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Cardiovascular disease is most frequently caused by the development and progression of atherosclerosis. When coronary arteries are afflicted, and the stenoses caused by atherosclerotic plaques are severe enough, the metabolic supply-and-offer balance is disturbed, leading to myocardial ischemia. If atherosclerotic plaques become unstable and local thrombosis develops, a myocardial infarction occurs. Sometimes, myocardial ischemia and infarction may result in significant and irreversible heart failure. To prevent severe complications, such as acute coronary syndromes and ischemia-related heart failure, extensive efforts have been made for developing biomarkers that would help identify patients at increased risk for cardiovascular events. In this two-part study, we attempted to provide a review of existing knowledge of blood biomarkers that may be used in this setting. The first part of this work was dedicated to conventional biomarkers, which are already used in clinical practice. In the second part, here presented, we discuss emerging biomarkers which have not yet become mainstream.

Atherosclerosis is the main cause of cardiovascular disease worldwide. The progression of coronary atherosclerosis leads to coronary artery disease, with impaired blood flow to the myocardium and subsequent development of myocardial ischemia. Acute coronary syndromes and post-myocardial infarction heart failure are two of the most common complications of coronary artery disease and are associated with worse outcomes. In order to improve the management of patients with coronary artery disease and avoid major cardiovascular events, several risk assessment tools have been developed. Blood and imaging biomarkers, as well as clinical risk scores, are now available and validated for clinical practice, but research continues. The purpose of the current paper is to provide a review of recent findings regarding the use of humoral biomarkers for risk assessment in patients with heart disease.

Abstract Background Emergency department (ED) professionals face a high risk of physical, mental, and emotional strain, which can lead to burnout. Burnout arises from chronic work-related stress, which negatively impacts ED professionals, the healthcare system, as well as patient outcomes. Effective adaptive emotion regulation strategies (ERS) are crucial for handling stress in healthcare workplaces and mitigating the risk of burnout. High-quality patient-professional relationships, including empathy, significantly improve patient compliance and disease outcomes. This cross-sectional exploratory study aims to investigate the relationship between mental distress, burnout, adaptive and maladaptive ERS among healthcare professionals in an ED. Methods A total of 159 ED workers participated in an online survey. Emotion regulation was assessed using the Cognitive Emotion Regulation Questionnaire, while burnout and mental distress were measured using the Maslach Burnout Inventory and the Depression, Anxiety, and Stress Scale. Results The findings revealed that more than 20% of participants displayed severe symptoms of stress (N = 35), anxiety (N = 36), and depression (N = 31), and only 10.7% (N = 27) had no signs of burnout. Despite these findings, it appears that the majority of ED professionals primarily utilise adaptive ERS (91.8%, N = 146). However, as burnout levels increased, the use of adaptive ERS declined compared to maladaptive strategies. Regression analysis identified several significant predictors of Emotional Exhaustion, Depersonalisation, and reduced Personal Accomplishment, including gender, age, physical exercise, smoking, sedative usage, stress, depression, maladaptive strategies and adaptive strategies such as positive reappraisal. Conclusions These study results highlight the urgent need to address workplace stress, burnout, and mental distress among healthcare professionals in EDs. Implementing effective strategies for adaptive emotion regulation and promoting a supportive work environment can help mitigate burnout and enhance the well-being of ED workers, ultimately benefiting patient care and outcomes.

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography–tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting “vesicular” proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at −80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of “vesicular” proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.

