Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 μM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

Long-duration manned space missions require that astronauts be equipped with analytical devices suitable for performing on-site clinical-chemistry analysis. In the frame of the “IN SITU Bioanalysis” project, an analytical device for measuring the steroid hormone cortisol (an indicator of chronic stress) in saliva has been developed. The device performed a Lateral Flow Immunoassay (LFIA) for cortisol employing disposable plastic cartridges produced by FDM (Fused Deposition Modeling) 3D printing technology, which encapsulated a sealed fluidic element containing the LFIA strip and the reagents required for the analysis. Detection was performed by chemiluminescence (CL) using a dedicated CL reader equipped with a cooled charge-coupled device (CCD) camera. The European Space Agency (ESA) astronaut Paolo Nespoli successfully used the “IN SITU Bioanalysis” analytical device during the mission “VITA”, a 6-months space mission onboard the International Space Station (ISS) in the framework of a dated bilateral agreement between the Italian Space Agency (ASI—Agenzia Spaziale Italiana) and NASA. The experiments proved the feasibility of CL-LFIA ultrasensitive (i.e., at the sub-ng/mL level) quantitative bioassays in space. In perspective, the “IN SITU Bioanalysis” analytical device could be adapted to the measurement of other biomarkers of diagnostic and prognostic relevance, even in a multiplexed fashion (i.e., detection of multiple biomarkers in a single analysis) to develop panels tests for diagnosis and follow-up of pathologies or monitoring health status of astronauts.

High polyphenols levels are one of the main quality characters of Extra Virgin Olive Oil (EVOO), leading to high antioxidant activity and contributing to the EVOO health beneficial effects. Analytical methods for the rapid assessment of EVOO polyphenols content, possibly directly at the place of production or distribution, are therefore important for the commercial enhancement of this product. Unfortunately, most analytical methods for quantification of polyphenols in EVOO require laboratory instrumentation and preliminary sample extraction procedures. In this paper we describe a paper-based chemosensor based on the well-known Folin-Ciocalteu colorimetric assay for the rapid (assay time 15 min) quantitative detection of polyphenols in EVOO without any preliminary sample extraction. Use of n-propanol for diluting the EVOO sample allows its direct analysis on paper supports loaded with the Folin-Ciocalteu reagent. The color change of the paper supports is measured using a smartphone camera. A disposable analytical cartridge containing all necessary reagents, including calibration standards, and accessories for performing the assay using a Samsung S8 smartphone have been developed to perform on-site analysis. Measurement of total polyphenol content of EVOO samples gave results in good agreement with the conventional Folin-Ciocalteu assay, and the limit of detection was 30 mu g gallic acid equivalents g(-1) EVOO. The same approach can be employed to measure polyphenols in other oils of vegetal origin.

Introduction: Bile acids (BAs) play an important role in lipid and glucose homeostasis.

Bile acids (BAs) are endogenous steroids involved in the transport of lipids in bile, acting also as molecular signaling hormones. Primary BAs synthesized in the liver undergo several metabolic pathways in the intestine by gut microbiota to produce secondary BAs. Together with secondary BAs, other metabolites have been recovered from human faeces, including many oxo-BA analogues produced in the colon through oxidation of BA hydroxy groups. However, the complete oxo-BA characterization in biospecimens (particularly intestinal content and faeces) has not been reported yet, hampering the assessment of their potential physiological role. Herein, we have developed and validated a new RP-HPLC-ESI-MS/MS method in negative ionization mode for the simultaneous analysis of 21 oxo-BAs and their 7 metabolic BAs precursors in human faeces. The elution was performed in gradient mode and 28 compounds, including primary, secondary BAs, and their oxo-derivatives, were separated within 50 min at 40 degrees C column temperature. The method is accurate (bias% <13%), precise (CV% <10%), with limits of quantification (LOQ<30 ng/mL(extract samples)). similar for all the studied compounds. The matrix effect does not significantly affect the analysis accuracy, allowing the use of standard solutions for the quantifications, without matrix-matched protocols. Thanks to the high detectability and the relatively high concentration of oxo-BAs (about mu g/g(wet faeces)), the method does not require a pre-analytical clean-up step. This method was used to identify and quantify oxo-BAs in human faecal samples from healthy subjects, serving as a proof of concept for application in patients with hepatobiliary disease and bacteria overgrowth. (C) 2018 Elsevier B.V. All rights reserved.

