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Supplementary Figures 1-4 from Ckap2 Regulates Aneuploidy, Cell Cycling, and Cell Death in a p53-Dependent Manner

We used DNA microarray screening to identify Ckap2 (cytoskeleton associated protein 2) as a novel p53 target gene in a mouse erythroleukemia cell line. DNA damage induces human and mouse CKAP2 expression in a p53-dependent manner and p53 activates the Ckap2 promoter. Overexpressed Ckap2 colocalizes with and stabilizes microtubules. In p53-null cells, overexpression of Ckap2 induces tetraploidy with aberrant centrosome numbers, suggesting disturbed mitosis and cytokinesis. In p53-competent cells, Ckap2 does not induce tetraploidy but activates p53-mediated cell cycle arrest and apoptosis. Our data suggest the existence of a functional positive feedback loop in which Ckap2 activates the G1 tetraploidy checkpoint and prevents aneuploidy.

Supplementary Methods from Ckap2 Regulates Aneuploidy, Cell Cycling, and Cell Death in a p53-Dependent Manner

This study examined differential item functioning (DIF) in the Center for Epidemiologic Studies Depression Scale (CES-D) between Chinese and White adolescents (aged 13 to 17 years) living in Canada. A series of ordinal logistic regressions were used to test for uniform and non-uniform DIF on items in the CES-D. The DIF analyses identified non-uniform DIF for Item 7 (“I felt that everything I did was an effort”). Controlling for gender and strength of ethnic identity in the DIF analyses did not alter the DIF results. The results of this study suggest that CES-D Item 7 does not appear to discriminate at higher levels of depression in Chinese adolescents. The results of this study hold notable implications for the use of the CES-D given that Chinese adolescents demonstrated a systematic difference in expression/experience of depression.

Little is known about the association between mental health and diminished food worry during the COVID-19 pandemic. This paper examines worry about having enough food to meet household needs and its association with mental health during the early months of the pandemic in Canada.Data are drawn from the first round of a multi-round mental health monitoring survey. Online surveys were administered between May 14 and 29, 2020, to a nationally representative sample of Canadian adults (n = 3000). Logistic regression models were used to examine associations between food worry and mental health indicators (anxious/worried, depressed, worse mental health compared with pre-pandemic, and suicidal thoughts/feelings), after adjusting for socio-demographic characteristics and pre-existing mental health conditions. Fully adjusted models explored the impact of controlling for financial worry due to the pandemic in the previous 2 weeks.Overall, 17.3% of the sample reported food worry due to the pandemic in the previous 2 weeks, with the highest prevalence found among those with a reported disability (29.3%), Indigenous identity (27.1%), or pre-existing mental health condition (25.3%). Compared with participants who did not report food worry, those who did had higher odds of reporting feeling anxious/worried (OR=1.36, 95% CI: 1.08-1.71) and suicidal thoughts/feelings (OR=1.87, 95% CI: 1.24-2.80) when controlling for socio-demographics, pre-existing mental health conditions, and financial worry.This paper provides insights about the associations between food worry and mental health in Canada during the COVID-19 pandemic and indicates the need for improved policies and social supports to mitigate food worry and associated mental health outcomes.RéSUMé: OBJECTIF: On en sait peu sur l’association entre la santé mentale et la diminution des inquiétudes liées à la nourriture durant la pandémie de COVID-19. Notre article porte sur l’inquiétude de ne pas avoir suffisamment de nourriture pour répondre aux besoins du ménage et son association avec la santé mentale au cours des premiers mois de la pandémie au Canada. MéTHODE: Nos données proviennent du premier cycle d’une enquête de surveillance de la santé mentale. Des sondages en ligne ont été administrés entre le 14 et le 29 mai 2020 à un échantillon national représentatif de Canadiennes et de Canadiens adultes (n = 3 000). Des modèles de régression logistique ont servi à examiner les associations entre les inquiétudes liées à la nourriture et quelques indicateurs de santé mentale (anxiété/inquiétude, dépression, santé mentale pire qu’avant la pandémie, pensées/sentiments suicidaires) après l’apport d’ajustements pour tenir compte du profil sociodémographique et des troubles de santé mentale préexistants. À l’aide de modèles entièrement ajustés, nous avons exploré l’effet de la prise en compte des soucis financiers dus à la pandémie au cours des deux semaines antérieures. RéSULTATS: Dans l’ensemble, le pourcentage de l’échantillon ayant fait état d’inquiétudes liées à la nourriture dues à la pandémie au cours des deux semaines antérieures était de 17,3 %, la plus forte prévalence ayant été constatée chez les répondants ayant un handicap déclaré (29,3 %), une identité autochtone (27,1 %) ou un trouble de santé mental préexistant (25,3 %). Comparativement aux participants n’ayant fait état d’aucune inquiétude liée à la nourriture, ceux qui ont fait état de telles inquiétudes présentaient une plus forte probabilité d’avoir déclaré des sentiments d’anxiété ou d’inquiétude (RC = 1,36, IC de 95 % : 1,08-1,71) et des pensées ou sentiments suicidaires (RC = 1,87, IC de 95 % : 1,24-2,80) après la prise en compte du profil sociodémographique, des troubles de santé mentale préexistants et des soucis financiers. CONCLUSION: Notre article jette un éclairage sur les associations entre les inquiétudes liées à la nourriture et la santé mentale au Canada durant la pandémie de COVID-19 et montre qu’il faudrait améliorer les politiques et les mesures de soutien social pour atténuer les inquiétudes liées à la nourriture et les résultats de santé mentale associés.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and the elderly. Here we investigated the impact of male sex and age by infecting adult male, aged male, and adult female ferrets with SARS-CoV-2. Aged male ferrets had a decrease in temperature which was accompanied by prolonged viral replication with increased pathology in the upper respiratory tract after infection. Transcriptome analysis of the nasal turbinates and lungs indicated that female ferrets had significant increases in interferon response genes (OASL, MX1, ISG15, etc.) on day 2 post infection which was delayed in aged males. In addition, genes associated with taste and smell such as RTP1, CHGA, and CHGA1 at later time points were upregulated in males but not in females. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.

Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford–AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites., Nature Communications, 11 (1), ISSN:2041-1723

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification., Nature Communications, 11 (1), ISSN:2041-1723

On 31 December 2019 the Wuhan Health Commission reported a cluster of atypical pneumonia cases that was linked to a wet market in the city of Wuhan, China. The first patients began experiencing symptoms of illness in mid-December 2019. Clinical isolates were found to contain a novel coronavirus with similarity to bat coronaviruses. As of 28 January 2020, there are in excess of 4,500 laboratory-confirmed cases, with > 100 known deaths. As with the SARS-CoV, infections in children appear to be rare. Travel-related cases have been confirmed in multiple countries and regions outside mainland China including Germany, France, Thailand, Japan, South Korea, Vietnam, Canada, and the United States, as well as Hong Kong and Taiwan. Domestically in China, the virus has also been noted in several cities and provinces with cases in all but one provinence. While zoonotic transmission appears to be the original source of infections, the most alarming development is that human-to-human transmission is now prevelant. Of particular concern is that many healthcare workers have been infected in the current epidemic. There are several critical clinical questions that need to be resolved, including how efficient is human-to-human transmission? What is the animal reservoir? Is there an intermediate animal reservoir? Do the vaccines generated to the SARS-CoV or MERS-CoV or their proteins offer protection against 2019-nCoV? We offer a research perspective on the next steps for the generation of vaccines. We also present data on the use of in silico docking in gaining insight into 2019-nCoV Spike-receptor binding to aid in therapeutic development. Diagnostic PCR protocols can be found at https://www.who.int/health-topics/coronavirus/laboratory-diagnostics-for-novel-coronavirus.

Eukaryotic cells deploy overlapping repair pathways to resolve DNA damage. Advancements in genome editing take advantage of these pathways to produce permanent genetic changes. Despite recent improvements, genome editing can produce diverse outcomes that can introduce risks in clinical applications. Although homology-directed repair is attractive for its ability to encode precise edits, it is particularly difficult in human cells. Here we discuss the DNA repair pathways that underlie genome editing and strategies to favour various outcomes.

