The Intermediate Filament Network in Cultured Human Keratinocytes Is Remarkably Extensible and Resilient

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes

Xylazine detected in unregulated opioids and drug administration equipment in Toronto, Canada: clinical and social implications

Abstract Background The North American opioid overdose crisis is driven in large part by the presence of unknown psychoactive adulterants in the dynamic, unregulated drug supply. We herein report the first detection of the psychoactive veterinary compound xylazine in Toronto, the largest urban center in Canada, by the city’s drug checking service. Methods Toronto’s Drug Checking Service launched in October 2019. Between then and February 2021, 2263 samples were submitted for analysis. The service is offered voluntarily at harm reduction agencies that include supervised consumption services. Samples were analyzed using gas chromatography–mass spectrometry or liquid chromatography-high resolution mass spectrometry. Targeted and/or untargeted screens for psychoactive substances were undertaken. Results In September 2020, xylazine was first detected by Toronto’s Drug Checking Service. Among samples analyzed from September 2020 to February 2021 expected to contain fentanyl in isolation (610) or in combination with methamphetamine (16), xylazine was detected in 46 samples (7.2% and 12.5% of samples, respectively). Samples were predominantly drawn from used drug equipment. Three of the samples containing xylazine (6.5%) were associated with an overdose. Conclusion We present the first detection of xylazine in Toronto, North America’s fourth-largest metropolitan area. The increased risk of overdose associated with use of xylazine and its detection within our setting highlights the importance of drug checking services in supporting rapid responses to the emergence of potentially harmful adulterants. These data also highlight the clinical challenges presented by the dynamic nature of unregulated drug markets and the concomitant need to establish regulatory structures to reduce their contribution to overdose morbidity and mortality

Diverse psychotropic substances detected in drug and drug administration equipment samples submitted to drug checking services in Toronto, Ontario, Canada, October 2019–April 2020

BackgroundThe overdose crisis has generated innovative harm reduction and drug market monitoring strategies. In Toronto, Ontario, Canada, a multi-site drug checking service (DCS) pilot project was launched in October 2019. The project provides people who use drugs with information on the chemical composition of their substances, thereby increasing their capacity to make more informed decisions about their drug use and avoid overdose. DCS also provides real-time market monitoring to identify trends in the unregulated drug supply.MethodsSample data were obtained through analyses of drug and used drug administration equipment samples submitted anonymously and free of charge to DCS in downtown Toronto from October 10, 2019, to April 9, 2020, representing the first six months of DCS implementation. Analyses were conducted in clinical laboratories using liquid chromatography- and/or gas chromatography-mass spectrometry (LC-MS, GC-MS) techniques.ResultsOverall, 555 samples were submitted, with 49% (271) of samples that were found to contain high-potency opioids, of which 87% (235) also contained stimulants. Benzodiazepine-type drugs were found in 21% (116) of all samples, and synthetic cannabinoids in 1% (7) of all samples. Negative effects (including overdose, adverse health events, and extreme sedation) were reported for 11% (59) of samples submitted for analysis.ConclusionsToronto's DCS identified a range of high-potency opioids with stimulants, benzodiazepine-type drugs, and a synthetic cannabinoid, AMB-FUBINACA. This information can inform a range of evidence-informed overdose prevention efforts

Diverse psychotropic substances detected in drug and drug administration equipment samples submitted to drug checking services in Toronto, Ontario, Canada, October 2019–April 2020

Abstract Background The overdose crisis has generated innovative harm reduction and drug market monitoring strategies. In Toronto, Ontario, Canada, a multi-site drug checking service (DCS) pilot project was launched in October 2019. The project provides people who use drugs with information on the chemical composition of their substances, thereby increasing their capacity to make more informed decisions about their drug use and avoid overdose. DCS also provides real-time market monitoring to identify trends in the unregulated drug supply. Methods Sample data were obtained through analyses of drug and used drug administration equipment samples submitted anonymously and free of charge to DCS in downtown Toronto from October 10, 2019, to April 9, 2020, representing the first six months of DCS implementation. Analyses were conducted in clinical laboratories using liquid chromatography- and/or gas chromatography-mass spectrometry (LC–MS, GC–MS) techniques. Results Overall, 555 samples were submitted, with 49% (271) of samples that were found to contain high-potency opioids, of which 87% (235) also contained stimulants. Benzodiazepine-type drugs were found in 21% (116) of all samples, and synthetic cannabinoids in 1% (7) of all samples. Negative effects (including overdose, adverse health events, and extreme sedation) were reported for 11% (59) of samples submitted for analysis. Conclusions Toronto’s DCS identified a range of high-potency opioids with stimulants, benzodiazepine-type drugs, and a synthetic cannabinoid, AMB-FUBINACA. This information can inform a range of evidence-informed overdose prevention efforts

Evaluating networked drug checking services in Toronto, Ontario : study protocol and rationale

Background: The increasing incidence of fatal opioid overdose is a public health crisis in Canada. Given growing consensus that this crisis is related to the presence of highly potent opioid adulterants (e.g., fentanyl) in the unregulated drug supply, drug checking services (DCS) have emerged as part of a comprehensive approach to overdose prevention. In Canada’s largest city, Toronto, a network of DCS launched in 2019 to prevent overdose and overdose-related risk behaviors. This network employs mass spectrometry technologies, with intake sites co-located with supervised consumption services (SCS) at three frontline harm reduction agencies. The protocol and rationale for assessing the impact of this multi-site DCS network in Toronto is described herein. The aims of this study are to (1) evaluate the impact of DCS access on changes in and factors influencing overdose and related risk behaviors, (2) investigate the perceived capacity of DCS to prevent overdose, and (3) identify composition (qualitative and quantitative) trends in Toronto’s unregulated drug supply. Methods: We will use a parallel-mixed-methods design with complementary data sources (including data from chemical analysis of drug samples, quantitative intake and post-test surveys, SCS, coroners, paramedic services, and qualitative interviews), followed by a meta-inference process wherein results from analyses are synthesized. Results: Whereas most DCS globally target “recreational drug users,” in Toronto, this networked DCS will primarily target marginalized people who use drugs accessing frontline services, many of whom use drugs regularly and by injection. This evolution in the application of DCS poses important questions that have not yet been explored, including optimal service delivery models and technologies, as well as unique barriers for this population. Increasing information on the unregulated drug supply may modify the risk environment for this population of people who use drugs. Conclusions: This study addresses evidence gaps on the emerging continuum of overdose prevention responses and will generate critical evidence on a novel approach to reducing the ongoing high incidence of drug-related morbidity and mortality in Canada and elsewhere.Medicine, Faculty ofNon UBCPopulation and Public Health (SPPH), School ofReviewedFacult