The Influence of Myeloid-Derived Suppressor Cell Expansion in Neuroinflammation and Neurodegenerative Diseases.

The neuro-immune axis has a crucial function both during physiological and pathological conditions. Among the immune cells, myeloid-derived suppressor cells (MDSCs) exert a pivotal role in regulating the immune response in many pathological conditions, influencing neuroinflammation and neurodegenerative disease progression. In chronic neuroinflammation, MDSCs could lead to exacerbation of the inflammatory state and eventually participate in the impairment of cognitive functions. To have a complete overview of the role of MDSCs in neurodegenerative diseases, research on PubMed for articles using a combination of terms made with Boolean operators was performed. According to the search strategy, 80 papers were retrieved. Among these, 44 papers met the eligibility criteria. The two subtypes of MDSCs, monocytic and polymorphonuclear MDSCs, behave differently in these diseases. The initial MDSC proliferation is fundamental for attenuating inflammation in Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS), but not in amyotrophic lateral sclerosis (ALS), where MDSC expansion leads to exacerbation of the disease. Moreover, the accumulation of MDSC subtypes in distinct organs changes during the disease. The proliferation of MDSC subtypes occurs at different disease stages and can influence the progression of each neurodegenerative disorder differently.

A real-world multicenter study on left atrial appendage occlusion: The Italian multi-device experience.

Background: Transcatheter left atrial appendage occlusion (LAAO) has emerged as an alternative treatment for stroke prevention in patients with atrial fibrillation (AF) at high risk of thromboembolism, who cannot tolerate long-term oral anticoagulation (OAC). Questions persist regarding effectiveness and safety of this treatment and the optimal post-interventional antithrombotic regimen after LAAO. Methods: We retrospectively gathered data from 428 patients who underwent percutaneous LAAO in 6 Italian high-volume centres, aimed at describing the real-world utilization, safety, and effectiveness of LAAO procedures, also assessing the clinical outcomes associated with different antithrombotic strategies. Results: Among the entire population, 20 (4.7 %) patients experienced a combination of pericardial effusion and periprocedural major bleeding: 8 (1.9 %) pericardial effusion, 1 (0.3 %) fatal bleeding, and 3 (0.7 %) non-fatal procedural major bleeding. Patients were discharged with different antithrombotic regimens: dual (DAPT) (27 %) or single (SAPT) (26 %) antiplatelet therapy, OAC (27 %), other antithrombotic regimens (14 %). Very few patients were not prescribed with antithrombotic drugs (6 %). At a medium 523 ± 58 days follow-up, 14 patients (3.3 %) experienced all-cause death, 6 patients (1.4 %) cardiovascular death, 3 patients (0.7 %) major bleeding, 10 patients (2.6 %) clinically relevant non-major bleeding, and 3 patients (0.7 %) ischemic stroke. At survival analysis, with DAPT as the reference group, OAC therapy was associated with better outcomes. Conclusions: Our findings confirm that LAAO is a safe procedure. Different individualized post-discharge antithrombotic regimens are now adopted, likely driven by the perceived thrombotic and hemorrhagic risk. The incidence of both ischemic and bleeding events tends to be low.

A Review on the Use of Deep Eutectic Solvents in Protection Reactions.

Given the recent research on the application of eco-sustainable methods in organic chemistry, we have focused our attention on the derivatization processes for fundamental functional groups in organic chemistry, such as amino, hydroxyl and carbonyl groups. Protection reactions are needed to temporarily block a certain reactive site on a molecule. The use of green solvents in this context has made an excellent contribution to the development of eco-sustainable methods. In recent years, deep eutectic solvents (DESs) have had great success as a new class of green solvents used in various chemical applications, such as extraction or synthetic processes. These solvents are biodegradable and nontoxic. In this framework, a list of relevant works found in the literature is described, considering DESs to be a good alternative to classic toxic solvents in the protection reactions of important functional groups.