Background and Aims. Ubiquitin-specific protease 18 (USP18) is involved in immunoregulation and response to interferon- (IFN-) based treatment in patients chronically infected with hepatitis C virus (HCV). We investigated whether and how its upregulation alters HCV infection. Methods. Overexpression of wild-type (USP18 WT) or catalytically inactive mutant (USP18 C64S) USP18 was examined for effects on HCV replication in the absence and presence of IFNα or IFNλ using both the HCV-infective model and replicon cells. The IFN signaling pathway was assessed via STAT1 phosphorylation (western blot) and downstream ISG expression (real-time PCR). Mechanistic roles were sought by quantifying microRNA-122 levels and J6/JFH1 infectivity of Huh7.5 cells. Results. We found that overexpression of either USP18 WT or USP18 C64S stimulated HCV production and blunted the anti-HCV effect of IFNα and IFNλ in the infective model but not in the replicon system. Overexpressed USP18 showed no effect on Jak/STAT signaling nor on microRNA-122 expression. However, USP18 upregulation markedly increased J6/JFH1 infectivity and promoted the expression of the key HCV entry factor CD81 on Huh7.5 cells. Conclusions. USP18 stimulates HCV production and blunts the effect of both type I and III IFNs by fostering a cellular environment characterized by upregulation of CD81, promoting virus entry and infectivity.

Background The SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2) has led to more than 165 million COVID-19 cases and >3.4 million deaths worldwide. Epidemiological analysis has revealed that the risk of developing severe COVID-19 increases with age. Despite a disproportionate number of older individuals and long-term care facilities being affected by SARS-CoV-2 and COVID-19, very little is understood about the immune responses and development of humoral immunity in the extremely old person after SARS-CoV-2 infection. Here we conducted a serological study to investigate the development of humoral immunity in centenarians following a SARS-CoV-2 outbreak in a long-term care facility. Methods Extreme aged individuals and centenarians who were residents in a long-term care facility and infected with or exposed to SARS-CoV-2 were investigated between April and June 2020 for the development of antibodies to SARS-CoV-2. Blood samples were collected from positive and bystander individuals 30 and 60 days after original diagnosis of SARS-CoV-2 infection. Plasma was used to quantify IgG, IgA, and IgM isotypes and subsequent subclasses of antibodies specific for SARS-CoV-2 spike protein. The function of anti-spike was then assessed by virus neutralization assays against the native SARS-CoV-2 virus. Findings Fifteen long-term care residents were investigated for SARS-CoV-2 infection. All individuals had a Clinical Frailty scale score ≥5 and were of extreme older age or were centenarians. Six women with a median age of 98.8 years tested positive for SARS-CoV-2. Anti-spike IgG antibody titers were the highest titers observed in our cohort with all IgG positive individuals having virus neutralization ability. Additionally, 5 out of the 6 positive participants had a robust IgA anti-SARS-CoV-2 response. In all 5, antibodies were detected after 60 days from initial diagnosis.

Background Evidence about the impact of the COVID-19 pandemic on the mental health of specific subpopulations, such as university students, is needed as communities prepare for future waves. Aims To study the association of proximity of COVID-19 with symptoms of anxiety and depression in university students. Method This trend study analysed weekly cross-sectional surveys of probabilistic samples of students from the University of British Columbia for 13 weeks, through the first wave of COVID-19. The main variable assessed was propinquity of COVID-19, defined as ‘knowing someone who tested positive for COVID-19’, which was specified at different levels: knowing someone anywhere globally, in Canada, in Vancouver, in their course or at home. Proximity was included in multivariable linear regressions to assess its association with primary outcomes, including 30-day symptoms of anxiety and/or depression. Results Of 1388 respondents (adjusted response rate of 50%), 5.6% knew someone with COVID-19 in Vancouver, 0.8% in their course and 0.3% at home. Ten percent were overwhelmed and unable to access help. Knowing someone in Vancouver was associated with an 11-percentage-point increase in the probability of 30-day anxiety symptoms (s.e. 0.05, P ≤ 0.05), moderated by gender, with a significant interaction of the exposure and being female (coefficient −20, s.e. 0.09, P ≤ 0.05). No association was found with depressive symptoms. Conclusions Propinquity of COVID-19 cases may increase the likelihood of anxiety symptoms in students, particularly among men. Most students reported coping well, but additional support is needed for an emotionally overwhelmed minority who report being unable to access help.

