In vitro insecticide resistance patterns in field strains of the sheep blowfly, Lucilia cuprina.

The control of the sheep blowfly relies on the use of insecticides. There have been several reports of in vitro and in vivo resistance to the most widely-used flystrike control chemical, dicyclanil. A recent report also described in vitro resistance to imidacloprid in a strain collected from a single property over three consecutive seasons that also showed resistance to dicyclanil. The present study aimed to use in vitro assays to examine five field-collected blowfly strains to determine if this co-occurrence of resistance to dicyclanil and imidacloprid was present more widely in field strains and to also measure resistance patterns to the other currently-used flystrike control chemicals. Each of the strains showed significant levels of resistance to both dicyclanil and imidacloprid: resistance factors at the IC50 of 9.1-23.8 for dicyclanil, and 8.7-14.1 for imidacloprid. Resistance factors at the IC95 ranged from 16.5 to 53.7, and 14.6-24.3 for dicyclanil and imidacloprid, respectively. Resistance factors were up to 8.5 for cyromazine at the IC95. Resistance to dicyclanil and imidacloprid was suppressed by co-treatment with the cytochrome P450 inhibitor, aminobenzotriazole, implicating this enzyme system in the observed resistances. We discuss the implications of the co-occurrence of resistance to dicyclanil and imidacloprid on insecticide rotation strategies for blowfly control. We also discuss the roles of insecticide resistance, environmental factors (e.g. rainfall), operational factors (e.g. insecticide application technique) and other animal health issues (e.g. scouring / diarrhoea) that together will impact on the likelihood of flystrike occurring at an earlier time point than expected after insecticide application.

Synthesis of PtNi Nanoparticles to Accelerate the Oxygen Reduction Reaction.

We report the synthesis of core-shell Ni-Pt nanoparticles (NPs) with varying degrees of crystallographic facets and surface layers rich in Pt via a seed-mediated thermolytic approach. Mixtures of different surfactants used during synthesis resulted in preferential surface passivation, which in turn dictated the size, chemical composition, and geometric evolution of these PtNi NPs. Electrochemical investigations of these pristine core-shell Ni-Pt structures in the oxygen reduction reaction (ORR) show that their catalytic functionalities outperform the commercial Pt/C reference catalyst. The enhanced electrocatalytic ORR performances of these Pt-based PtNi NPs are correlated with the weakened oxygen binding strength or surface-adsorbed hydroxyl (OH) species on active Pt surface sites induced by the downshift of the d-band center as a result of compressive strain effects. Our studies offer a robust synthetic approach for the development of core-shell nanostructures for enhanced ORR catalysis.

Osteopontin Activation and Microcalcification in Venous Grafts Can Be Modulated by Dexamethasone.

BACKGROUND: Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin. METHODS: We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography. RESULTS: The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake. CONCLUSIONS: Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.

Promoting disability employment with avail® - a pilot study for feasibility and preliminary efficacy.

PURPOSE: Supported employment is a promising vocational rehabilitation intervention for improving disability employment. Its implementation, however, is not without challenges. Lack of funding and the heavy caseload of job coaches significantly constrain its impacts. A mobile software, avail® by CentralReach, was created based on the individual placement and support model for assisting individuals with disabilities to perform their job tasks. The purpose of this pilot study was to explore the feasibility and preliminary efficacy of avail. METHOD: A total of 17 participants with disabilities were recruited in a Midwestern state from the United States to try avail for a period of time ranging from 40 to 348 days. Participants' experiences, including perceived usability, acceptance, and satisfaction, were collected to determine the feasibility of avail. In addition, a pre- and post-intervention analysis was conducted to identify its effects on users' job satisfaction, self-efficacy, and self-determination. RESULTS: Participants of this study expressed positive user experience for avail. Specifically, they felt that avail was helpful for their job performance. The results also showed a statistically significant result for improving participants' self-determination. CONCLUSION: avail is a feasible and potentially promising application for assisting workers with disabilities in their job performance. Directly available on mobile devices, avail has several advantages, such as real-time support and good portability. Suggestions and future directions were discussed for further software development.

