.1 and Omicron BA.2, with a slightly lower rate of 85% for Delta (p < 0.01). Elevated relative abundance of Alpha, Delta, Omicron BA.1 and BA.2 were each associated with increased COVID-19 positivity rate. Alpha, Delta and Omicron BA.2 reached 90% relative abundance in wastewater within 80, 111 and 62 days after their initial detection, respectively. Omicron BA.1 increased more rapidly, reaching a 90% relative abundance in wastewater after 35 days. Our results from VOC surveillance in wastewater correspond with clinical observations that Omicron is the VOC with highest disease burden over the shortest period in Alberta to date. The findings suggest that changes in relative abundance of a VOC in wastewater can be used as a supplementary indicator to track and perhaps predict COVID-19 burden in a population.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays.

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

Adaptation of transgene mRNA translation boosts the anticancer efficacy of oncolytic HSV1.

BACKGROUND: Transgenes deliver therapeutic payloads to improve oncolytic virus immunotherapy. Transgenes encoded within oncolytic viruses are designed to be highly transcribed, but protein synthesis is often negatively affected by viral infection, compromising the amount of therapeutic protein expressed. Studying the oncolytic herpes simplex virus-1 (HSV1), we found standard transgene mRNAs to be suboptimally translated in infected cells. METHODS: Using RNA-Seq reads, we determined the transcription start sites and 5'leaders of HSV1 genes and uncovered the US11 5'leader to confer superior activity in translation reporter assays. We then incorporated this 5'leader into GM-CSF expression cassette in oncolytic HSV1 and compared the translationally adapted oncolytic virus with the conventional, leaderless, virus in vitro and in mice. RESULTS: Inclusion of the US11 5'leader in the GM-CSF transgene incorporated into HSV1 boosted translation in vitro and in vivo. Importantly, treatment with US11 5'leader-GM-CSF oncolytic HSV1 showed superior antitumor immune activity and improved survival in a syngeneic mouse model of colorectal cancer as compared with leaderless-GM-CSF HSV1. CONCLUSIONS: Our study demonstrates the therapeutic value of identifying and integrating platform-specific cis-acting sequences that confer increased protein synthesis on transgene expression.

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities.

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) will have greater value as an important diagnostic tool. An in-depth analysis and understanding of the metrics derived from WWS is required to interpret and utilize WWS-acquired data effectively (McClary-Gutierrez et al., 2021; O'Keeffe, 2021). In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven cities in Canada over periods ranging from 8 to 21 months. This work demonstrates that significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing (resulting in a reduction to testing access and a reduction in the number of daily tests) in these communities, despite increases in the wastewater signal. Furthermore, the WC ratio decreased significantly in 6 of the 7 studied locations, serving as a potential signal of the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community (40-60 % allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community (40-60 % allelic proportion). Finally, a significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant's greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when community immunity was high. The WC ratio, used as an additional monitoring metric, could complement clinical case counts and wastewater signals as individual metrics in its potential ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.

Municipal and neighbourhood level wastewater surveillance and subtyping of an influenza virus outbreak.

Recurrent influenza epidemics and pandemic potential are significant risks to global health. Public health authorities use clinical surveillance to locate and monitor influenza and influenza-like cases and outbreaks to mitigate hospitalizations and deaths. Currently, global integration of clinical surveillance is the only reliable method for reporting influenza types and subtypes to warn of emergent pandemic strains. The utility of wastewater surveillance (WWS) during the COVID-19 pandemic as a less resource intensive replacement or complement for clinical surveillance has been predicated on analyzing viral fragments in wastewater. We show here that influenza virus targets are stable in wastewater and partitions favorably to the solids fraction. By quantifying, typing, and subtyping the virus in municipal wastewater and primary sludge during a community outbreak, we forecasted a citywide flu outbreak with a 17-day lead time and provided population-level viral subtyping in near real-time to show the feasibility of influenza virus WWS at the municipal and neighbourhood levels in near real time using minimal resources and infrastructure.

Understanding the dynamic relation between wastewater SARS-CoV-2 signal and clinical metrics throughout the pandemic.