BackgroundThe SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2) has led to more than 114 million COVID-19 cases and >2.5 million deaths worldwide. Epidemiological analysis has revealed that the risk of developing severe COVID-19 increases with age. Despite a disproportionate number of older individuals and long-term care facilities being affected by SARS-CoV-2 and COVID-19, very little is understood about the immune responses and development of humoral immunity in the extremely old person after SARS-CoV-2 infection. Here we investigated the development of humoral immunity in centenarians following a SARS-CoV-2 outbreak in a long-term care facility.MethodsExtreme aged individuals and centenarians who were residents in a long-term care facility and infected with or exposed to SARS-CoV-2 were investigated for the development of antibodies to SARS-CoV-2. Blood samples were collected from positive and bystander individuals 30 and 60 days after original diagnosis of SARS-CoV-2 infection. Plasma was used to quantify IgG, IgA, and IgM isotypes and subsequent subclasses of antibodies specific for SARS-CoV-2 spike protein. The function of anti-spike was then assessed by virus neutralization assays against the native SARS-CoV-2 virus.FindingsFifteen long-term care residents were investigated for SARS-CoV-2 infection. All individuals had a Clinical Frailty scale score ≥5 and were of extreme older age or were centenarians. Six women with a median age of 98.8 years tested positive for SARS-CoV-2. Anti-spike IgG antibody titers were the highest titers observed in our cohort with all IgG positive individuals having virus neutralization ability. Additionally, 5 out of the 6 positive participants had a robust IgA anti-SARS-CoV-2 response. In all 5, antibodies were detected after 60 days from initial diagnosis.InterpretationExtreme older frail individuals and centenarians were able to elicit robust IgG and IgA antibodies directed toward SARS-CoV-2 spike protein. The antibodies were able to neutralize the virus. Humoral responses were still detectable after 60 days from initial diagnosis. Together, these data suggest that recovered participants who are of extreme old age would be protected if re-exposed to the same SARS-CoV-2 viral variant. Considering the threat of SARS-CoV-2 and COVID-19 to older age groups and long-term care facilities, the humoral responses to SARS-CoV-2 in older age groups is of public health importance and has implications to vaccine responses.FundingCanadian Institutes of Health Research (CIHR), NIH/NIAID, Genome Atlantic. VIDO receives operational funding from the Canada Foundation for Innovation through the Major Science Initiatives Fund and by Government of Saskatchewan through Innovation Saskatchewan.

Multiple studies have reported a doubling in risk of Coronavirus Disease-2019 (COVID-19) among cancer patients. Here, we examine the potential biological rationale behind this recurrent epidemiological observation. By leveraging large-scale genome-wide transcriptional data of normal and malignant tissues from adults and children, we found evidence of increased expression of SARS-CoV-2 viral entry genes in the cancer state, particularly in respiratory, gastrointestinal, and genitourinary tract tissues, with decreased expression in pediatric vs. adult samples. Additionally, by interrogating the temporal effects of radiotherapy on human peripheral blood mononuclear and mucosal cells, we observed important treatment-related alterations in host innate immunity, specifically type I interferon responses. Overall, cancers enhance expression of critical viral entry genes, and innate viral defenses can be dysregulated transiently during radiation treatments. These factors may contribute to the observed increased susceptibility to SARS-CoV-2 entry and severity of COVID-19 in cancer patients.

Oncolytic viruses (OVs) and phytochemical ursolic acid (UA) are two efficacious therapeutic candidates in development against breast cancer, the deadliest women&rsquo, s cancer worldwide. However, as single agents, OVs and UA have limited clinical efficacies. As a common strategy of enhancing monotherapeutic anticancer efficacy, we explored the combinatorial chemovirotherapeutic approach of combining oncolytic measles virus (MV), which targets the breast tumor marker Nectin-4, and the anticancer UA against breast adenocarcinoma. Our findings revealed that in vitro co-treatment with UA synergistically potentiated the killing of human breast cancer cells by oncolytic MV, without UA interfering the various steps of the viral infection. Mechanistic studies revealed that the synergistic outcome from the combined treatment was mediated through UA&rsquo, s potentiation of apoptotic killing by MV. To circumvent UA&rsquo, s poor solubility and bioavailability and strengthen its clinical applicability, we further developed UA nanoparticles (UA-NP) by nanoemulsification. Compared to the non-formulated UA, UA-NP exhibited improved drug dissolution property and similarly synergized with oncolytic MV in inducing apoptotic breast cancer cell death. This oncolytic potentiation was partly attributed to the enhanced autophagic flux induced by the UA-NP and MV combined treatment. Finally, the synergistic effect from the UA-NP and MV combination was also observed in BT-474 and MDA-MB-468 breast cancer cells. Our study thus highlights the potential value of oncolytic MV and UA-based chemovirotherapy for further development as a treatment strategy against breast cancer, and the feasibility of employing nanoformulation to enhance UA&rsquo, s applicability.

