Assessing the Impact of Polyethylene Nano/Microplastic Exposure on Human Vaginal Keratinocytes.

The global rise of single-use throw-away plastic products has elicited a massive increase in the nano/microplastics (N/MPLs) exposure burden in humans. Recently, it has been demonstrated that disposable period products may release N/MPLs with usage, which represents a potential threat to women's health which has not been scientifically addressed yet. By using polyethyl ene (PE) particles (200 nm to 9 µm), we showed that acute exposure to a high concentration of N/MPLs induced cell toxicity in vaginal keratinocytes after effective cellular uptake, as viability and apoptosis data suggest, along with transmission electron microscopy (TEM) observations. The internalised N/MPLs altered the expression of junctional and adherence proteins and the organisation of the actin cortex, influencing the level of genes involved in oxidative stress signalling pathways and that of miRNAs related to epithelial barrier function. When the exposure to PE N/MPLs was discontinued or became chronic, cells were able to recover from the negative effects on viability and differentiation/proliferation gene expression in a few days. However, in all cases, PE N/MPL exposure prompted a sustained alteration of DNA methyltransferase and DNA demethylase expression, which might impact epigenetic regulation processes, leading to accelerated cell ageing and inflammation, or the occurrence of malignant transformation.

Gene expression profiles of oral soft tissue-derived fibroblast from healing wounds: correlation with clinical outcome, autophagy activation and fibrotic markers expression.

AIM: Our aim was to evaluate gene expression profiling of fibroblasts from human alveolar mucosa (M), buccal attached gingiva (G) and palatal (P) tissues during early wound healing, correlating it with clinical response. MATERIALS AND METHODS: M, G and P biopsies were harvested from six patients at baseline and 24 hr after surgery. Clinical response was evaluated through Early wound Healing Score (EHS). Fibrotic markers expression and autophagy were assessed on fibroblasts isolated from those tissues by Western blot and qRT-PCR. Fibroblasts from two patients were subjected to RT2 profiler array, followed by network analysis of the differentially expressed genes. The expression of key genes was validated with qRT-PCR on all patients. RESULTS: At 24 hr after surgery, EHS was higher in P and G than in M. In line with our clinical results, no autophagy and myofibroblast differentiation were observed in G and P. We observed significant variations in mRNA expression of key genes: RAC1, SERPINE1 and TIMP1, involved in scar formation; CDH1, ITGA4 and ITGB5, contributing to myofibroblast differentiation; and IL6 and CXCL1, involved in inflammation. CONCLUSIONS: We identified some genes involved in periodontal soft tissue clinical outcome, providing novel insights into the molecular mechanisms of oral repair (ClinicalTrial.gov-NCT04202822).

Effect of Chlorhexidine Digluconate in Early Wound Healing of Human Gingival Tissues. A Histological, Immunohistochemical and Biomolecular Analysis.

Chlorhexidine digluconate (CHX) is considered the gold standard for oral cavity antiseptic treatment. Nevertheless, several in vitro studies have reported detrimental effects in oral tissue repair. The aim of the present study was to evaluate the in vivo effect of post-surgical CHX mouth rinse on gingival tissue (G) 24 h after injury. G biopsies were obtained in three patients 24 h after surgery with the indication of post-surgical 0.12% CHX use and were compared with those obtained from the same patients without any antiseptic use. Changes in collagen production, cell proliferation, and apoptosis were examined by histological and Ki-67/P53 immunohistochemical analysis. Fibrotic markers (COL1A1, αSMA), proapoptotic protein (BAX) expression, and wound healing-related gene modulation (RAC1, SERPINE1, TIMP1) were analyzed by quantitative real-time PCR analysis. CHX was able to reduce cellular proliferation and increase collagen deposition, proapoptotic molecule and fibrotic marker expression, and myofibroblast differentiation, reduce expression of RAC1 and trigger expression of SERPINE1 and TIMP1, showing "scar wound healing response" pattern. This study assessed for the first time the in vivo effects of CHX on gingival tissue. The demonstration of a CHX-induced fibrotic transformation, leading to scar repair, supports the need for new post-surgical clinical protocols based on a strategic and personalized use of CHX.

Sex-Related Factors in Cardiovascular Complications Associated to COVID-19.