Mechanisms of action of mineral fibres in a placental syncytiotrophoblast model: An in vitro toxicology study.

Asbestos has been widely used due to its unique characteristics. It is known that exposure to asbestos causes serious damage to health but one species, chrysolite, is still used because it is considered less toxic and not biopersistent in some countries. The aim of our study was to investigate if cellular process underlying the proliferation, differentiation and cell death of placental tissues could be modify in presence of asbestos fibres (50 µg/ml final concentration), long chrysolite fibres (CHR-L) and short chrysolite fibres (CHR-S), using BeWo cell line, an in vitro model that mimics the syncytiotrophoblast (STB), the outer layer of placental villi. Our data demonstrated that none of the fibres analysed alter syncytiotrophoblast formation but all of them induce ROS formation and reduced cell proliferation. Moreover, we showed that only CHR-L fibre induced was able to induce irreversible DNA alterations that carried cells to apoptosis. In fact, BeWo cells exposed to CHR-L fibre showed a significant increase in cleaved CASP3 protein, a marker of apoptosis. These data suggest that CHR-L may induce death of the placental villi leading to impaired placental development. The impairment of placental development is the basis of many gestational pathologies such as preeclampsia and intrauterine growth retardation. Since these pathologies are very dangerous for foetal and maternal life, we suggest to the gynaecologists to carefully evaluate the area of maternal residence, the working environment, the food used, and the materials used daily to avoid contact with these fibres as much as possible.

Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1/PD-L1 blockade strategies.

BACKGROUND: Previous studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy. METHODS: Reprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models. RESULTS: Oleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression. DISCUSSION: Oleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells.

Low circulating levels of miR-17 and miR-126-3p are associated with increased mortality risk in geriatric hospitalized patients affected by cardiovascular multimorbidity.

MultiMorbidity (MM), defined as the co-occurrence of two or more chronic conditions, is associated with poorer health outcomes, such as recurrent hospital readmission and mortality. As a group of conditions, cardiovascular disease (CVD) exemplifies several challenges of MM, and the identification of prognostic minimally invasive biomarkers to stratify mortality risk in patients affected by cardiovascular MM is a huge challenge. Circulating miRNAs associated to inflammaging and endothelial dysfunction, such as miR-17, miR-21-5p, and miR-126-3p, are expected to have prognostic relevance. We analyzed a composite profile of circulating biomarkers, including miR-17, miR-21-5p, and miR-126-3p, and routine laboratory biomarkers in a sample of 246 hospitalized geriatric patients selected for cardiovascular MM from the Report-AGE INRCA database and BioGER INRCA biobank, to evaluate the association with all-cause mortality during 31 days and 12 and 24 months follow-up. Circulating levels of miR-17, miR-126-3p, and some blood parameters, including neutrophil to lymphocyte ratio (NLR) and eGFR, were significantly associated with mortality in these patients. Overall, our results suggest that in a cohort of geriatric hospitalized patients affected by cardiovascular MM, lower circulating miR-17 and miR-126-3p levels could contribute to identify patients at higher risk of short- and medium-term mortality.

The cell line models to study tyrosine kinase inhibitors in non-small cell lung cancer with mutations in the epidermal growth factor receptor: A scoping review.

Non-Small Cell Lung Cancer (NSCLC) represents ∼85% of all lung cancers and ∼15-20% of them are characterized by mutations affecting the Epidermal Growth Factor Receptor (EGFR). For several years now, a class of tyrosine kinase inhibitors was developed, targeting sensitive mutations affecting the EGFR (EGFR-TKIs). To date, the main burden of the TKIs employment is due to the onset of resistance mutations. This scoping review aims to resume the current situation about the cell line models employed for the in vitro evaluation of resistance mechanisms induced by EGFR-TKIs in oncogene-addicted NSCLC. Adenocarcinoma results the most studied NSCLC histotype with the H1650, H1975, HCC827 and PC9 mutated cell lines, while Gefitinib and Osimertinib the most investigated inhibitors. Overall, data collected frame the current advancement of this topic, showing a plethora of approaches pursued to overcome the TKIs resistance, from RNA-mediated strategies to the innovative combination therapies.