Background and aims: Oxidized LDL (oxLDL) or pro-inflammatory stimuli lead to increased oxidative stress linked to endothelial dysfunction and atherosclerosis. The oxLDL receptor-1 (LOX1) is elevated within atheromas and cholesterol-lowering statins inhibit LOX1 expression. Berberine (BBR), an alkaloid extracted from plants of gender Berberis, has lipidlowering and anti-inflammatory activity. However, its role in regulating LOX1-mediated signaling is still unknown. The aim of this study was to investigate the effect of BBR on oxLDLand TNFa-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs) and to compare it with that of lovastatin (LOVA).Methods and results: Cytotoxicity was determined by lactate dehydrogenase assay. Antioxidant capacity was measured with chemiluminescent and fluorescent method and intracellular ROS levels through a fluorescent dye. Gene and protein expression levels were assayed by qRT-PCR and western blot, respectively.HUVECs exposure to oxLDL (30 mu g/ml) or TNF alpha (10 ng/ml) for 24 h led to a significant increase in LOX1 expression, effect abrogated by BBR (5 mM) and LOVA (5 mM). BBR but not LOVA treatment abolished the TNF alpha-induced cytotoxicity and restored the activation of Akt signaling. In spite of a low direct antioxidant capacity, both compounds reduced intracellular ROS levels generated by treatment of TNF alpha but only BBR inhibited NOX2 expression, MAPK/Erk1/2 signaling and subsequent NF-kappa B target genes VCAM and ICAM expression, induced by TNF alpha.Conclusions: These findings demonstrated for the first time that BBR could prevent the oxLDL and TNFa -induced LOX1 expression and oxidative stress, key events that lead to NOX, MAPK/Erk1/2 and NF-kappa B activation linked to endothelial dysfunction.Chemical compounds studied in this article: Berberine (PubChem CID: 2353); Lovastatin (PubChem CID: 53232). (C) 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Background and Aims: Indocyanine Green retention test is a liver function test reflecting total blood flow and functional reserve; it is able to predict clinical decompensation and survival in compensated patients undergoing liver surgery and in patients with advanced liver cirrhosis.The aim of our study is to evaluate ICG clearance as a predictor of clinical decompensation and mortality in patients with compensated cirrhosis.Material and Methods: From Jan-2010 to Jan-2011 we prospectively enrolled compensated cirrhotic patients (MELD <15; Bilirubin <2 mg/dL; INR <1.5); present or previous decompensation and HCC were excluded from the study.All underwent lab-tests, gastroscopy, HVPG and ICG clearance measurement and completed at least 18 months of follow up during which we recorded all liverrelated events, liver transplant and death.Patients who underwent liver transplantation were censored.Cumulative incidence and predictors of clinical outcomes were determined by Kaplan-Meier analysis and Cox regression.Results: 110 patients (75 male; 35 female; age 60.6±11.6 yrs) were followed up for 29 (19-34) months.46 out of 110 (41.8%) developed decompensation, 6 (5.5%) underwent liver transplant and 9 (8.2%) died during follow-up.Together with MELD values [P 0.041; HR 1.137 (1.004-1.287)]multivariate analysis identified ICG-r15 as an independent predictor of decompensation [P 0.0001; HR 1.042 (1.020-1.064)].Time-dependent ROC curve identified a cutoff value of 23% of ICG-r15 for the development of decompensation (Sensitivity 87.0%; Specificity 65.6%; +LR 2.53; -LR 0.20; AUROC 0.885) and mortality (Sensitivity 90.9%; Specificity 81.4%; +LR 4.90; -LR 0.11; AUROC 0.869).Kaplan-Meier analysis showed an increased risk of decompensation for patients with ICG-r15 >23% ; P < 0.0001].Presence of Large Esophageal varices (P = 0.01), HVPG value (P = 0.02) and ICG-r15 (P = 0.0004) were independently related to the risk of bleeding.Finally, patients with a basal ICG-r15 >23% showed an increased mortality ); P 0.0051]. Conclusion:Together with MELD, ICG-r15 appear to be a useful predictor of liver decompensation; moreover, ICG clearance is significantly correlated to the risk of bleeding and survival among patients with compensated liver cirrhosis.

Background and aim: Although the lack of extensive data in humans, bile acids (BAs) and particularly CDCA a potent FXR and TGR5 ligand, are signaling molecules in glucose, lipid and energy metabolism. Aim of our study was to evaluate serum BA profile including Glycine (G), Taurine (T) and Unconjugated (U)-Bas in blood donors (BD) and to identify correlations between individual BA concentration in normal and insulin-resistant (IR) subjects. Material and methods:We prospectively enrolled 160 blood donors from our Transfusion Center, from January to June 2011. Exclusion criteria were: alteration in blood tests, history of liver or GI disease, previous abdominal surgery and treatment with metabolic and GI medications. All patients underwent biochemistry tests (fasting glucose and insulin, total cholesterol, HDL, LDL, triglycerides (TG), ALT, GammaGT, ALP) and serum BA profile measurement (HPLC-ESI-MS/MS). All patients underwent an abdominal ultrasound within a week from enrollment. Medical history, laboratory parameters and ultrasonographic findings of all subjects were reviewed from our physicians and subjects were classified as Healthy (H, n=120), IR (n=28) and gallstone subjects (GS, n=12, excluded from analysis). Results: 148 subjects (84 male/64 female; mean age 41.7±12.6) met the inclusion criteria. BMI was 25.1±3.7; total cholesterol 194.6±35.6; HDL 60.5±15.2; TG 91.8±48.7; ALT, GammaGt, ALP and other mean biochemical index were within normal limits. In IR subjects, total serum BAs (5.02±2.39 vs 3.56±2.55 μmol/l; P= 0.0017), serum unconjugated BAs (1.95±1.25 vs 1.40±1.17 μmol/l; P= 0.025) and CDCA (2.28±1.39 vs 1.63±1.43; P=0.03) were significantly higher than in healthy subjects. On multivariate analysis, BMI (r=0.35; P<0.0001), CDCA (r= 0.194; P= 0.0033) and TG (r=0.26; P=0.0134) were independently and directly related to the HOMA-IR. No significant correlation were found between serum cholesterol or triglycerides levels and serum BA profile. Conclusions: Total serum Bas (U-Bas, G-Bas, T-Bas) and particularly CDCA are significantly increased in patients with IR. CDCA, together with BMI and TG are independently related to insulin-resistance.

