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As key regulators of cytoskeletal dynamics, Rho GTPases coordinate a wide range of cellular processes, including cell polarity, cell migration, and cell cycle progression. The adoption of a pro-migratory phenotype enables cancer cells to invade the stroma surrounding the primary tumor and move toward and enter blood or lymphatic vessels. Targeting these early events could reduce the progression to metastatic disease, the leading cause of cancer-related deaths. Rho GTPases play a key role in the formation of dynamic actin-rich membrane protrusions and the turnover of cell-cell and cell-extracellular matrix adhesions required for efficient cancer cell invasion. Here, we discuss the roles of Rho GTPases in cancer, their validation as therapeutic targets and the challenges of developing clinically viable Rho GTPase inhibitors. We review other therapeutic targets in the wider Rho GTPase signaling network and focus on the four best characterized effector families: p21-activated kinases (PAKs), Rho-associated protein kinases (ROCKs), atypical protein kinase Cs (aPKCs), and myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs).

Determining whether potential cancer therapies effectively kill cancer cells is important for informing effective therapeutic choice for patients. Here we describe a rapid label-free method for testing drug efficacy in vitro that evaluates cellular viability from sub-cellular fluctuation imaging (SCFI). We used staurosporine and paclitaxel as known cytotoxic drugs at different concentrations, and four different human cancer-derived cell lines: PC3 (prostate), Caco-2 (colorectal), Calu-3 (lung) and A549 (lung). Both drugs caused a rapid decrease in sub-cellular fluctuations within 1 to 3 h except when the specific cell line was known to be resistant to one of the drugs. We also demonstrated that the method is able to differentiate between treated and untreated PC3 cells within 3 to 4 h after cells have been plated, thus eliminating the need for overnight incubation, and further decreasing the total time needed to evaluate drug efficacy. SCFI is therefore able to identify reliably if drugs are cytotoxic within 3 h of addition, which is considerably faster than current commonly used techniques.

Access equity has been raised as a fundamental concern with virtual care, both as it was used during the SARS-CoV-2 pandemic as well as its future applications within health systems. These concerns have not yet been substantiated with quantifiable data. We conducted a comparison of healthcare utilization and access across all dimensions of the Ontario Marginalization Index between virtual care and in-person care in the province of Ontario. We conducted a retrospective observational study using ICES databases in the Province of Ontario between March 14, 2020, and March 13,2022. We identified all virtual and in-person visits using billing codes. All visits were linked to their individual postal dissemination area for which there was census data from the Ontario Marginalization Index. Dissemination areas were divided, according to their categorization within each marginalization dimension, and visit rates were calculated for both populations. A total of 93,363,194 visits were included as part of the final analysis. Significant differences in virtual healthcare utilization were noted between the most and least marginalized populations within each dimension. This effect was not observed by visits for in-person care. The only exception was that racialized, and newcomer populations had higher virtual care utilization among the most marginalized. This data is the first that uses a large retrospective dataset and seems to confirm concerns for access inequity among the most marginalized populations. These differences much be part of policy considerations for the future of virtual care use.

The downregulation of Pleckstrin Homology-Like Domain family A member 1 (PHLDA1) expression mediates resistance to targeted therapies in receptor tyrosine kinase-driven cancers. The restoration and maintenance of PHLDA1 levels in cancer cells thus constitutes a potential strategy to circumvent resistance to inhibitors of receptor tyrosine kinases. Through a pharmacological approach, we identify the inhibition of MAPK signalling as a crucial step in PHLDA1 downregulation. Further ChIP-qPCR analysis revealed that MEK1/2 inhibition produces significant epigenetic changes at the PHLDA1 locus, specifically a decrease in the activatory marks H3Kme3 and H3K27ac. In line with this, we show that treatment with the clinically relevant class I histone deacetylase (HDAC) inhibitor 4SC-202 restores PHLDA1 expression in lapatinib-resistant human epidermal growth factor receptor-2 (HER2)+ breast cancer cells. Critically, we show that when given in combination, 4SC-202 and lapatinib exert synergistic effects on 2D cell proliferation and colony formation capacity. We therefore propose that co-treatment with 4SC-202 may prolong the clinical efficacy of lapatinib in HER2+ breast cancer patients.