Assessment of Mediterranean Citrus Peel Flavonoids and Their Antioxidant Capacity Using an Innovative UV-Vis Spectrophotometric Approach.

Citrus fruits exert various beneficial health effects due to the large amount of polyphenols they contain. Citrus peels, often considered food waste, contain several health-promoting polyphenols. Among these, flavonoids have long been quantified through colorimetric assays which, if not adequately applied, can lead to conflicting results. Flavonoids possess strong antioxidant properties and can decrease circulating free radicals, thereby reducing oxidative stress phenomena. Quantifying flavonoids and properly estimating their antioxidant capacity allows us to predict plausible beneficial effects of citrus fruits on human health. The aim of this research was to analyze the advantageous phenolic compounds found in the peels of citrus fruits commonly found in the Mediterranean region. The objective was to measure their antioxidant capacity and ability to neutralize free radicals. To achieve this purpose, UV-visible spectrophotometric analyses, liquid chromatography (LC) and Electron Paramagnetic Spectroscopy (EPR) were utilized and compared, finally suggesting an innovative approach for assessing the overall flavonoid content by the nitrite-aluminum assay. HPLC data demonstrated that hesperidin was the most abundant flavonoid in all peel extracts except for orange peels, in which naringin was the predominant flavonoid. The total flavonoid content was greater than 1.3 mg/mL in all extracts, with tangerine and orange yielding the best results. Citrus peel polyphenols exerted strong antioxidant and free radical scavenging effects, inhibiting up to 75% of the free radicals used as reference in the EPR analyses.

Storage Effects on Bioactive Phenols in Calabrian Monovarietal Extra Virgin Olive Oils Based on the EFSA Health Claim.

The beneficial properties of extra virgin olive oil (EVOO) on lipids blood levels were recognized by the European Food Safety Authority (EFSA) with a health claim, specifically referring to EVOOs containing at least 5 mg of hydroxytyrosol and its secoiridoids derivatives per 20 g of oil. The main purpose of the work was to characterize the phenolic profile of two commercially available Calabrian monovarietal EVOOs (Nocellara del Belice, VN; Dolce di Rossano, VDR), and to study the effect of one-year storage on secoiridoids composition, by monthly controls. A new UHPLC-ESI-HRMS method was developed and validated, thus facilitating the EFSA claim application and allowing producers to valorize their products. Seven biologically active compounds were chosen: tyrosol, hydroxytyrosol, oleocanthal, oleacein, oleuropein aglycone, verbascoside, and oleuropein. LODs and LOQs were 0.001-0.02 mg g-1 and 0.002-0.08 mg g-1, respectively. The variation coefficients were ≤20% and the percentage of recovery was between 89-109%. During the 12-month storage period, the concentration of selected compounds ranged between 1258.78-1478.91 mg Kg-1 for VN, and 1408.22-2071.45 mg Kg-1 for VDR, with a decrease of 15% and 32% respectively. The method allows an accurate quantification of EVOO phenols thus being useful to certify the nutraceutical properties of olive oil.

Serum levels of soluble suppression of tumorigenicity 2 (sST2) and heart-type fatty acid binding protein (H-FABP) independently predict in-hospital mortality in geriatric patients with COVID-19.