Coronavirus disease 2019 (COVID-19), the pandemic infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents with an extremely heterogeneous spectrum of symptoms and signs. The clinical manifestations seem to be correlated with disease severity. COVID-19 susceptibility and mortality show a significant sex imbalance, with men being more prone to infection and showing a higher rate of hospitalization and mortality compared to women. Such variability can be ascribed to both sex-related biological factors and gender-related behavioral cues. This review will discuss the potential mechanisms accounting for sex/gender influence in vulnerability to COVID-19. Cardiovascular diseases play a central role in determining COVID-19 outcome, whether they are pre-existent or arose upon infection. We will pay particular attention to the impact of sex and gender on cardiovascular manifestations related to COVID-19. Finally, we will discuss the sex-dependent variability in some biomarkers for the evaluation of COVID-19 infection and prognosis. The aim of this work is to highlight the significance of gendered medicine in setting up personalized programs for COVID-19 prevention, clinical evaluation and treatment.

Altered Expression of Candidate Genes in Mayer-Rokitansky-Küster-Hauser Syndrome May Influence Vaginal Keratinocytes Biology: A Focus on Protein Kinase X.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare and complex disease defined by congenital aplasia of the vagina and uterus in 46,XX women, often associated with kidney and urinary tract anomalies. The aetiopathogenesis of MRKH syndrome is still largely unknown. Herein, we investigated the role of selected candidate genes in the aetiopathogenesis of MRKH syndrome, with a focus on PRKX, which encodes for protein kinase X. Through RT-qPCR analyses performed on vaginal dimple samples from patients, and principal component analysis (PCA), we highlighted a phenotype-related expression pattern of PRKX, MUC1, HOXC8 and GREB1L in MRKH patients. By using an in vitro approach, we proved that PRKX ectopic overexpression in a cell model of vaginal keratinocytes promotes cell motility through epithelial-to-mesenchymal transition (EMT) activation, a fundamental process in urogenital tract morphogenesis. Moreover, our findings showed that PRKX upregulation in vaginal keratinocytes is able to affect transcriptional levels of HOX genes, implicated in urinary and genital tract development. Our study identified the dysregulation of PRKX expression as a possible molecular cause for MRKH syndrome. Moreover, we propose the specific role of PRKX in vaginal keratinocyte biology as one of the possible mechanisms underlying this complex disease.

Calcineurin Gamma Catalytic Subunit PPP3CC Inhibition by miR-200c-3p Affects Apoptosis in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) outpaces all the other forms of the female reproductive system malignancies. MicroRNAs have emerged as promising predictive biomarkers to therapeutic treatments as their expression might characterize the tumor stage or grade. In EOC, miR-200c is considered a master regulator of oncogenes or tumor suppressors. To investigate novel miR-200c-3p target genes involved in EOC tumorigenesis, we evaluated the association between this miRNA and the mRNA expression of several potential target genes by RNA-seq data of both 46 EOC cell lines from Cancer Cell line Encyclopedia (CCLE) and 456 EOC patient bio-specimens from The Cancer Genome Atlas (TCGA). Both analyses showed a significant anticorrelation between miR-200c-3p and the protein phosphatase 3 catalytic subunit γ of calcineurin (PPP3CC) levels involved in the apoptosis pathway. Quantitative mRNA expression analysis in patient biopsies confirmed the inverse correlation between miR-200c-3p and PPP3CC levels. In vitro regulation of PPP3CC expression through miR-200c-3p and RNA interference technology led to a concomitant modulation of BCL2- and p-AKT-related pathways, suggesting the tumor suppressive role of PPP3CC in EOC. Our results suggest that inhibition of high expression of miR-200c-3p in EOC might lead to overexpression of the tumor suppressor PPP3CC and subsequent induction of apoptosis in EOC patients.

MiR-200c-3p maintains stemness and proliferative potential in adipose-derived stem cells by counteracting senescence mechanisms.