Wastewater surveillance (WWS) of SARS-CoV-2 was proven to be a reliable and complementary tool for population-wide monitoring of COVID-19 disease incidence but was not as rigorously explored as an indicator for disease burden throughout the pandemic. Prior to global mass immunization campaigns and during the spread of the wildtype COVID-19 and the Alpha variant of concern (VOC), viral measurement of SARS-CoV-2 in wastewater was a leading indicator for both COVID-19 incidence and disease burden in communities. As the two-dose vaccination rates escalated during the spread of the Delta VOC in Jul. 2021 through Dec. 2021, relations weakened between wastewater signal and community COVID-19 disease incidence and maintained a strong relationship with clinical metrics indicative of disease burden (new hospital admissions, ICU admissions, and deaths). Further, with the onset of the vaccine-resistant Omicron BA.1 VOC in Dec. 2021 through Mar. 2022, wastewater again became a strong indicator of both disease incidence and burden during a period of limited natural immunization (no recent infection), vaccine escape, and waned vaccine effectiveness. Lastly, with the populations regaining enhanced natural and vaccination immunization shortly prior to the onset of the Omicron BA.2 VOC in mid-Mar 2022, wastewater is shown to be a strong indicator for both disease incidence and burden. Hospitalization-to-wastewater ratio is further shown to be a good indicator of VOC virulence when widespread clinical testing is limited. In the future, WWS is expected to show moderate indication of incidence and strong indication of disease burden in the community during future potential seasonal vaccination campaigns.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

miR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages.

BACKGROUND: A significant burden of atherosclerotic disease is driven by inflammation. Recently, microRNAs (miRNAs) have emerged as important factors driving and protecting from atherosclerosis. miR-223 regulates cholesterol metabolism and inflammation via targeting both cholesterol biosynthesis pathway and NFkB signaling pathways; however, its role in atherosclerosis has not been investigated. We hypothesize that miR-223 globally regulates core inflammatory pathways in macrophages in response to inflammatory and atherogenic stimuli thus limiting the progression of atherosclerosis. METHODS AND RESULTS: Loss of miR-223 in macrophages decreases Abca1 gene and protein expression as well as cholesterol efflux to apoA1 (Apolipoprotein A1) and enhances proinflammatory gene expression. In contrast, overexpression of miR-223 promotes the efflux of cholesterol and macrophage polarization toward an anti-inflammatory phenotype. These beneficial effects of miR-223 are dependent on its target gene, the transcription factor Sp3. Consistent with the antiatherogenic effects of miR-223 in vitro, mice receiving miR223-/- bone marrow exhibit increased plaque size, lipid content, and circulating inflammatory cytokines (ie, IL-1ß). Deficiency of miR-223 in bone marrow-derived cells also results in an increase in circulating pro-atherogenic cells (total monocytes and neutrophils) compared with control mice. Furthermore, the expression of miR-223 target gene (Sp3) and pro-inflammatory marker (Il-6) are enhanced whereas the expression of Abca1 and anti-inflammatory marker (Retnla) are reduced in aortic arches from mice lacking miR-223 in bone marrow-derived cells. In mice fed a high-cholesterol diet and in humans with unstable carotid atherosclerosis, the expression of miR-223 is increased. To further understand the molecular mechanisms underlying the effect of miR-223 on atherosclerosis in vivo, we characterized global RNA translation profile of macrophages isolated from mice receiving wild-type or miR223-/- bone marrow. Using ribosome profiling, we reveal a notable upregulation of inflammatory signaling and lipid metabolism at the translation level but less significant at the transcription level. Analysis of upregulated genes at the translation level reveal an enrichment of miR-223-binding sites, confirming that miR-223 exerts significant changes in target genes in atherogenic macrophages via altering their translation. CONCLUSIONS: Our study demonstrates that miR-223 can protect against atherosclerosis by acting as a global regulator of RNA translation of cholesterol efflux and inflammation pathways.

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Transcriptional profiling of macrophages reveals distinct parasite stage-driven signatures during early infection by Leishmania donovani.

Macrophages undergo swift changes in mRNA abundance upon pathogen invasion. Herein we describe early remodelling of the macrophage transcriptome during infection by amastigotes or promastigotes of Leishmania donovani. Approximately 10-16% of host mRNAs were differentially modulated in L. donovani-infected macrophages when compared to uninfected controls. This response was partially stage-specific as a third of changes in mRNA abundance were either exclusively driven by one of the parasite forms or significantly different between them. Gene ontology analyses identified categories associated with immune functions (e.g. antigen presentation and leukocyte activation) among significantly downregulated mRNAs during amastigote infection while cytoprotective-related categories (e.g. DNA repair and apoptosis inhibition) were enriched in upregulated transcripts. Interestingly a combination of upregulated (e.g. cellular response to IFNß) and repressed (e.g. leukocyte activation, chemotaxis) immune-related transcripts were overrepresented in the promastigote-infected dataset. In addition, Ingenuity Pathway Analysis (IPA) associated specific mRNA subsets with a number of upstream transcriptional regulators predicted to be modulated in macrophages infected with L. donovani amastigotes (e.g. STAT1 inhibition) or promastigotes (e.g. NRF2, IRF3, and IRF7 activation). Overall, our results indicate that early parasite stage-driven transcriptional remodelling in macrophages contributes to orchestrate both protective and deleterious host cell responses during L. donovani infection.