A job coach is needed in the individual placement and support model (IPS) to provide on-site job support for individuals with disabilities. However, job coaches were typically overwhelmed by the heavy caseloads.A job coach application such as the avail was created, and this study was to test its feasibility and preliminary efficacy.The study showed that avail was appropriate for individuals with disabilities to use at work, and they generally enjoyed using the application.The preliminary efficacy showed that avail improved their job performances and overall self-determination.

Evaluating Demographic Representation in Clinical Trials: Use of the Adaptive Coronavirus Disease 2019 Treatment Trial (ACTT) as a Test Case.

Background: Clinical trials initiated during emerging infectious disease outbreaks must quickly enroll participants to identify treatments to reduce morbidity and mortality. This may be at odds with enrolling a representative study population, especially when the population affected is undefined. Methods: We evaluated the utility of the Centers for Disease Control and Prevention's COVID-19-Associated Hospitalization Surveillance Network (COVID-NET), the COVID-19 Case Surveillance System (CCSS), and 2020 United States (US) Census data to determine demographic representation in the 4 stages of the Adaptive COVID-19 Treatment Trial (ACTT). We compared the cumulative proportion of participants by sex, race, ethnicity, and age enrolled at US ACTT sites, with respective 95% confidence intervals, to the reference data in forest plots. Results: US ACTT sites enrolled 3509 adults hospitalized with COVID-19. When compared with COVID-NET, ACTT enrolled a similar or higher proportion of Hispanic/Latino and White participants depending on the stage, and a similar proportion of African American participants in all stages. In contrast, ACTT enrolled a higher proportion of these groups when compared with US Census and CCSS. The proportion of participants aged ≥65 years was either similar or lower than COVID-NET and higher than CCSS and the US Census. The proportion of females enrolled in ACTT was lower than the proportion of females in the reference datasets. Conclusions: Although surveillance data of hospitalized cases may not be available early in an outbreak, they are a better comparator than US Census data and surveillance of all cases, which may not reflect the population affected and at higher risk of severe disease.

Baricitinib Treatment of Coronavirus Disease 2019 Is Associated With a Reduction in Secondary Infections.

We performed a secondary analysis of the National Institutes of Health-sponsored Adaptive COVID-19 Treatment Trial (ACTT-2) randomized controlled trial and found that baricitinib was associated with a 50% reduction in secondary infections after controlling for baseline and postrandomization patient characteristics. This finding provides a novel mechanism of benefit for baricitinib and supports the safety profile of this immunomodulator for the treatment of coronavirus disease 2019.

The Role of Preservation Solutions upon Saphenous Vein Endothelial Integrity and Function: Systematic Review and UK Practice Survey.

The long saphenous vein is the most used conduit in cardiac surgery, but its long-term patency is limited by vein graft disease (VGD). Endothelial dysfunction is a key driver of VGD; its aetiology is multi-factorial. However emerging evidence identifies vein conduit harvest technique and preservation fluids as causal in their onset and propagation. This study aims to comprehensively review published data on the relationship between preservation solutions, endothelial cell integrity and function, and VGD in human saphenous veins harvested for CABG. The review was registered with PROSPERO (CRD42022358828). Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until August 2022. Papers were evaluated in line with registered inclusion and exclusion criteria. Searches identified 13 prospective, controlled studies for inclusion in the analysis. All studies used saline as a control solution. Intervention solutions included heparinised whole blood and saline, DuraGraft, TiProtec, EuroCollins, University of Wisconsin (UoW), buffered, cardioplegic and Pyruvate solutions. Most studies demonstrated that normal saline appears to have negative effects on venous endothelium and the most effective preservation solutions identified in this review were TiProtec and DuraGraft. The most used preservation solutions in the UK are heparinised saline or autologous whole blood. There is substantial heterogeneity both in practice and reporting of trials evaluating vein graft preservation solutions, and the quality of existing evidence is low. There is an unmet need for high quality trials evaluating the potential for these interventions to improve long-term patency in venous bypass grafts.

Disturbed flow increases endothelial inflammation and permeability via a Frizzled-4-ß-catenin-dependent pathway.

