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Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer (p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly (p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling.
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Aterosclerose , Doença da Artéria Coronariana , Placa Aterosclerótica , Humanos , Microscopia de Força Atômica , Estudos Retrospectivos , Aterosclerose/patologia , Módulo de Elasticidade , Colágeno , LipídeosRESUMO
Pluripotent cells are subject to much interest as a source of differentiated cellular material for research models, regenerative medical therapies and novel applications such as lab-cultured meat. Greater understanding of the pluripotent state and control over its differentiation is therefore desirable. The role of biomechanical properties in directing cell fate and cell behavior has been increasingly well described in recent years. However, many of the mechanisms which control cell morphology and mechanical properties in somatic cells are absent from pluripotent cells. We leveraged naturally occurring variation in biomechanical properties and expression of pluripotency genes in murine ESCs to investigate the relationship between these parameters. We observed considerable variation in a Rex1-GFP expression reporter line and found that this variation showed no apparent correlation to cell spreading morphology as determined by circularity, Feret ratio, phase contrast brightness or cell spread area, either on a parameter-by-parameter basis, or when evaluated using a combined metric derived by principal component analysis from the four individual criteria. We further confirmed that cell volume does not co-vary with Rex1-GFP expression. Interestingly, we did find that a subpopulation of cells that were readily detached by gentle agitation collectively exhibited higher expression of Nanog, and reduced LmnA expression, suggesting that elevated pluripotency gene expression may correlate with reduced adhesion to the substrate. Furthermore, atomic force microscopy and quantitative fluorescent imaging revealed a connection between cell stiffness and Rex1-GFP reporter expression. Cells expressing high levels of Rex1-GFP are consistently of a relatively low stiffness, while cells with low levels of Rex1-GFP tend toward higher stiffness values. These observations indicate some interaction between pluripotency gene expression and biomechanical properties, but also support a strong role for other interactions between the cell culture regime and cellular biomechanical properties, occurring independently of the core transcriptional network that supports pluripotency.
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The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.
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INTRODUCTION: Potential impacts of climate change on health are increasingly studied due to the diversity of the associated risks (heatwaves, air pollution, water- and vector-borne diseases). Consequently, adaptation and mitigation strategies, including tools, have been developed by different cities, states, and organizations to assess the effects of climate change on health. OBJECTIVE: Health impact assessment (HIA) is a tool that could be used to assess the potential health impacts of climate change policies before their implementation. The objective of this study is therefore to analyze the way HIA is used in the development of these policies. METHOD: A scoping review of grey and scientific literature in French and English (period: 1990-2019) allowed us to identify 35 articles and reports, with 6 using HIA specifically. The areas of HIA application related to transport, urban planning or the building sector. The main health issues addressed in these HIAs concerned air, noise, physical activity, urban heat islands, green spaces, and functional diversity. RESULTS: These studies have shown that HIA is an approach that can facilitate cross-sectoral collaboration, and its flexibility allows for its application to adaptation and mitigation policies, as well as at several spatial scales (cities, regions). DISCUSSION: The principal limitation in this approach relates to uncertainties associated with quantifying projected impacts.
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Avaliação do Impacto na Saúde , Temperatura Alta , Cidades , Planejamento de Cidades , Política de Saúde , HumanosRESUMO
Climate change represents a serious threat to the health and well-being of populations. Today, many countries, regions, and cities around the world are implementing policies and strategies to adapt to climate change and mitigate its effects. A scoping review was performed to identify tools and methods that help integrate health into climate change adaptation and mitigation policies and strategies. The literature search includes scientific and grey literature. The scientific literature was conducted using PubMed, Elsevier Embase, and Web of Science databases. A grey literature web search was performed to complement the results. A total of 35 studies (28 from the scientific literature and 7 from the grey literature) were finally included. A large majority of research articles (24/28) and almost all reports (6/7) from the grey literature were published after 2010. Results show that the tools that were found most frequently are the nested models (12/35), health impact assessment (6/35), vulnerability and adaptation assessment (3/35), conceptual frameworks (3/35), and mixed methods (3/35). This review shows an increasing interest in the topic of developing tools to better manage health issues in adaptation and mitigation strategies, with a recent increase in the number of publications. Additional analyses of tools' effectiveness should be conducted in further studies.
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Aclimatação , Mudança Climática , PolíticasRESUMO
The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction.
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The mechanical state of cells is a critical part of their healthy functioning and it is controlled primarily by cytoskeletal networks (actin, microtubules and intermediate filaments). Drug-based strategies targeting the assembly of a given cytoskeletal network are often used to pinpoint their role in cellular function. Unlike actin and microtubules, there has been limited interest in the role of intermediate filaments, and fewer drugs have thus been identified and characterised as modulators of its assembly. Here, we evaluate whether Withaferin-A (WFA), an established disruptor of vimentin filaments, can also be used to modulate keratin filament assembly. Our results show that in keratinocytes, which are keratin-rich but vimentin-absent, Withaferin-A disrupts keratin filaments. Importantly, the dosages required are similar to those previously reported to disrupt vimentin in other cell types. Furthermore, Withaferin-A-induced keratin disassembly is accompanied by changes in cell stiffness and migration. Therefore, we propose that WFA can be repurposed as a useful drug to disrupt the keratin cytoskeleton in epithelial cells.