Hepatocellular carcinoma (HCC), including hepatitis C virus (HCV)-induced HCC, is a deadly disease highly refractory to chemotherapy, thus requiring the continuous identification of novel treatment strategies. Berberine (BBR) has been previously reported to inhibit hepatoma cell growth, but the main type of cell death elicited by BBR, and whether the alkaloid can inhibit hepatoma cells carrying HCV genomes, is unclear. Herein, we show that BBR treatment induced a biphasic cell death irrespective of the presence of HCV subgenomic replicon RNA, first triggering apoptosis that then progressed to necrosis between 24 and 48 h post-treatment. Furthermore, BBR treatment potentiated the HCV replicon-induced reactive oxygen species (ROS) production, inhibition of which with an antioxidant attenuated the cell death that was elicited by BBR in these cells. Moreover, BBR dampened the autophagic response in HCV RNA-positive or negative hepatoma cells, and pharmacological inhibition of autophagy conversely augmented the BBR-induced cell death. Finally, BBR inhibited the growth of Huh-7 cells that were persistently infected with the full-length genome HCV particles, and concomitant pharmacological inhibition of autophagy potentiated the killing of these cells by BBR. Our findings suggest that combining BBR with the inhibition of autophagy could be an attractive treatment strategy against HCC, irrespective of the presence of the HCV genome.

Lesbian, gay, bisexual, trans, other queer, and Two-Spirit (LGBTQ2+) people are particularly at risk for the psycho-social consequences of the COVID-19 pandemic, though population-tailored research within this context remains limited. This study examines the extent of, and associations between, increased alcohol and cannabis use and deteriorating mental health among LGBTQ2+ adults in Canada during the COVID-19 pandemic. Data are drawn from LGBTQ2+ respondents to a repeated, cross-sectional survey administered to adults living in Canada (May 2020–January 2021). Bivariate cross-tabulations and multivariable logistic regression models were utilized to examine associations between increased alcohol and cannabis use, and self-reported mental health, overall coping, and suicidal thoughts. Five-hundred and two LGBTQ2+ participants were included in this analysis. Of these, 24.5% reported increased alcohol use and 18.5% reported increased cannabis use due to the pandemic. In the adjusted analyses, increased alcohol use was associated with poor overall coping (OR = 2.28, 95% CI = 1.28–4.07) and worse self-reported mental health (OR = 1.98, 95% CI = 1.21–3.25), whereas increased cannabis use was associated with suicidal thoughts (OR = 2.30, 95% CI = 1.16–4.55). These findings underscore the need for population-tailored, integrated substance use and mental health supports to address interrelated increases in alcohol/cannabis use and worsening mental health among LGBTQ2+ adults, in the context of the COVID-19 pandemic and beyond.

SummaryCas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9’s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

BackgroundPaediatric mental health-related visits to the emergency department are rising. However, few tools exist to identify concerns early and connect youth with appropriate mental healthcare. Our objective was to develop a digital youth psychosocial assessment and management tool (MyHEARTSMAP) and evaluate its inter-rater reliability when self-administered by a community-based sample of youth and parents.MethodsWe conducted a multiphasic, multimethod study. In phase 1, focus group sessions were used to inform tool development, through an iterative modification process. In phase 2, a cross-sectional study was conducted in two rounds of evaluation, where participants used MyHEARTSMAP to assess 25 fictional cases.ResultsMyHEARTSMAP displays good face and content validity, as supported by feedback from phase 1 focus groups with youth and parents (n=38). Among phase 2 participants (n=30), the tool showed moderate to excellent agreement across all psychosocial sections (κ=0.76–0.98).ConclusionsOur findings show that MyHEARTSMAP is an approachable and interpretable psychosocial assessment and management tool that can be reliably applied by a diverse community sample of youth and parents.