Multidirectional or disturbed flow promotes endothelial dysfunction and is associated with early atherogenesis. Here we investigated the role of Wnt signalling in flow-mediated endothelial dysfunction. The expression of Frizzled-4 was higher in cultured human aortic endothelial cells (ECs) exposed to disturbed flow compared to that seen for undisturbed flow, obtained using an orbital shaker. Increased expression was also detected in regions of the porcine aortic arch exposed to disturbed flow. The increased Frizzled-4 expression in cultured ECs was abrogated following knockdown of R-spondin-3. Disturbed flow also increased the nuclear localisation and activation of ß-catenin, an effect that was dependent on Frizzled-4 and R-spondin-3. Inhibition of ß-catenin using the small-molecule inhibitor iCRT5 or knockdown of Frizzled-4 or R-spondin-3 resulted in reduced expression of pro-inflammatory genes in ECs exposed to disturbed flow, as did inhibition of WNT5A signalling. Inhibition of the canonical Wnt pathway had no effect. Inhibition of ß-catenin also reduced endothelial paracellular permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. These data suggest the presence of an atypical Frizzled-4-ß-catenin pathway that promotes endothelial dysfunction in response to disturbed flow.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

BACKGROUND: Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. METHODS: PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. RESULTS: PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. CONCLUSIONS: We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Large animal model of vein grafts intimal hyperplasia: A systematic review.

Coronary artery bypass grafting remains the treatment of choice for a large cohort of patients with significant coronary disease. Despite the increased use of arterial grafts, the long saphenous vein remains the most commonly used conduit. Long-term graft patency continues to be the Achilles heel of saphenous vein grafts. This is due to the development of intimal hyperplasia, a chronic inflammatory disease that results in the narrowing and occlusion of a significant number of vein grafts. Research models for intimal hyperplasia are essential for a better understanding of pathophysiological processes of this condition. Large animal models resemble human anatomical structures and have been used as a surrogate to study disease development and prevention over the years. In this paper, we systematically review all published studies that utilized large animal models of vein graft disease with a focus on the type of model and any therapeutic intervention, specifically the use of external stents/mesh.

Longitudinal Resting-State Functional Magnetic Resonance Imaging Study: A Seed-Based Connectivity Biomarker in Patients with Ischemic and Intracerebral Hemorrhage Stroke.

Objective: The primary aim of the research was to compare the impact of postischemic and hemorrhagic stroke on brain connectivity and recovery using resting-state functional magnetic resonance imaging. Methods and Procedures: We serially imaged 20 stroke patients, 10 with ischemic stroke (IS) and 10 with intracerebral hemorrhage (ICH), at 1, 3, and 12 months (1M, 3M, and 12M) after ictus. Data from 10 healthy volunteers were obtained from a publically available imaging data set. All functional and structural images underwent standard processing for brain extraction, realignment, serial registration, unwrapping, and denoising using SPM12. A seed-based group analysis using CONN software was used to evaluate the default mode network and the sensorimotor network connections by applying bivariate correlation and hemodynamic response function weighting. Results: In comparison with healthy controls, both IS and ICH exhibited disrupted interactions (decreased connectivity) between these two networks at 1M. Interactions then increased by 12M in each group. Temporally, each group exhibited a minimal increase in connectivity at 3M compared with 12M. Overall, the ICH patients exhibited a greater magnitude of connectivity disruption compared with IS patients, despite a significant intrasubject reduction in hematoma volume. We did not observe any significant correlation between change in connectivity and recovery as measured on the National Institutes of Health Stroke Scale (NIHSS) at any time point. Conclusions: These findings demonstrate that the largest changes in functional connectivity occur earlier (3M) rather than later (12M) and show subtle differences between IS and ICH during recovery and should be explored further in larger samples.

Mechanobiology of the endothelium in vascular health and disease: in vitro shear stress models.