Detection of the Omicron (B.1.1.529) variant of SARS-CoV-2 in aircraft wastewater.

On the 26th of November 2021, the World Health Organization (WHO) designated the newly detected B.1.1.529 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the Omicron Variant of Concern (VOC). The genome of the Omicron VOC contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and potential to evade immune responses developed for previous VOCs such as Alpha and Delta. In the days since the designation of B.1.1.529 as a VOC, infections with the lineage have been reported in countries around the globe and many countries have implemented travel restrictions and increased border controls in response. We putatively detected the Omicron variant in an aircraft wastewater sample from a flight arriving to Darwin, Australia from Johannesburg, South Africa on the 25th of November 2021 via positive results on the CDC N1, CDC N2, and del(69-70) RT-qPCR assays per guidance from the WHO. The Australian Northern Territory Health Department detected one passenger onboard the flight who was infected with SARS-CoV-2, which was determined to be the Omicron VOC by sequencing of a nasopharyngeal swab sample. Subsequent sequencing of the aircraft wastewater sample using the ARTIC V3 protocol with Nanopore and ATOPlex confirmed the presence of the Omicron variant with a consensus genome that clustered with the B.1.1.529 BA.1 sub-lineage. Our detection and confirmation of a single onboard Omicron infection via aircraft wastewater further bolsters the important role that aircraft wastewater can play as an independent and unintrusive surveillance point for infectious diseases, particularly coronavirus disease 2019.

The highs and lows of ionizing radiation and its effects on protein synthesis.

Ionizing radiation (IR) is a constant feature of our environment and one that can dramatically affect organismal health and development. Although the impacts of high-doses of IR on mammalian cells and systems have been broadly explored, there are still challenges in accurately quantifying biological responses to IR, especially in the low-dose range to which most individuals are exposed in their lifetime. The resulting uncertainty has led to the entrenchment of conservative radioprotection policies around the world. Thus, uncovering long-sought molecular mechanisms and tissue responses that are targeted by IR could lead to more informed policymaking and propose new therapeutic avenues for a variety of pathologies. One often overlooked target of IR is mRNA translation, a highly regulated cellular process that consumes more than 40% of the cell's energy. In response to environmental stimuli, regulation of mRNA translation allows for precise and rapid changes to the cellular proteome, and unsurprisingly high-dose of IR was shown to trigger a severe reprogramming of global protein synthesis allowing the cell to conserve energy by preventing the synthesis of unneeded proteins. Nonetheless, under these conditions, certain mRNAs encoding specific proteins are translationally favoured to produce the factors essential to repair the cell or send it down the path of no return through programmed cell death. Understanding the mechanisms controlling protein synthesis in response to varying doses of IR could provide novel insights into how this stress-mediated cellular adaptation is regulated and potentially uncover novel targets for radiosensitization or radioprotection. Here, we review the current literature on the effects of IR at both high- and low-dose on the mRNA translation machinery.

Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity.

COVID-19 wastewater surveillance in rural communities: Comparison of lagoon and pumping station samples.

Wastewater-based epidemiology/wastewater surveillance has been a topic of significant interest over the last year due to its application in SARS-CoV-2 surveillance to track prevalence of COVID-19 in communities. Although SARS-CoV-2 surveillance has been applied in more than 50 countries to date, the application of this surveillance has been largely focused on relatively affluent urban and peri-urban communities. As such, there is a knowledge gap regarding the implementation of reliable wastewater surveillance in small and rural communities for the purpose of tracking rates of incidence of COVID-19 and other pathogens or biomarkers. This study examines the relationships existing between SARS-CoV-2 viral signal from wastewater samples harvested from an upstream pumping station and from an access port at a downstream wastewater treatment lagoon with the community's COVID-19 rate of incidence (measured as percent test positivity) in a small, rural community in Canada. Real-time quantitative polymerase chain reaction (RT-qPCR) targeting the N1 and N2 genes of SARS-CoV-2 demonstrate that all 24-h composite samples harvested from the pumping station over a period of 5.5 weeks had strong viral signal, while all samples 24-h composite samples harvested from the lagoon over the same period were below the limit of quantification. RNA concentrations and integrity of samples harvested from the lagoon were both lower and more variable than from samples from the upstream pumping station collected on the same date, indicating a higher overall stability of SARS-CoV-2 RNA upstream of the lagoon. Additionally, measurements of PMMoV signal in wastewater allowed normalizing SARS-CoV-2 viral signal for fecal matter content, permitting the detection of actual changes in community prevalence with a high level of granularity. As a result, in sewered small and rural communities or low-income regions operating wastewater lagoons, samples for wastewater surveillance should be harvested from pumping stations or the sewershed as opposed to lagoons.

Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.