Coxsackievirus A16 (CVA16) is an etiologic agent of hand, foot, and mouth disease (HFMD) that affects young children, and although typically self-limited, severe complications, and fatal cases have been reported. Due to the lack of specific medication and vaccines against CVA16, there is currently a need to develop effective antivirals to better control CVA16 infections in epidemic areas. In this study, we identified the tannins chebulagic acid (CHLA) and punicalagin (PUG) as small molecules that can efficiently disrupt the CVA16 infection of human rhabdomyosarcoma cells. Both compounds significantly reduced CVA16 infectivity at micromolar concentrations without apparent cytotoxicity. A mechanistic analysis revealed that the tannins particularly targeted the CVA16 entry phase by inactivating cell-free viral particles and inhibiting viral binding. Further examination by molecular docking analysis pinpointed the targets of the tannins in the fivefold axis canyon region of the CVA16 capsid near the pocket entrance that functions in cell surface receptor binding. We suggest that CHLA and PUG are efficient antagonists of CVA16 entry and could be of value as antiviral candidates or as starting points for developing molecules to treat CVA16 infections.

Evidence on the population-level mental health impacts of COVID-19 are beginning to amass; however, to date, there are significant gaps in our understandings of whose mental health is most impacted, how the pandemic is contributing to widening mental health inequities, and the coping strategies being used to sustain mental health. The first wave of a repeated cross-sectional monitoring survey was conducted between May 14-29, 2020 to assess the mental health impacts of the pandemic and to identify the disproportionate impacts on populations or groups identified as experiencing increased risks due to structural vulnerability and pre-existing health and social inequities. Respondents included a nationally representative probability sample (n = 3000) of Canadian adults 18 years and older. Overall, Canadian populations are experiencing a deterioration in mental health and coping due to the pandemic. Those who experience health, social, and/or structural vulnerabilities due to pre-existing mental health conditions, disability, income, ethnicity, sexuality, and/or gender are more likely to endorse mental health deterioration, challenging emotions, and difficulties coping. This monitoring study highlights the differential mental health impacts of the pandemic for those who experience health, social, and structural inequities. These data are critical to informing responsive, equity-oriented public health, and policy responses in real-time to protect and promote the mental health of those most at risk during the pandemic and beyond.

Summary Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9’s activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Eukaryotic cells deploy overlapping repair pathways to resolve DNA damage. Advancements in genome editing take advantage of these pathways to produce permanent genetic changes. Despite recent improvements, genome editing can produce diverse outcomes that can introduce risks in clinical applications. Although homology-directed repair is attractive for its ability to encode precise edits, it is particularly difficult in human cells. Here we discuss the DNA repair pathways that underlie genome editing and strategies to favour various outcomes.

Repair of double strand DNA breaks (DSBs) can result in gene disruption or precise gene modification via homology directed repair (HDR) from a templating donor DNA. During genome editing, altering cellular responses to DSBs may be an effective strategy to rebalance editing outcomes towards HDR and away from other repair pathways. To identify factors that regulate HDR from a double-stranded DNA donor (dsDonor), we utilized a pooled screen to define the consequences of thousands of individual gene knockdowns during Cas9-initiated HDR from a double strand plasmid donor. We find that templated dsDonor repair pathways are mostly genetically distinct from single strand donor DNA (ssDonor) repair but share aspects that include dependency upon the Fanconi Anemia (FA) pathway. We also identified several factors whose knockdown increases HDR and thus act as repressors of gene modification. Screening available small molecule inhibitors of these repressors revealed that the cell division cycle 7-related protein kinase (CDC7) inhibitor XL413 increases the efficiency of HDR by 2-3 fold in many contexts, including primary T-cells. XL413 stimulates HDR through cell cycle regulation, inducing an early S-phase cell cycle arrest that, to the best of our knowledge, is uncharacterized for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for boosting the efficiency of gene modification.