In recent years, there has been growing evidence that vascular pathologies arise in sites experiencing an altered haemodynamic environment. Fluid shear stress (FSS) is an important contributor to vascular homeostasis and regulates endothelial cell (EC) gene expression, morphology, and behaviour through specialised mechanosensitive signalling pathways. The presence of an altered FSS profile is a pathological characteristic of many vascular diseases, with the most established example being the preferential localisation of atherosclerotic plaque development. However, the precise haemodynamic contributions to other vascular pathologies including coronary artery vein graft failure remains poorly defined. To evaluate potential novel therapeutics for the treatment of vascular diseases via targeting EC behaviour, it is important to undertake in vitro experiments using appropriate culture conditions, particularly FSS. There are a wide range of in vitro models used to study the effect of FSS on the cultured endothelium, each with the ability to generate FSS flow profiles through which the investigator can control haemodynamic parameters including flow magnitude and directionality. An important consideration for selection of an appropriate model of FSS exposure is the FSS profile that the model can generate, in comparison to the physiological and pathophysiological haemodynamic environment of the vessel of interest. A resource bringing together the haemodynamic environment characteristic of atherosclerosis pathology and the flow profiles generated by in vitro methods of applying FSS would be beneficial to researchers when selecting the appropriate model for their research. Consequently, here we summarise the widely used methods of exposing cultured endothelium to FSS, the flow profile they generate and their advantages and limitations in investigating the pathological contribution of altered FSS to vascular disease and evaluating novel therapeutic targets for the treatment and prevention of vascular disease.

Temporal Improvements in COVID-19 Outcomes for Hospitalized Adults: A Post Hoc Observational Study of Remdesivir Group Participants in the Adaptive COVID-19 Treatment Trial.

BACKGROUND: The COVID-19 standard of care (SOC) evolved rapidly during 2020 and 2021, but its cumulative effect over time is unclear. OBJECTIVE: To evaluate whether recovery and mortality improved as SOC evolved, using data from ACTT (Adaptive COVID-19 Treatment Trial). DESIGN: ACTT is a series of phase 3, randomized, double-blind, placebo-controlled trials that evaluated COVID-19 therapeutics from February 2020 through May 2021. ACTT-1 compared remdesivir plus SOC to placebo plus SOC, and in ACTT-2 and ACTT-3, remdesivir plus SOC was the control group. This post hoc analysis compared recovery and mortality between these comparable sequential cohorts of patients who received remdesivir plus SOC, adjusting for baseline characteristics with propensity score weighting. The analysis was repeated for participants in ACTT-3 and ACTT-4 who received remdesivir plus dexamethasone plus SOC. Trends in SOC that could explain outcome improvements were analyzed. (ClinicalTrials.gov: NCT04280705 [ACTT-1], NCT04401579 [ACTT-2], NCT04492475 [ACTT-3], and NCT04640168 [ACTT-4]). SETTING: 94 hospitals in 10 countries (86% U.S. participants). PARTICIPANTS: Adults hospitalized with COVID-19. INTERVENTION: SOC. MEASUREMENTS: 28-day mortality and recovery. RESULTS: Although outcomes were better in ACTT-2 than in ACTT-1, adjusted hazard ratios (HRs) were close to 1 (HR for recovery, 1.04 [95% CI, 0.92 to 1.17]; HR for mortality, 0.90 [CI, 0.56 to 1.40]). Comparable patients were less likely to be intubated in ACTT-2 than in ACTT-1 (odds ratio, 0.75 [CI, 0.53 to 0.97]), and hydroxychloroquine use decreased. Outcomes improved from ACTT-2 to ACTT-3 (HR for recovery, 1.43 [CI, 1.24 to 1.64]; HR for mortality, 0.45 [CI, 0.21 to 0.97]). Potential explanatory factors (SOC trends, case surges, and variant trends) were similar between ACTT-2 and ACTT-3, except for increased dexamethasone use (11% to 77%). Outcomes were similar in ACTT-3 and ACTT-4. Antibiotic use decreased gradually across all stages. LIMITATION: Unmeasured confounding. CONCLUSION: Changes in patient composition explained improved outcomes from ACTT-1 to ACTT-2 but not from ACTT-2 to ACTT-3, suggesting improved SOC. These results support excluding nonconcurrent controls from analysis of platform trials in rapidly changing therapeutic areas. PRIMARY FUNDING SOURCE: National Institute of Allergy and Infectious Diseases.