Adipose-derived mesenchymal stem cells (ASCs) are promising therapeutic tools in regenerative medicine because they possess self-renewal, differentiation and immunomodulatory capacities. After isolation, ASCs are passaged multiple times in vitro passages to obtain a sufficient amount of cells for clinical applications. During this time-consuming procedure, ASCs become senescent and less proliferative, compromising their clinical efficacy. Here, we sought to investigate how in vitro passages impact ASC proliferation/senescence and expression of immune regulatory proteins. MicroRNAs are pivotal regulators of ASC physiology. Particularly, miR-200c is known to maintain pluripotency and targets the immune checkpoint Programmed death-ligand 1 (PD-L1). We therefore investigated its involvement in these critical characteristics of ASCs during in vitro passages. We found that when transiently expressed, miR-200c-3p promotes proliferation, maintains stemness, and contrasts senescence in late passaged ASCs. Additionally, this miRNA modulates PD-L1 and Indoleamine 2,3-Dioxygenase (IDO1) expression, thus most likely interfering with the immunoregulatory capacity of ASCs. Based on our results, we suggest that expression of miR-200c-3p may prime ASC towards a self-renewing phenotype by improving their in vitro expansion. Contrarily, its inhibition is associated with senescence, reduced proliferation and induction of immune regulators. Our data underline the potential use of miR-200c-3p as a switch for ASCs reprogramming and their clinical application.

MiR-200c sensitizes Olaparib-resistant ovarian cancer cells by targeting Neuropilin 1.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Treatment with PARP inhibitors (PARPi), such as Olaparib, has been recently introduced for OC patients, but resistance may occur and underlying mechanisms are still poorly understood. The aim of this study is to identify target genes within the tumor cells that might cause resistance to Olaparib. We focused on Neuropilin 1 (NRP1), a transmembrane receptor expressed in OC and correlated with poor survival, which has been also proposed as a key molecule in OC multidrug resistance. METHODS: Using three OC cell lines (UWB, UWB-BRCA and SKOV3) as model systems, we evaluated the biological and molecular effects of Olaparib on OC cell growth, cell cycle, DNA damage and apoptosis/autophagy induction, through MTT and colony forming assays, flow cytometry, immunofluorescence and Western blot analyses. We evaluated NRP1 expression in OC specimens and cell lines by Western blot and qRT-PCR, and used RNA interference to selectively inhibit NRP1. To identify miR-200c as a regulator of NRP1, we used miRNA target prediction algorithms and Pearsons' correlation analysis in biopsies from OC patients. Then, we used a stable transfection approach to overexpress miR-200c in Olaparib-resistant cells. RESULTS: We observed that NRP1 is expressed at high levels in resistant cells (SKOV3) and is upmodulated in partially sensitive cells (UWB-BRCA) upon prolonged Olaparib treatment, leading to poor drug response. Our results show that the selective inhibition of NRP1 is able to overcome Olaparib resistance in SKOV3 cells. Moreover, we demonstrated that miR-200c can target NRP1 in OC cells, causing its downmodulation, and that miR-200c overexpression is a valid approach to restore Olaparib sensitivity in OC resistant cells. CONCLUSIONS: These data demonstrate that miR-200c significantly enhanced the anti-cancer efficacy of Olaparib in drug-resistant OC cells. Thus, the combination of Olaparib with miRNA-based therapy may represent a promising treatment for drug resistant OC, and our data may help in designing novel precision medicine trials for optimizing the clinical use of PARPi.

Regulation of myoblast differentiation by metabolic perturbations induced by metformin.

The metabolic perturbation caused by calorie restriction enhances muscle repair by playing a critical role in regulating satellite cell availability and activity in the muscles of young and old mice. To clarify the underlying mechanisms we asked whether myoblast replication and differentiation are affected by metformin, a calorie restriction-mimicking drug. C2C12, a mouse myoblast cell line, readily differentiate in vitro and fuse to form myotubes. However, when incubated with metformin, C2C12 slow their replication and do not differentiate. Interestingly, lower doses of metformin promote myogenic differentiation. We observe that metformin treatment modulates the expression of cyclins and cyclin inhibitors thereby inducing a cell cycle perturbation that causes a delay in the G2/M transition. The effect of metformin treatment is reversible since after drug withdrawal, myoblasts can re-enter the cell cycle and/or differentiate, depending on culture conditions. Myoblasts cultured under metformin treatment fail to up-regulate MyoD and p21cip1, a key step in cell cycle exit and terminal differentiation. Although the details of the molecular mechanisms underlying the effect of the drug on myoblasts still need to be clarified, we propose that metformin negatively affects myogenic differentiation by inhibiting irreversible exit from the cell cycle through reduction of MyoD and p21cip1 levels.