With the introduction of CNC-based milling systems, it became possible to more uniformly deconstruct silicon devices for through-silicon electrical fault isolation techniques that feature either a convex or concave geometry or by thermally relaxing the device. This paper presents a novel methodology for uniform mechanical deprocessing of stacked memory modules using a CNC milling system. The PCB (FR4) substrate has been identified as the primary mechanism influencing both the profile of the device and relaxation that occurs during device deconstruction. Examples showing this effect are provided on both pre- and post-processed devices where a difference in symmetry can be observed. Utilizing this new strategy, it is possible to restore a more symmetrical profile that is easier to process with a CNC milling machine.

Oncolytic virotherapy represents an emerging development in anticancer therapy. Although it has been tested against a variety of cancers, including breast cancer, the efficacy of oncolytic viral vectors delivered as a monotherapy is limited. Enhancing viral oncolytic therapies through combination treatment with anticancer agents is a feasible strategy. In this study, we considered a chemovirotherapeutic approach for treating breast adenocarcinoma using oncolytic measles virus (MV) and the chemotherapeutic agent camptothecin (CPT). Our results demonstrated that co-treatment of MV with CPT yielded enhanced cytotoxicity against breast cancer cells. Low dosage CPT combined with MV was also found to elicit the same therapeutic effect as high doses of CPT. At the lower dosage used, CPT did not inhibit the early stages of MV entry, nor reduce viral replication. Further studies revealed that co-treatment induced significantly enhanced apoptosis of the breast cancer cells compared to either MV or CPT alone. Overall, our findings demonstrate the potential value of MV plus CPT as a novel chemovirotherapeutic treatment against breast cancer and as a strategy to enhance MV oncolytic activity.

CRISPR–Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.

Antiviral development is plagued by drug resistance and genetic barriers to resistance are needed. For HIV and hepatitis C virus (HCV), combination therapy has proved life-saving. The targets of direct-acting antivirals for HCV infection are NS3/4A protease, NS5A phosphoprotein and NS5B polymerase. Differential visualization of drug-resistant and -susceptible RNA genomes within cells revealed that resistant variants of NS3/4A protease and NS5A phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. Confocal microscopy revealed that RNA replication complexes are genome-specific, rationalizing the non-interaction of wild-type and variant products. No HCV antivirals yet display the dominance of drug susceptibility shown for capsid proteins of other viruses. However, effective inhibitors of HCV polymerase exact such high fitness costs for drug resistance that stable genome selection is not observed. Barriers to drug resistance vary with target biochemistry and detailed analysis of these barriers should lead to the use of fewer drugs., eLife digest Viruses are simple organisms that consist of genetic information and a few types of proteins. They cannot replicate on their own, and instead hijack the molecular machinery of a host cell to produce more of themselves. Inside an infected cell, the genetic information of the virus is replicated and ‘read’ to create viral proteins. These components are then assembled to form a new generation of viruses. During this process, genetic errors may occur that lead to modifications in the viral proteins, and help the virus become resistant to treatment. For instance, a viral protein that used to be targeted by a drug can change slightly and not be recognized anymore. Currently, the most efficient way to fight drug resistance is to use combination therapy, where several drugs are given at the same time. This strategy is successful, for example to treat infections with the hepatitis C virus, but it is also expensive, especially for developing countries. An alternative approach is dominant-drug targeting, which exploits the fact that both drug-resistant and drug-susceptible viruses are ‘born’ in the same cell. There, the susceptible viruses can overwhelm and ‘mask’ the benefits of the resistant ones. For example, proteins from resistant strains, which are no longer detected by a treatment, can bind to proteins from susceptible viruses; drugs will still be able to recognize these resulting viral structures. The proteins that operate in such ways are potential dominant-drug targets. However, resistant and susceptible strains can also cohabit without any contacts if their proteins do not interact with each other. Now, van Buuren et al. screen several viral proteins, including one called NS5A, to test whether a dominant drug target exists for the hepatitis C virus. Only a few molecules of a drug that targets NS5A can stop the virus from growing. In theory, drug-bound NS5A proteins could block their non-drug-bound neighbors, but when these drugs have been used on their own, resistance quickly emerged. Experiments showed that NS5A is not a dominant drug target because the drug-resistant and drug-susceptible proteins do not mix. Unless ‘forced’ in the laboratory, NS5A proteins only bind to the ones produced by the same strain of virus. This explains why resistant viruses quickly take over when NS5A drugs are the sole treatment. However, other hepatitis C proteins, such as the HCV core protein, are known to mix during the assembly of the virus, and thus are likely be dominant drug targets.