Development and Preliminary Testing of Porcine Blood-Derived Endothelial-like Cells for Vascular Tissue Engineering Applications: Protocol Optimisation and Seeding of Decellularised Human Saphenous Veins.

Functional endothelial cells (EC) are a critical interface between blood vessels and the thrombogenic flowing blood. Disruption of this layer can lead to early thrombosis, inflammation, vessel restenosis, and, following coronary (CABG) or peripheral (PABG) artery bypass graft surgery, vein graft failure. Blood-derived ECs have shown potential for vascular tissue engineering applications. Here, we show the development and preliminary testing of a method for deriving porcine endothelial-like cells from blood obtained under clinical conditions for use in translational research. The derived cells show cobblestone morphology and expression of EC markers, similar to those seen in isolated porcine aortic ECs (PAEC), and when exposed to increasing shear stress, they remain viable and show mRNA expression of EC markers similar to PAEC. In addition, we confirm the feasibility of seeding endothelial-like cells onto a decellularised human vein scaffold with approximately 90% lumen coverage at lower passages, and show that increasing cell passage results in reduced endothelial coverage.

Resistance to dicyclanil and imidacloprid in the sheep blowfly, Lucilia cuprina, in Australia.

BACKGROUND: The sheep blowfly, Lucila cuprina, is a myiasis-causing parasite responsible for significant production losses and welfare issues for the Australian sheep industry. Control relies largely on the use of insecticides. The pyrimidine compound, dicyclanil, is the predominant control chemical, although other insecticides also are used, including imidacloprid, ivermectin, cyromazine and spinosad. We investigated in vitro resistance patterns and mechanisms in field-collected blowfly strains. RESULTS: The Walgett 2019 strain showed significant levels of resistance to both dicyclanil and imidacloprid, with resistance factors at the IC50 of 26- and 17-fold, respectively, in in vitro bioassays. Co-treatment with the cytochrome P450 inhibitor, aminobenzotriazole, resulted in significant levels of synergism for dicyclanil and imidacloprid (synergism ratios of 7.2- and 6.1-fold, respectively), implicating cytochrome P450 in resistance to both insecticides. Cyp12d1 transcription levels were increased up to 40-fold throughout the larval life stages in the resistant strain compared to a reference susceptible strain, whereas transcription levels of some other cyp genes (6g1, 4d1, 28d1) did not differ between the strains. Similar resistance levels also were observed in flies collected from the same property in two subsequent years. CONCLUSION: This study indicates that in vitro resistance to both dicyclanil and imidacloprid in this field-collected blowfly strain is likely mediated by cytochrome P450, with Cyp12d1 implicated as the enzyme responsible; however, it remains possible that another P450 also may be involved. A common resistance mechanism for the two drugs has important implications for drug rotation strategies designed to prolong the useful life of flystrike control chemicals. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Hybrid coronary revascularization versus coronary artery bypass grafting for multivessel coronary artery disease: A systematic review and meta-analysis.

