Role of Protein Tyrosine Phosphatase Receptor, Type Gamma (PTPRG) in the regulation of endothelial permeability.

The vascular endothelium is a selective barrier that regulates, through paracellular and/or transcellular routes, the transport of macro-molecules and cells across the vessels. Modifications of endothelial cell barrier permeability are involved in many physio-pathological events, such as acute inflammatory response and cancer. Several signalling molecules regulate endothelial permeability, regulating actin cytoskeleton dynamics and/or junctional complex disassembly. Although the role of protein kinases in the regulation of endothelial permeability during physio-pathological conditions is well established, the involvement of protein phosphatases in endothelial cells function still remains poorly defined. In a recent study, our laboratory showed that the Protein Tyrosine Phosphatase Receptor, type gamma (PTPRG), which belongs to the protein tyrosine phosphatases receptor-like family, has a negative role on the regulation of recruitment of human primary monocytes. Indeed, activation of PTPRG tyr-phosphatase activity by means of two Trojan fusion proteins, namely TAT-ICD, that encompasses the complete intracellular active enzymatic domain of PTPRG, and P1-WD (P1-wedge domain), which activates the endogenous PTPRG activity, inhibits signalling pathways controlling integrin activation by chemoattractants. In this study, by taking advantage of TAT-ICD and P1-WD, we investigated the role of PTPRG in the regulation of endothelial permeability. We provided evidence showing that PTPRG activation induces a time- and dose-dependent increase of permeability of endothelial cell monolayers. To corroborate these data, we evaluated the function of ZO-1, a tight junction-associated protein. The data indicated a dose-dependent reduction of ZO-1 expression upon PTPRG activation. Since ZO-1 protein expression critically regulates the stability of tight junctions, these data support the role of PTPRG in the regulation of endothelial permeability. We speculated that PTPRG activity may modulate, directly or indirectly, the phosphorylation state of signalling events controlling the expression and function of junctional proteins involved in the control of endothelial permeability

Direct Monitoring of the Calcium Concentration in the Sarcoplasmic and Endoplasmic Reticulum of Skeletal Muscle Myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed

Optimization and validation of a LC-HRMS method for aflatoxins determination in urine samples

Mycotoxins’ exposure by inhalation and/or dermal contact can occur in different branches of industry especially where heavily dusty settings are present and the handling of dusty commodities is performed. This study aims to explore the possible contribution of the occupational exposure to aflatoxins by analysing urine samples for the presence of aflatoxins B1 and M1 and aflatoxin B1-N7-guanine adduct. The study was conducted in 2017 on two groups of volunteers, the workers group, composed by personnel employed in an Italian feed plant (n = 32), and a control group (n = 29), composed by the administrative employees of the same feed plant; a total of 120 urine samples were collected and analysed. A screening method and a quantitative method with high-resolution mass spectrometry determination were developed and fully validated. Limits of detections were 0.8 and 1.5 pg/mLurine for aflatoxin B1 and M1, respectively. No quantitative determination was possible for the adduct aflatoxin B1-N7-guanine. Aflatoxin B1 and its adduct were not detected in the analysed samples, and aflatoxin M1, instead, was found in 14 samples (12%) within the range 1.9–10.5 pg/mLurine. Only one sample showed a value above the limit of quantification (10.5 pg/mLurine). The absence of a statistical difference between the mean values for workers and the control group which were compared suggests that in this specific setting, no professional exposure occurs. Furthermore, considering the very low level of aflatoxin M1 in the collected urine samples, the contribution from the diet to the overall exposure is to be considered negligible

Biomonitoring data for assessing aflatoxins and ochratoxin a exposure by italian feedstuffs workers

Mycotoxins exposure by inhalation and/or dermal contact is possible in different branches of industry especially where heavily dusty settings are present and the handling of dusty commodities is performed. This study aims to explore the validity of the biomonitoring as a tool to investigate the intake of mycotoxins in a population of workers operating in an Italian feed plant. Serum samples were collected for the determination of aflatoxins B1 (AFB1), AFB1-Lysine adduct and ochratoxin A (OTA). A method based on liquid-liquid extraction coupled with high resolution mass spectrometry determination was developed and fully validated. For AFB1, a high number of non-detected samples (90%) was found and no statistical difference was observed comparing workers and control group. None of the analyzed samples showed the presence of AFB1-Lysine adduct. For OTA, the 100% of the analyzed samples was positive with a 33% of the samples showing a concentration higher than the limit of quantification (LOQ), but no statistical difference was highlighted between the average levels of exposed and control groups. In conclusion, the presence of AFB1 and OTA in serum cannot be attributable to occupational exposure

Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma.The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs.RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC).The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p<0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas.Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients

Consumption patterns of energy drinks in university students. A systematic review and meta-analysis

Energy Drinks (EDs) use is promoted to stimulate mental and/or physical activity, and over the years their popularity increased, especially among young people. However, the use of EDs is often improper and can induce some adverse effects for human health. The purpose of this systematic review and metaanalysis was to analyse the literature to characterize prevalence of ED consumption and motivations to use among undergraduate students. Furthermore, adverse effects and health-related behaviors associated to ED use were explored. This systematic review was carried out according to the PRISMA Statement and the databases PubMed, Scopus and Web of Science were used for data research. 71 articles published between 2007 and 2021 met the inclusion criteria and were included in the review. The estimated overall prevalence of ED consumption was 42.9% in undergraduate students (95% CI:42.5%-43.3%), with significant heterogeneity among studies (Q test: p&lt;0.001; I2 =99.4%) . The main reasons for their use were engagement in study, projects or examinations, to stay awake or alert, physical activity/sport engagement. Sleep disturbance and increased heart rate or blood pressure were the most commonly reported adverse effects. ED consumption was frequently associated with alcohol use and smoking. These findings suggest that inappropriate use of EDs and related unhealthy behaviors should be early identified and addressed through effective educational interventions

Alcohol mixed with Energy Drinks (AmED) use among University Students:A Systematic Review and Meta-analysis

Abstract: In the last decades, there has been a huge increase in the consumption of both Energy Drinks (EDs) and alcohol and, concurrently, these two trends generated the additional practice of mixing ED with alcohol, known as Alcohol mixed with Energy Drink (AmED). One of the most important group of AmED consumers is represented by young. Indeed, the study population of the researches in this field are mainly represented by college students and the results evidence a great range of negative consequences for health. The purpose of the systematic review was to explore the prevalence of AmED consumption among undergraduate students, together with motivations to their use, adverse effects and health-related behaviors associated to AmEDs use. The review was conducted according to the PRISMA Statement and PubMed, Scopus and Web of Science were interrogated. 42 articles, published from 2008 to 2021, were included in the review. An overall prevalence rate of 37% was estimated for AmEDs use in undergraduates, with geographical differences. Although a decrease in consumption was observed throughout the studied period, a continuous monitoring on this phenomenon is needed for identifying those risk groups that could develop risky behaviors related to AmEDs consumption and provide them targeted educational intervention