The endoplasmic reticulum (ER) plays a central role in the co- and post-translational modification of many proteins. Disruption of these processes can lead to the accumulation of misfolded proteins in the endoplasmic reticulum - a condition known as endoplasmic reticulum stress. In recent years, the association of endoplasmic reticulum stress with a number of disease pathologies has increased interest in the study of this condition. Current methods to detect endoplasmic reticulum stress are indirect and retrospective. Here we describe a new method to detect and quantify endoplasmic reticulum stress in live cells using Thioflavin T (ThT), a small molecule that exhibits enhanced fluorescence when it binds to protein aggregates. We show that enhanced ThT-fluorescence correlates directly with established indicators of unfolded protein response activation. Furthermore, enhanced ThT-fluorescence can be detected in living cells within 20 min of application of an endoplasmic reticulum stress-inducing agent. ThT is capable of detecting endoplasmic reticulum stress induced by distinctly different conditions and compounds, in different cultured cell types as well as in mouse tissue samples. Pre-treatment with a potent endoplasmic reticulum stress-reducing agent, 4-phenylbutyric acid, mitigates the enhanced ThT signal. This new tool will be useful in future research investigating the role of protein misfolding in the development and/or progression of human diseases.

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.

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.

A therosclerosis is the main underlying pathology of cardiovascular disease, which accounts for the majority of deaths in developed nations.1 Myeloid lineage cells are critical mediators in the development of atherosclerosis and account for the majority of a lesion's cellular bulk.2 Within the atherosclerotic lesion, macrophages phagocytose modified lipid particles becoming lipid engorged foam cells. Foam cells exacerbate disease progression through the secretion of proinflammatory cytokines and growth factors. In advanced lesions, foam cells undergo apoptosis leading to the formation of a lipid-rich, acellular, and highly thrombotic necrotic core. The underlying molecular mechanisms that regulate myeloid cell behavior during atherosclerosis remain poorly defined.Increasing evidence suggests that myeloid cell subpopulations are heterogeneous and have distinctive phenotypes that play unique roles in disease states. Macrophages are often broadly classified as having a classical (M1) or an alternative (M2) phenotype. M1 macrophages, elicited by toll-like receptor or interferon-γ receptor stimulation, are the most prominent macrophages at sites of inflammation and exacerbate the inflammatory response through the secretion of proinflammatory cytokines and chemoatractants.3,4 M1 macrophages promote atherosclerotic lesion development and complexity.5-7 M2 macrophages patrol tissue, perform reparative and immunoregulatory functions, efferocytose debris, and are antiatherogenic. 3,4,8 Other macrophage subtypes have been identified in atherosclerotic lesions, including M4, Mox, and Mhem; however, their roles are less well characterized. 3,5,9,10 Our understanding of the cellular signaling networks that regulate macrophage polarization in the context of atherosclerosis and the relative contribution of each phenotype to the progression and development of atherosclerosis is limited.Glycogen synthase kinase (GSK)-3 α and β are homologous serine/threonine kinases encoded by separate genes. 11GSK3α and GSK3β share 98% amino acid homology within their kinase domain but only 36% homology in the C-terminal domain. GSK3α (51 kDa) is 5 kDa larger than GSK3β (46 kDa) because of an N-terminal glycine-rich domain with an © 2015 American Heart Association, Inc. Objective-Glycogen synthase kinase (GSK)-3α/β has been implicated in the pathogenesis of diabetes mellitus, cancer, Alzheimer, and atherosclerosis. The tissue-and homolog-specific functions of GSK3α and β in atherosclerosis are unknown. This study examines the effect of hepatocyte or myeloid cell deletion of GSK3α or GSK3β on atherosclerosis in low-density lipoprotein receptor (LDLR) −/− mice. Approach and Results-We ablated GSK3α or GSK3β expression in hepatic or myeloid cells of LDLR −/− mice, and mice were fed a high-fat diet for 10 weeks. GSK3α or GSK3β deficiency in hepatic or myeloid cells did not affect metabolic parameters, including plasma lipid levels. Hepatic deletion of GSK3α or GSK3β did not affect the development of atherosclerosis or hepatic lipid content. Myeloid ...