Elevation of cardiac damage biomarkers is associated with adverse clinical outcomes and increased mortality in COVID-19 patients. This study assessed the association of admission serum levels of sST2 and H-FABP with in-hospital mortality in 191 geriatric patients (median age 86 yrs., IQR 82-91 yrs.) with COVID-19 and available measures of hs-cTnT and NT-proBNP at admission. Cox proportional hazards models were utilized to predict in-hospital mortality, considering clinical/biochemical confounders as covariates. A composite cardiac score was calculated to improve predictive accuracy. Patients deceased during their hospital stay (26%) exhibited higher levels of all biomarkers, which demonstrated good discrimination for in-hospital mortality. Addition of sST2 and H-FABP significantly improved the discriminatory power of hs-cTnT and NT-proBNP. The composite cardiac score (AUC=0.866) further enhanced the predictive accuracy. Crude and adjusted Cox regressions models revealed that both sST2 and H-FABP were independently associated with in-hospital mortality (HR for sST2 ≥129 ng/mL, 4.32 [1.48-12.59]; HR for H-FABP ≥18 ng/mL, 7.70 [2.12-28.01]). The composite cardiac score also independently correlated with in-hospital mortality (HR for 1-unit increase, 1.47 [1.14-1.90]). In older patients with COVID-19, sST2 and H-FABP demonstrated prognostic value, improving the predictive accuracy of the routinely assessed biomarkers hs-cTnT and NT-proBNP.

Anti-Inflammatory Effects of Olive Leaf Extract and Its Bioactive Compounds Oleacin and Oleuropein-Aglycone on Senescent Endothelial and Small Airway Epithelial Cells.

Olive tree by-products have been deeply studied as an invaluable source of bioactive compounds. Several in vitro and in vivo studies showed that olive leaf extract (OLE) has anti-inflammatory and antioxidant properties. Here, we wanted to assess the valuable benefits of two less-studied OLE components-3,4-DHPEA-EDA (Oleacin, OC) and 3,4-DHPEA-EA (Oleuropein-Aglycone, OA)-directly purified from OLE using a cost-effective and environmentally sustainable method, in line with the principles of circular economy. OLE, OC and OA were then tested in human cellular models involved in acute and chronic inflammation and in the pathogenesis of viral infections, i.e., lipopolysaccharide (LPS)-treated monocyte/macrophages (THP-1) and endothelial cells (HUVECs), senescent HUVECs and Poly(I:C)-treated small airway epithelial cells (hSAECs). Results showed that OC and OA are efficient in ameliorating almost all of the pro-inflammatory readouts (IL-1ß, TNF-α, IL-8, ICAM, VCAM) and reducing the release of IL-6 in all the cellular models. In hSAECs, they also modulate the expression of SOD2, NF-kB and also ACE2 and TMPRSS2, whose expression is required for SARS-CoV-2 virus entry. Overall, these data suggest the usefulness of OLE, OC and OA in controlling or preventing inflammatory responses, in particular those associated with viral respiratory infections and aging.

Anti-Inflammatory Effects of Nutritionally Relevant Concentrations of Oleuropein and Hydroxytyrosol on Peripheral Blood Mononuclear Cells: An Age-Related Analysis.

Immunosenescence and inflammaging facilitate the insurgence of chronic diseases. The Mediterranean diet is a non-invasive intervention to improve the chronic low-grade inflammatory status associated with aging. Olive oil oleuropein (OLE) and hydroxytyrosol (HT) demonstrated a controversial modulatory action on inflammation in vitro when tested at concentrations exceeding those detectable in human plasma. We studied the potential anti-inflammatory effects of OLE and HT at nutritionally relevant concentrations on peripheral blood mononuclear cells (PBMCs) as regards cell viability, frequency of leukocyte subsets, and cytokine release, performing an age-focused analysis on two groups of subjects: Adult (age 18-64 years) and Senior (age ≥ 65 years). OLE and HT were used alone or as a pre-treatment before challenging PBMCs with lipopolysaccharide (LPS). Both polyphenols had no effect on cell viability irrespective of LPS, but 5 µM HT had an LPS-like effect on monocytes, reducing the intermediate subset in Adult subjects. OLE and HT had no effect on LPS-triggered release of TNF-α, IL-6 and IL-8, but 5 µM HT reduced IL-10 secretion by PBMCs from Adult vs. Senior group. In summary, nutritionally relevant concentrations of OLE and HT elicit no anti-inflammatory effect and influence the frequency of immune cell subsets with age-related different outcomes.