The inhibitors carbobenzoxy (Z)- d -Phe- l -Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z- d -Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z- d -Phe- l -Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z- d -Phe- l -Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

CRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.

Background: Although injection drug use is known to result in a range of health-related harms, including transmission of HIV and fatal overdose, little is known about the possible role of synthetic drugs in injection initiation. We sought to determine the effect of crystal methamphetamine use on risk of injection initiation among street-involved youth in a Canadian setting. Methods: We used Cox regression analyses to identify predictors of injection initiation among injection-naive street-involved youth enrolled in the At-Risk Youth Study, a prospective cohort study of street-involved youth in Vancouver, British Columbia. Data on circumstances of first injection were also obtained. Results: Between October 2005 and November 2010, a total of 395 drug injection–naive, street-involved youth provided 1434 observations, with 64 (16.2%) participants initiating injection drug use during the follow-up period, for a cumulative incidence of 21.7 (95% confidence interval [CI] 1.7–41.7) per 100 person-years. In multivariable analysis, recent noninjection use of crystal methamphetamine was positively associated with subsequent injection initiation (adjusted hazard ratio 1.93, 95% CI 1.31–2.85). The drug of first injection was most commonly reported as crystal methamphetamine (14/31 [45%]). Interpretation: Noninjection use of crystal methamphetamine predicted subsequent injection initiation, and crystal methamphetamine was the most commonly used drug at the time of first injection. Evidence-based strategies to prevent transition to injection drug use among crystal methamphetamine users are urgently needed.

Little information exists on the use of condoms as protective barriers to sexually transmitted infections (STIs) among indigenous people in Canada. This study explores risk factors of inconsistent condom use (during consensual sex) over time among participants in the Cedar Project, a prospective cohort study of indigenous young people living in Vancouver and Prince George, British Columbia, who use drugs. Due to the serial measurements for each study subject, generalized estimating equations modeling with logit link was used to accommodate the temporal correlation within subjects. For young women, inconsistent condom use over time was predicted by having a recent STI (adjusted odds ratio [AOR] = 1.76, 95% confidence interval [CI] [1.12, 2.79]), smoking crack daily (AOR = 1.63, 95%CI [1.02, 2.61]), and having experienced recent sexual abuse (AOR = 2.07, 95%CI [1.20, 3.56]). Among young men, living in Prince George (AOR = 1.63, 95%CI [1.14, 2.39]) and daily crack smoking (AOR = 1.56, 95%CI [1.02, 2...

Electrical failure analysis toolsets continue to develop at a pace significantly faster than sample preparation techniques. New solid immersion lenses (SIL) have been developed that require tighter requirements in remaining silicon thickness (RST) variation. Due to the SIL requirements, well established sample preparation techniques are quickly becoming ineffective, and new techniques/tools are required. In this paper, we will present a new methodology for preparing contoured devices by utilizing a contour capable milling system with an incorporated spectral reflectance measurement tool to enable contour correction milling based on remaining silicon thickness. This technique has demonstrated the capability of meeting 50 μm (or other desired target thickness) ±5 μm remaining silicon thickness requirement as requested by the SIL manufacturers.

Canine distemper virus (CDV) was shown to use dog nectin-4 as a receptor to gain entry into epithelial cells. RNA from dog placenta or MDCK kidney cells was isolated and cDNAs were prepared. Two splice variants of dog nectin-4 were identified. A deletion of 25 amino acids was found in the cytoplasmic domain of dog nectin-4 from MDCK cells, corresponding to a splice variant that is also seen in murine nectin-4, and did not affect its role as a receptor. Both dog nectin-4 and human nectin-4 could function as an entry factor for CDV containing an EGFP reporter gene. Inhibition of dog nectin-4 expression by RNAi or nectin-4 antibodies decreased CDV titers and EGFP fluorescence. Finally, dog nectin-4 also promotes syncytia formation, which could be inhibited by siRNA treatment. These data confirm that dog nectin-4 can be used by CDV to gain entry into epithelial cells, and facilitate virus spread.