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Actinas/metabolismo , Epiderme/fisiologia , Filamentos Intermediários/fisiologia , Queratinócitos/fisiologia , Queratinas/metabolismo , Vitanolídeos/farmacologia , Células Cultivadas , Epiderme/efeitos dos fármacos , Humanos , Filamentos Intermediários/efeitos dos fármacos , Queratinócitos/efeitos dos fármacosRESUMO
Ezrin, a member of the ERM (ezrin/radixin/moesin) family of proteins, serves as a crosslinker between the plasma membrane and the actin cytoskeleton. By doing so, it provides structural links to strengthen the connection between the cell cortex and the plasma membrane, acting also as a signal transducer in multiple pathways during migration, proliferation, and endocytosis. In this study, we investigated the role of ezrin phosphorylation and its intracellular localization on cell motility, cytoskeleton organization, and cell stiffness, using fluorescence live-cell imaging, image quantification, and atomic force microscopy (AFM). Our results show that cells expressing constitutively active ezrin T567D (phosphomimetic) migrate faster and in a more directional manner, especially when ezrin accumulates at the cell rear. Similarly, image quantification results reveal that transfection with ezrin T567D alters the cell's gross morphology and decreases cortical stiffness. In contrast, constitutively inactive ezrin T567A accumulates around the nucleus, and although it does not impair cell migration, it leads to a significant buildup of actin fibers, a decrease in nuclear volume, and an increase in cytoskeletal stiffness. Finally, cell transfection with the dominant negative ezrin FERM domain induces significant morphological and nuclear changes and affects actin, microtubules, and the intermediate filament vimentin, resulting in cytoskeletal fibers that are longer, thicker, and more aligned. Collectively, our results suggest that ezrin's phosphorylation state and its intracellular localization plays a pivotal role in cell migration, modulating also biophysical properties, such as membrane-cortex linkage, cytoskeletal and nuclear organization, and the mechanical properties of cells.
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Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Fosfotreonina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Fenômenos Biomecânicos , Núcleo Celular/metabolismo , Forma do Núcleo Celular , Proteínas do Citoesqueleto/genética , Camundongos , Mutação/genética , Células NIH 3T3 , Fosforilação , Tubulina (Proteína)/metabolismo , Vimentina/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Live-imaging techniques are at the forefront of biology research to explore behaviour and function from sub-cellular to whole organism scales. These methods rely on intracellular fluorescent probes to label specific proteins, which are commonly assumed to only introduce artefacts at concentrations far-exceeding routine use. Lifeact, a small peptide with affinity for actin microfilaments has become a gold standard in live cell imaging of the cytoskeleton. Nevertheless, recent reports have raised concerns on Lifeact-associated artefacts at the molecular and whole organism level. We show here that Lifeact induces dose-response artefacts at the cellular level, impacting stress fibre dynamics and actin cytoskeleton architecture. These effects extend to the microtubule and intermediate filament networks as well as the nucleus, and ultimately lead to altered subcellular localization of YAP, reduced cell migration and abnormal mechanical properties. Our results suggest that reduced binding of cofilin to actin filaments may be the underlying cause of the observed Lifeact-induced cellular artefacts.
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Actinas/metabolismo , Fenômenos Biofísicos , Forma Celular , Células/citologia , Células/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Peptídeos/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Células COS , Movimento Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoesqueleto/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Fenótipo , Ligação ProteicaRESUMO
The regulation of nuclear state by the cytoskeleton is an important part of cellular function. Actomyosin stress fibres, microtubules and intermediate filaments have distinct and complementary roles in integrating the nucleus into its environment and influencing its mechanical state. However, the interconnectedness of cytoskeletal networks makes it difficult to dissect their individual effects on the nucleus. We use simple image analysis approaches to characterize nuclear state, estimating nuclear volume, Poisson's ratio, apparent elastic modulus and chromatin condensation. By combining them with cytoskeletal quantification, we assess how cytoskeletal organization regulates nuclear state. We report for a number of cell types that nuclei display auxetic properties. Furthermore, stress fibres and intermediate filaments modulate the mechanical properties of the nucleus and also chromatin condensation. Conversely, nuclear volume and its gross morphology are regulated by intracellular outward pulling forces exerted by myosin. The modulation exerted by the cytoskeleton onto the nucleus results in changes that are of similar magnitude to those observed when the nucleus is altered intrinsically, inducing chromatin decondensation or cell differentiation. Our approach allows pinpointing the contribution of distinct cytoskeletal proteins to nuclear mechanical state in physio- and pathological conditions, furthering our understanding of a key aspect of cellular behaviour.