Ground-level falls are common among older adults and are the most frequent cause of traumatic intracranial bleeding. The aim of this study was to derive a clinical decision rule that safely excludes clinically important intracranial bleeding in older adults who present to the emergency department after a fall, without the need for a computed tomography (CT) scan of the head. This prospective cohort study in 11 emergency departments in Canada and the United States enrolled patients aged 65 years or older who presented after falling from standing on level ground, off a chair or toilet seat, or out of bed. We collected data on 17 potential predictor variables. The primary outcome was the diagnosis of clinically important intracranial bleeding within 42 days of the index emergency department visit. An independent adjudication committee, blinded to baseline data, determined the primary outcome. We derived a clinical decision rule using logistic regression. The cohort included 4308 participants, with a median age of 83 years; 2770 (64%) were female, 1119 (26%) took anticoagulant medication and 1567 (36%) took antiplatelet medication. Of the participants, 139 (3.2%) received a diagnosis of clinically important intracranial bleeding. We developed a decision rule indicating that no head CT is required if there is no history of head injury on falling; no amnesia of the fall; no new abnormality on neurologic examination; and the Clinical Frailty Scale score is less than 5. Rule sensitivity was 98.6% (95% confidence interval [CI] 94.9%–99.6%), specificity was 20.3% (95% CI 19.1%–21.5%) and negative predictive value was 99.8% (95% CI 99.2%–99.9%). We derived a Falls Decision Rule, which requires external validation, followed by clinical impact assessment. Trial registration: ClinicalTrials. gov, no. NCT03745755.

Chronic Chagasic cardiomyopathy (CCC), caused by the protozoan Trypanosoma cruzi, is the most severe manifestation of Chagas disease.CCC is characterized by cardiac inflammation and fibrosis caused by a persistent inflammatory response. Following infection, macrophages secrete inflammatory mediators such as IL-1β, IL-6, and TNF-α to control parasitemia. Although this response contains parasite infection, it causes damage to the heart tissue. Thus, the use of immunomodulators is a rational alternative to CCC. Rho-associated kinase (ROCK) 1 and 2 are RhoA-activated serine/threonine kinases that regulate the actomyosin cytoskeleton. Both ROCKs have been implicated in the polarization of macrophages towards an M1 (pro-inflammatory) phenotype. Statins are FDA-approved lipid-lowering drugs that reduce RhoA signaling by inhibiting geranylgeranyl pyrophosphate (GGPP) synthesis. This work aims to identify the effect of statins on U937 macrophage polarization and cardiac tissue inflammation and its relationship with ROCK activity during T. cruzi infection. PMA-induced, wild-type, GFP-, CA-ROCK1- and CA-ROCK2-expressing U937 macrophages were incubated with atorvastatin, or the inhibitors Y-27632, JSH-23, TAK-242, or C3 exoenzyme incubated with or without T. cruzi trypomastigotes for 30 min to evaluate the activity of ROCK and the M1 and M2 cytokine expression and secretion profiling. Also, ROCK activity was determined in T. cruzi-infected, BALB/c mice hearts. In this study, we demonstrate for the first time in macrophages that incubation with T. cruzi leads to ROCK activation via the TLR4 pathway, which triggers NF-κB activation. Inhibition of ROCKs by Y-27632 prevents NF-κB activation and the expression and secretion of M1 markers, as does treatment with atorvastatin. Furthermore, we show that the effect of atorvastatin on the NF-kB pathway and cytokine secretion is mediated by ROCK. Finally, statin treatment decreased ROCK activation and expression, and the pro-inflammatory cytokine production, promoting anti-inflammatory cytokine expression in chronic chagasic mice hearts. These results suggest that the statin modulation of the inflammatory response due to ROCK inhibition is a potential pharmacological strategy to prevent cardiac inflammation in CCC.