BACKGROUND: Hybrid coronary revascularization (HCR) combines the benefits of a left internal mammary artery to left anterior descending artery anastomosis, via a mini thoracotomy, with percutaneous coronary intervention (PCI) for other diseased coronaries. AIMS: The aim of this meta-analysis is to compare the short- and long-term outcomes of HCR with those of coronary artery bypass grafting (CABG) for multi-vessel coronary artery disease (MCAD). METHODS: We performed a meta-analysis with a primary outcome of short-term mortality and secondary outcomes of mid-term survival, length of hospital stay, stroke, renal failure and mid-term MACE rate. RESULTS: 3399 patients (HCR = 1164, CABG = 2235) were included, with no significant difference in short-term mortality between groups (OR = 1.50, 95% CI = [0.90,2.49], p = 0.11), although a higher mortality rate was seen in the HCR group (0.73% vs 0.64%). The average length of stay in intensive care unit was significantly shorter following HCR than CABG (mean difference = -15.52 h, CI = [-22.47,-8.59], p˂0.001) and overall hospital stay was also shorter in this group, although not statistically significant (mean difference = -3.15 days, 95% CI = [-6.55, 0.25], p = 0.07). HCR was associated with a reduced odds of blood transfusion (OR = 0.34, 95% CI = [0.22,0.54], p < 0.001). There was not a significant difference in mid-term survival (OR = 0.86, 95% CI = [0.62,1.21], p = 0.39) or MACE rate (OR = 0.82, 95% CI = [0.55,1.23], p = 0.34). No differences were found between HCR and CABG for post-operative stroke (OR = 1.36, 95% CI = [0.87, 2.13], p = 0.16) or renal failure (OR = 0.71, 95% CI = [0.43,1.16], p = 0.14). CONCLUSIONS: HCR has a higher incidence of short-term mortality compared to CABG in patients with MCAD, although this difference is not statistically significant. Similar rates of mid-term survival and other short term post-operative complications were found between the two groups. HCR has a shorter ICU stays and reduced requirement for blood transfusion.

Next-Generation and Single-Cell Sequencing Approaches to Study Atherosclerosis and Vascular Inflammation Pathophysiology: A Systematic Review.

Background and Aims: Atherosclerosis is a chronic inflammatory disease that remains the leading cause of morbidity and mortality worldwide. Despite decades of research into the development and progression of this disease, current management and treatment approaches remain unsatisfactory and further studies are required to understand the exact pathophysiology. This review aims to provide a comprehensive assessment of currently published data utilizing single-cell and next-generation sequencing techniques to identify key cellular and molecular contributions to atherosclerosis and vascular inflammation. Methods: Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until February 2022. A narrative synthesis of all included studies was performed for all included studies. Quality assessment and risk of bias analysis was evaluated using the ARRIVE and SYRCLE checklist tools. Results: Thirty-four studies were eligible for narrative synthesis, with 16 articles utilizing single-cell exclusively, 10 utilizing next-generation sequencing and 8 using a combination of these approaches. Studies investigated numerous targets, ranging from exploratory tissue and plaque analysis, cell phenotype investigation and physiological/hemodynamic contributions to disease progression at both the single-cell and whole genome level. A significant area of focus was placed on smooth muscle cell, macrophage, and stem/progenitor contributions to disease, with little focus placed on contributions of other cell types including lymphocytes and endothelial cells. A significant level of heterogeneity exists in the outcomes from single-cell sequencing of similar samples, leading to inter-sample and inter-study variation. Conclusions: Single-cell and next-generation sequencing methodologies offer novel means of elucidating atherosclerosis with significantly higher resolution than previous methodologies. These approaches also show significant potential for translatability into other vascular disease states, by facilitating cell-specific gene expression profiles between disease states. Implementation of these technologies may offer novel approaches to understanding the disease pathophysiology and improving disease prevention, management, and treatment.Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021229960, identifier: CRD42021229960.

Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro.

Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.

Monitoring Cellular Proliferation and Apoptosis in Atherosclerosis Plaques and Intimal Thickenings.

Immunohistochemistry for specific proteins characteristic of proliferative or apoptotic cells allows for monitoring of these cell behaviors in biological tissues samples, including atherosclerotic plaques and intimal thickenings. Proliferating cell nuclear antigen (PCNA) and Ki-67 are widely used markers of cell proliferation and cleaved caspase-3 is a well-established marker of apoptosis that can be detected in tissue samples using immunohistochemistry. This technique enables quantification of the abundance of these proteins and provides information on the distribution of these biomarkers in tissues. By combining with immunohistochemistry for specific cell type markers, it is also possible to determine which cell types are proliferating or undergoing apoptosis. Here, we detail protocols for immunohistochemistry of PCNA, Ki-67, and cleaved caspase-3 for evaluation of cellular proliferation and apoptosis in atherosclerotic plaques in vivo. In addition, we outline methods for the quantification and localization of cell proliferation using bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) and ethynyldeoxyuridine/5-ethynyl-2 ́-deoxyuridine(EdU) labeled tissue samples collected from animals exposed to BrdU or EdU.