Background: Current guidelines recommend that glycoprotein IIb/IIIa inhibitor (GPI) and manual aspiration thrombectomy should not be routinely used in patients with ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (pPCI), although there is a lack of dedicated studies. The aim of this study was to examine the impact of combined usage of a potent P2Y12 inhibitor, GPI, and manual aspiration thrombectomy on long-term survival after STEMI.Methods: All STEMI patients treated by pPCI in a tertiary center who have been included prospectively in the local PCI registry between January 2016 and December 2022 were analyzed in this study. Patients were excluded if they required oral anticoagulation or bridging between clopidogrel or ticagrelor during hospitalization.Results: A total of 1,210 patients were included in the present study, with a median follow-up of 2.78 (1.00–4.88) years. Ticagrelor significantly reduced all-cause and cardiovascular-cause mortality [HR = 0.27 (0.21–0.34), p < 0.0001 and HR = 0.23 (0.17–0.30), p < 0.0001, respectively]. Eptifibatide significantly reduced all-cause and cardiovascular-cause mortality [HR = 0.72 (0.57–0.92), p = 0.002, and HR = 0.68 (0.52–0.89), p = 0.001, respectively]. Manual thrombus aspiration had no significant effect on both all-cause and cardiovascular-cause mortality. In multivariate Cox regression, all-cause mortality was reduced by ticagrelor, while eptifibatide or manual thrombus aspiration had no significant effect. However, cardiovascular-cause mortality was reduced by both ticagrelor and eptifibatide, while manual thrombus aspiration had no significant effect.Conclusion: Ticagrelor consistently reduced cardiovascular and all-cause mortality, while eptifibatide reduced only cardiovascular mortality. Manual thrombus aspiration provided no long-term benefit. Our findings support the current guideline recommendation that GPI and manual aspiration thrombectomy should not be routinely used in treatment of STEMI with pPCI.

Background and Objectives: Progressive supranuclear palsy (PSP) is a neurodegenerative disease, a tauopathy, which results in a wide clinical spectrum of neurological symptoms. The diagnosis is mostly based on clinical signs and neuroimaging; however, possible biomarkers for screening have been under investigation, and the role of the gut microbiome is unknown. The aim of our study was to identify potential blood biomarkers and observe variations in the gut microbiome within a PSP discordant monozygotic twin pair. Materials and Methods: Anthropometric measurements, neuropsychological tests, and the neurological state were evaluated. Blood was collected for metabolic profiling and for the detection of neurodegenerative and vascular biomarkers. Both the gut microbiome and brain MRI results were thoroughly examined. Results: We found a relevant difference between alpha-synuclein levels and moderate difference in the levels of MMP-2, MB, Apo-A1, Apo-CIII, and Apo-H. With respect to the ratios, a small difference was observed for ApoA1/SAA and ApoB/ApoA1. Using a microbiome analysis, we also discovered a relative dysbiosis, and the MRI results revealed midbrain and frontoparietal cortical atrophy along with a reduction in overall brain volumes and an increase in white matter lesions in the affected twin. Conclusions: We observed significant differences between the unaffected and affected twins in some risk factors and blood biomarkers, along with disparities in the gut microbiome. Additionally, we detected abnormalities in brain MRI results and alterations in cognitive functions.

Abstract Stem cell-based therapies raise hope for cell replacement and provide opportunity for cardiac regenerative medicine and tumor therapy. Extracellular vesicles are a membrane-enclosed intercellular delivery system with the potential to improve the therapeutic efficacy of the treatment of a variety of disorders. As the incidence of breast cancer continues to rise, radiotherapy has emerged as a leading treatment modality. Radiotherapy also increases the risk of coronary heart disease and cardiac mortality. In a chest-irradiated mouse model of cardiac injury, we investigated the effects of local irradiation. We found an increased lethality after 16 Gy irradiation. Importantly, radio-detoxified LPS (RD-LPS) treatment prolonged the survival significantly. By flow cytometry, we demonstrated that upon administration of RD-LPS, the number of bone marrow-derived endothelial progenitor cells increased in the bone marrow and, in particular, in the circulation. Furthermore, mass spectrometry analysis showed that RD-LPS altered the proteomic composition of bone marrow cell-derived small extracellular vesicles (sEVs). RD-LPS treatment increased interferon-induced transmembrane protein-3 (IFITM3) expression markedly both in bone marrow cells and in bone marrow cell-derived small extracellular vesicles. This is the first study to demonstrate that radio-detoxified LPS treatment induces an increase of circulating endothelial progenitor cells (EPCs) in parallel with a reduced radiotherapy-related mortality. While the total number of bone marrow-derived extracellular vesicles was significantly increased 24 h after treatment in the RD-LPS groups, the number of endothelial progenitor cells was reduced in animals injected with GW4896 (a chemical inhibitor of exosome biogenesis) as compared with controls. In contrast to these in vivo results, in vitro experiments did not support the effect of sEVs on EPCs. Our data raise the intriguing possibility that IFITM3 may serve as a marker of the radio-detoxified LPS treatment.