Patients with type 2 hepatorenal syndrome (HRS) may develop type-1 HRS; however, the frequency and risk factors associated with the occurrence of this complication are currently unknown.With the aim of studying the risk factors associated with the development of type-1 HRS, 164 consecutive patients with cirrhosis and type-2 HRS were evaluated.The mean age was 62±10 years, 70% were male and 47% had alcoholic cirrhosis.At diagnosis, serum creatinine value was 1.77±0.25 mg/dL, 57% patients had refractory ascites and 40% hyponatremia (serum sodium 131±5 mEq/L).Patients showed a marked deterioration in systemic hemodynamics and liver function, as evidenced by low mean arterial pressure (78±11 mmHg), an increase in endogenous vasoactive systems activity (plasma renin activity 10.7±9 ng/ml*h) and high values for MELD and MELD-sodium (18±5 and 23±5, respectively).At one year, only 34 patients were still alive, 29 had been transplanted and 92 had died.Thirty-nine patients (24%) developed type-1 HRS within a median of 65 days (95% CI 15-117).Mean serum creatinine value at the time of diagnosis was 3.77±1.17mg/dL, which corresponded to mean an increase of 117% respect to baseline (95% CI 100-137%).No precipitating event was identified in 22 patients (56%).Bacterial infections were the most common precipitating factor of type-1 HRS (observed in 14 patients (37%).On multivariate analysis, MELD-Sodium was the only factor independently associated with survival and development of type-1 HRS, and the best cut-off point was 22.At 6 months, the incidence of type 1 HRS was significantly higher in patients with MELD-Sodium greater than or equal to 22, when compared to patients with MELD-Sodium lower than 22 (29 vs. 9%, p = 0.002).In conclusion, development of type-1 HRS is a frequent event in patients with type-2 HRS and occurs in absence of any precipitating event in almost half of cases.MELD-Sodium is a useful tool to predict both survival and development of type-1 HRS in this population.These results should be accounted not only on prediction of prognosis of these patients, but also for design of possible future therapeutic strategies.

The endogenous estradiol metabolite, estradiol 17b-D-glucuronide (E17G), induces an acute cholestasis in rat liver due in part to retrieval of canalicular transporters such as the bile salt export pump (Bsep, Abcc11) in a process that involves PKC alpha (Hepatology 2008(Hepatology , 48: 1885-95)-95).Recently, estrogen receptors (ER) have been implicated in the chronic estrogen cholestasis model induced by ethynylestradiol (JBC 2006, 281:16625-31).The aim of this study was to evaluate the involvement of ER in E17Ginduced changes in bile salt canalicular excretion.Methods: Isolated rat hepatocytes couplets were cultured for 5 h, exposed to the estrogen receptor inhibitor ICI-182,780 (ICI, 1 mM) for 15 min and then incubated with E17G (50 mM) for 20 min.Other couplet preparations were additionally incubated with PKC inhibitor Gö 6976 (Gö, 1 mM, 15 min) before E17G incorporation.Finally, all preparations were incubated with cholyl-lysylfluorescein (CLF) a fluorescent bile salt substrate of Bsep.Couplets accumulating CLF in their vacuole were counted in a fluorescent microscope and informed as a percentage.Results were compared by ANOVA followed by Student-Newman-Keuls test.a significantly different from Control (p < 0.01); b significantly different from Control and E17G (p < 0.05).n = 3.Conclusions: ER inhibition prevents E17G-induced alteration in bile salt excretion.Selective blockage of PKC alpha activation with Gö6976 protects from E17G effects but did not increased the estrogen inhibitor action.These findings support a role for ER in E17G-induced cholestasis probably in a pathway that involves PKC alpha activation since the effects were not additive.

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16(INK4A) (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.