Testing for high-sensitivity cardiac troponin (hs-cTn) may assist triage and clinical decision-making in patients presenting to the emergency department with symptoms of acute coronary syndrome; however, this could result in the misclassification of risk because of analytical variation or laboratory error. We sought to evaluate a new laboratory-based risk-stratification tool that incorporates tests for hs-cTn, glucose level and estimated glomerular filtration rate to identify patients at risk of myocardial infarction or death when presenting to the emergency department. We constructed the clinical chemistry score (CCS) (range 0–5 points) and validated it as a predictor of 30-day myocardial infarction (MI) or death using data from 4 cohort studies involving patients who presented to the emergency department with symptoms suggestive of acute coronary syndrome. We calculated diagnostic parameters for the CCS score separately using high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT). For the combined cohorts (n = 4245), 17.1% of participants had an MI or died within 30 days. A CCS score of 0 points best identified low-risk participants: the hs-cTnI CCS had a sensitivity of 100% (95% confidence interval [CI] 99.5%–100%), with 8.9% (95% CI 8.1%–9.8%) of the population classified as being at low risk of MI or death within 30 days; the hs-cTnT CCS had a sensitivity of 99.9% (95% CI 99.2%–100%), with 10.5% (95% CI 9.6%–11.4%) of the population classified as being at low risk. The CCS had better sensitivity than hs-cTn alone (hs-cTnI < 5 ng/L: 96.6%, 95% CI 95.0%–97.8%; hs-cTnT < 6 ng/L: 98.2%, 95% CI 97.0%–99.0%). A CCS score of 5 points best identified patients at high risk (hs-cTnI CCS: specificity 96.6%, 95% CI 96.0%–97.2%; 11.2% [95% CI 10.3%–12.2%] of the population classified as being at high risk; hs-cTnT CCS: specificity 94.0%, 95% CI 93.1%–94.7%; 13.1% [95% CI 12.1%–14.1%] of the population classified as being at high risk) compared with using the overall 99th percentiles for the hs-cTn assays (specificity of hs-cTnI 93.2%, 95% CI 92.3–94.0; specificity of hs-cTnT 73.8%, 95% CI 72.3–75.2). The CCS score at the chosen cut-offs was more sensitive and specific than hs-cTn alone for risk stratification of patients presenting to the emergency department with suspected acute coronary syndrome. Study registration: ClinicalTrials.gov, nos. NCT01994577; NCT02355457

[This corrects the article DOI: 10.1371/journal.pdig.0000708.].

The objective was to compare specialty-specific 7- and 30-day outcomes between virtual care visits and in-person visits which occurred during the SARS-CoV-2 pandemic. Using administrative data from provincial databases in Ontario, ambulatory care visits occurring virtually and in-person during specific timeframes within the pandemic were analyzed. Virtual care visits were matched with corresponding in-person visits based on multiple baseline patient characteristics. We assessed short-term patient outcomes at 7 and 30 days, including subsequent visits, hospital and ICU admissions, surgeries, and mortality and compared them using multivariate logistic regression. Odds ratios were calculated as measures of association between populations. For statistical significance, we used 99% confidence intervals to account for the increased likelihood of chance findings due to the multiple comparisons conducted. Overall, 9,340,519 visits were compared between populations using a 1:1 match on a 20% random sample of the available eligible visits. Over 70% of patients included were seen by a General Practitioner. With few exceptions and across almost all specialties, revisits, ED visits, admissions, ICU and OR use, and mortality were found to be more frequent for patients seen in person. When using the administrative data available to policy makers, there is no evidence to suggest that, in the short-term, virtual care is less safe than in person care. The causes for worse in-person outcomes are not yet clear although are likely related to the streaming of more acutely unwell patients towards in-person care.

IntroductionFalling on level ground is now the most common cause of traumatic intracranial bleeding worldwide. Older adults frequently present to the emergency department (ED) after falling. It can be challenging for clinicians to determine who requires brain imaging to rule out traumatic intracranial bleeding, and often head injury decision rules do not apply to older adults who fall. The goal of our study is to derive a clinical decision rule, which will identify older adults who present to the ED after a fall who do not have clinically important intracranial bleeding.Methods and analysisThis is a prospective cohort study enrolling patients aged 65 years or older, who present to the ED of 11 hospitals in Canada and the USA within 48 hours of having a fall. Patients are included if they fall on level ground, off a chair, toilet seat or out of bed. The primary outcome is the diagnosis of clinically important intracranial bleeding within 42 days of the index ED visit. An independent adjudication committee will determine the primary outcome, blinded to all other data. We are collecting data on 17 potential predictor variables. The treating physician completes a study data form at the time of initial assessment, prior to brain imaging. Data extraction is supplemented by an independent, structured electronic medical record review. We will perform binary recursive partitioning using Classification and Regression Trees to derive a clinical decision rule.Ethics and disseminationThe study was initially approved by the Hamilton Integrated Research Ethics Committee and subsequently approved by the research ethics boards governing all participating sites. We will disseminate our results by journal publication, presentation at international meetings and social media.Trial registration numberNCT03745755.