Background. tert-butylhydroquinone (tBHQ) is an antioxidant commonly used as a food additive. Studies suggest that tBHQ could modulate immune responses to influenza and SARS-CoV-2 infection. In our transcriptomic analysis we explored the molecular mechanisms behind tBHQ’s modulatory properties and the relationships to respiratory viral infections. Methods. tBHQ was administered per os to BALB/c mice (1.5% [w/w]) for 20 days. Splenic T cells were isolated with magnetic separation and subjected to transcriptomic analysis. Gene-set enrichment analysis and g:Profiler was conducted to provide a functional interpretation of significantly changed genes. Further analysis for AHR/NRF2 binding sites was performed with GeneHancer. Results. In CD4+ cells, we found significantly altered expression of 269 genes by tBHQ. Of them, many had relevance in influenza infection such as genes responsible for virus entry (Anxa1/2, Cd14), interferon signaling (Dusp10, Tnfsf13), or prostaglandin synthesis (Ptgs1/2). In SARS-CoV-2 infections, interferon signaling (Ifitm1), proteolytic enzymes (CtsB), and also cell-surface proteins (Cd14, Cd151) were among the prominent alterations after tBHQ exposure. Of these genes, many had one or more binding sites for AHR and NRF2, two major xenosensors triggered by tBHQ. Conclusions. Our results strongly suggest that a common food additive, tBHQ, can modulate virus-dependent processes in both influenza and SARS-CoV-2 infections.

The prevalence of isolated right coronary artery (RCA) absence ranges from 0.014% to 0.066% in the general population, but its combination with an absent left main (dual ostium left anterior descending [LAD] and super-dominant left circumflex [LCx]) has not been previously described. We report the case of a rare coronary artery anomaly: an absent RCA with LAD and LCx coronary arteries arising separately from the left coronary sinus. A 53-year-old male with recent COVID-19 infection was referred to our service for coronary computed tomography angiography (CCTA) due to the recent onset of atypical chest pain. The RCA was absent, with no vessel leaving the right or non-coronary sinus. The LAD and LCx emerged from the left coronary sinus, with a “double-barrel” appearance. The LAD was unremarkable, with small, non-stenosed calcified plaque. The LCx had a 3 mm diameter, arching downward in the left atrioventricular groove, passing through the crux cordis, continuing into the right atrioventricular groove, and ending as a left acute artery and sinonodal artery. No significant stenosis was found on any of the vessels, ruling out atherosclerotic coronary disease.

Abstract Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

Abstract There is no available blood marker for the preoperative diagnosis of adrenocortical malignancy. The objective of this study was to investigate the expression of extracellular vesicle-associated microRNAs and their diagnostic potential in plasma samples of patients suffering from adrenocortical tumors. Extracellular vesicles were isolated either by using Total Exosome Isolation Kit or by differential centrifugation/ultracentrifugation. Preoperative plasma extracellular vesicle samples of 6 adrenocortical adenomas (ACA) and 6 histologically verified adrenocortical cancer (ACC) were first screened by Taqman Human Microarray A-cards. Based on the results of screening, two miRNAs were selected and validated by targeted quantitative real-time PCR. The validation cohort included 18 ACAs and 16 ACCs. Beside RNA analysis, extracellular vesicle preparations were also assessed by transmission electron microscopy, flow cytometry and dynamic light scattering. Significant overexpression of hsa-miR-101 and hsa-miR-483-5p in ACC relative to ACA samples has been validated. Receiver operator characteristics of data revealed dCT hsa-miR-483-5p normalized to cel-miR-39 to have the highest diagnostic accuracy (area under curve 0.965), the sensitivity and the specifity were 87.5 and 94.44, respectively. Extracellular vesicle-associated hsa-miR-483-5p thus appears to be a promising minimally invasive biomarker in the preoperative diagnosis of ACC but needs further validation in larger cohorts of patients.

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 μm (mean±S.D.: 1.17 ± 0.34 μm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro, HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.