Functional Compounds of Cold-Pressed Pomegranate Seed Oil: Fatty Acids and Phytosterols Profile as Quality Biomarkers for Origin Discrimination.

Cold-pressed pomegranate seed oil (PSO) is a product of the extraction of non-edible pomegranate seeds. Its unique chemical composition in terms of both polyunsaturated fatty acids, especially punicic acid (PA), and secondary metabolites, such as phytosterols, tocopherols and phenols, make it an interesting functional ingredient for food enrichment. It is not clear if the biomarkers profile of PSO depends to factors connected to the geographical origin of seeds. This work presents a statistical comparative analysis, concerning biomolecules composition and geographical origin of 32 commercial cold-pressed PSOs, performed by principal component analysis. The study discriminates between Turkish and Italian PSOs, on the base of the fatty acid profile and phytosterols, and not on the tocopherols and phenols. These results confirmed PA as the main characteristic biomarker of oil genuineness and, for the first time, disclosed a statistically relevant variability of phytosterols, which can be proposed as quality biomarkers for discrimination of geographical origins.

Sensitivity and specificity of in vivo COVID-19 screening by detection dogs: Results of the C19-Screendog multicenter study.

Trained dogs can recognize the volatile organic compounds contained in biological samples of patients with COVID-19 infection. We assessed the sensitivity and specificity of in vivo SARS-CoV-2 screening by trained dogs. We recruited five dog-handler dyads. In the operant conditioning phase, the dogs were taught to distinguish between positive and negative sweat samples collected from volunteers' underarms in polymeric tubes. The conditioning was validated by tests involving 16 positive and 48 negative samples held or worn in such a way that the samples were invisible to the dog and handler. In the screening phase the dogs were led by their handlers to a drive-through facility for in vivo screening of volunteers who had just received a nasopharyngeal swab from nursing staff. Each volunteer who had already swabbed was subsequently tested by two dogs, whose responses were recorded as positive, negative, or inconclusive. The dogs' behavior was constantly monitored for attentiveness and wellbeing. All the dogs passed the conditioning phase, their responses showing a sensitivity of 83-100% and a specificity of 94-100%. The in vivo screening phase involved 1251 subjects, of whom 205 had a COVID-19 positive swab and two dogs per each subject to be screened. Screening sensitivity and specificity were respectively 91.6-97.6% and 96.3-100% when only one dog was involved, whereas combined screening by two dogs provided a higher sensitivity. Dog wellbeing was also analyzed: monitoring of stress and fatigue suggested that the screening activity did not adversely impact the dogs' wellbeing. This work, by screening a large number of subjects, strengthen recent findings that trained dogs can discriminate between COVID-19 infected and healthy human subjects and introduce two novel research aspects: i) assessement of signs of fatigue and stress in dogs during training and testing, and ii) combining screening by two dogs to improve detection sensitivity and specificity. Using some precautions to reduce the risk of infection and spillover, in vivo COVID-19 screening by a dog-handler dyad can be suitable to quickly screen large numbers of people: it is rapid, non-invasive and economical, since it does not involve actual sampling, lab resources or waste management, and is suitable to screen large numbers of people.

A Data-Mining Approach to Identify NF-kB-Responsive microRNAs in Tissues Involved in Inflammatory Processes: Potential Relevance in Age-Related Diseases.

The nuclear factor NF-kB is the master transcription factor in the inflammatory process by modulating the expression of pro-inflammatory genes. However, an additional level of complexity is the ability to promote the transcriptional activation of post-transcriptional modulators of gene expression as non-coding RNA (i.e., miRNAs). While NF-kB's role in inflammation-associated gene expression has been extensively investigated, the interplay between NF-kB and genes coding for miRNAs still deserves investigation. To identify miRNAs with potential NF-kB binding sites in their transcription start site, we predicted miRNA promoters by an in silico analysis using the PROmiRNA software, which allowed us to score the genomic region's propensity to be miRNA cis-regulatory elements. A list of 722 human miRNAs was generated, of which 399 were expressed in at least one tissue involved in the inflammatory processes. The selection of "high-confidence" hairpins in miRbase identified 68 mature miRNAs, most of them previously identified as inflammamiRs. The identification of targeted pathways/diseases highlighted their involvement in the most common age-related diseases. Overall, our results reinforce the hypothesis that persistent activation of NF-kB could unbalance the transcription of specific inflammamiRNAs. The identification of such miRNAs could be of diagnostic/prognostic/therapeutic relevance for the most common inflammatory-related and age-related diseases.

Inflammation and DNA damage: cause, effect or both.

Inflammation is a biological response involving immune cells, blood vessels and mediators induced by endogenous and exogenous stimuli, such as pathogens, damaged cells or chemicals. Unresolved (chronic) inflammation is characterized by the secretion of cytokines that maintain inflammation and redox stress. Mitochondrial or nuclear redox imbalance induces DNA damage, which triggers the DNA damage response (DDR) that is orchestrated by ATM and ATR kinases, which modify gene expression and metabolism and, eventually, establish the senescent phenotype. DDR-mediated senescence is induced by the signalling proteins p53, p16 and p21, which arrest the cell cycle in G1 or G2 and promote cytokine secretion, producing the senescence-associated secretory phenotype. Senescence and inflammation phenotypes are intimately associated, but highly heterogeneous because they vary according to the cell type that is involved. The vicious cycle of inflammation, DNA damage and DDR-mediated senescence, along with the constitutive activation of the immune system, is the core of an evolutionarily conserved circuitry, which arrests the cell cycle to reduce the accumulation of mutations generated by DNA replication during redox stress caused by infection or inflammation. Evidence suggests that specific organ dysfunctions in apparently unrelated diseases of autoimmune, rheumatic, degenerative and vascular origins are caused by inflammation resulting from DNA damage-induced senescence.

MiR-422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal stem cells.

Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with its 3' UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2-targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in patients with osteoporosis-a condition characterized by increased marrow adiposity-demonstrating that its levels are negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis.

Genome-Wide Methylation Changes Associated with Replicative Senescence and Differentiation in Endothelial and Bone Marrow Mesenchymal Stromal Cells.

Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/ß-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent cells.

The Double-Edged Sword of Oleuropein in Ovarian Cancer Cells: From Antioxidant Functions to Cytotoxic Effects.

Oleuropein plays a key role as a pro-oxidant as well as an antioxidant in cancer. In this study, the activity of oleuropein, in an in vitro model of ovarian (OCCs) and breast cancer cells (BCCs) was investigated. Cell viability and cell death were analyzed. Oxidative stress was measured by CM-H2DCFDA flow cytometry assay. Mitochondrial dysfunction was evaluated based on mitochondrial reactive oxygen species (ROS) and GPX4 protein levels. Further, the effects on iron metabolism were analyzed by measuring the intracellular labile iron pool (LIP). We confirmed that high doses of oleuropein show anti-proliferative and pro-apoptotic activity on HEY and MCF-7 cells. Moreover, our results indicate that low doses of oleuropein impair cell viability without affecting the mortality of cells, and also decrease the LIP and ROS levels, keeping them unchanged in MCF-7 cells. For the first time, our data show that low doses of oleuropein reduce erastin-mediated cell death. Interestingly, oleuropein decreases the levels of intracellular ROS and LIP in OCCs treated with erastin. Noteworthily, we observed an increased amount of ROS scavenging enzyme GPX4 together with a consistent reduction in mitochondrial ROS, confirming a reduction in oxidative stress in this model.