Study of the effects of High Hydrostatic Pressure (HHP) and Pulsed Light (PL) on BSA structure and hydrolysis

Non-thermal technologies, such as High Hydrostatic Pressure (HHP) and Pulsed light (PL), affect protein inducing modifications in its conformational structure. For this reason the hydrolysis reaction of the protein can be modulated if it is conducted in combination with these technologies which are able to change the availability of peptide bonds exposed to the enzymatic action. The aim is to study the effects of HHP and PL on the structure and the extent of hydrolysis reaction of a target protein: the Bovine Serum Albumin (BSA). BSA protein (5 mg/mL) in sodium phosphate buffer (50 mM, pH = 8) were treated with PL and HHP at different processing conditions, namely pressure level and treatment time in the case of HHP and treatment time and energy input in the case of PL. Structural modification of the protein solutions were analyzed by determining the sulphidrilic groups and the changes of the secondary structure. The effect of the two treatments on the hydrolysis degree (HD) at 37 °C was also evaluated by OPA method. Chymotrypsin and trypsin (E/S ratio = 1/10) were used to hydrolyze the BSA protein solutions. The hydrolysis was carried out in HHP assisted or PL assisted conditions or the protein solutions were treated with HHP or PL processes and immediately after hydrolyzed with the enzymes. Results obtained so far demonstrated that the two technologies tested are able to induce protein modifications and the occurrence and importance of this phenomenon depends on processing parameters causing protein unfolding, namely pressure level and number of pulses. When the maximum protein unfolding is obtained, higher HD values are detected. The highest HD value is obtained in HHP assisted hydrolysis with longer treatment time, and when, before undergoing hydrolysis, the PL treatment is applied to the solution placed at the higher distance from the lamp

Acid hydrolysis of spent coffee grounds: effects on possible prebiotic activity of oligosaccharides

Abstract Background Spent coffee grounds (SCG) are a promising source of natural by-products which can be used for different purposes. In this work, a possible use of oligosaccharides isolated from SCG as functional ingredients was investigated. SCGs were treated with an acid hydrolysis at high temperature (200 °C) in a closed reactor setting reaction time of 30, 60 and 90 s depending on the sample (original or defatted). A comprehensive study of the resulted water-soluble hydrolysate using a high-resolution mass spectrometry analysis was performed. Additionally, the growth of four Lactobacillus strains was tested to assess the prebiotic potential of the hydrolysate. Results Oligosaccharide chains formed by hexoses with a degree of polymerization ranging from 3 to 6 were identified and characterized. Regardless of the composition and the reaction time of hydrolysis, the bacterial activity of SCG extracts exhibited significantly higher values than the well-known versatile carbohydrate used by food industry, i.e., inulin. Conclusions The results pave the way toward the use of hydrolysate SCG as an innovative ingredient intended to fortify food formulations. The diversity in coffee oligosaccharides composition suggests their selective prebiotic activity for specific bacterial strains. Graphical Abstrac

Development of iron-rich whey protein hydrogels following application of ohmic heating Effects of moderate electric fields

The influence that ohmic heating technology and its associated moderate electric fields (MEF) have upon production of whey protein isolate cold-set gels mediated by iron addition was investigated. Results have shown that combining heating treatments (90 °C, 5 min) with different MEF intensities let hydrogels with distinctive micro and macro properties i.e. particle size distribution, physical stability, rheological behavior and microstructure. Resulting hydrogels were characterized (at nano-scale) by an intensity-weighted mean particle diameter of 145 nm, a volume mean of 240 nm. Optimal conditions for production of stable whey protein gels were attained when ohmic heating treatment at a MEF of 3 V  cm 1 was combined with a cold gelation step using 33 mmol  L 1 of Fe2 +. The consistency index of hydrogels correlated negatively to MEF intensity, but a shear thickening behavior was observed when MEF intensity was increased up to 10 V  cm 1. According to transmission electron microscopy, ohmic heating gave rise to a more homogenous and compact fine-stranded whey protein-iron microstructure. Ohmic heating appears to be a promising technique, suitable to tailor properties of whey protein gels and with potential for development of innovative functional foods.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Rui M. Rodrigues, Oscar L. Ramos and Ricardo N. Pereira and, gratefully acknowledge to FCT their financial grants with references SFRH/BD/ 110723/2015, SFRH/BPD/80766/2011 and SFRH/BPD/81887/2011, respectively.info:eu-repo/semantics/publishedVersio

The chemical composition of the aerial parts of Stachys spreitzenhoferi (Lamiaceae) growing in Kythira Island (Greece), and their antioxidant, antimicrobial, and antiproliferative properties

The Stachys L. genus has been used in traditional medicine to treat skin inflammations, stomach disorders, and stress. The aim of this study was to investigate the chemical profile and biological activity of the methanolic extract of Stachys spreitzenhoferi Heldr. (Lamiaceae) aerial parts, collected on the island of Kythira, South Greece. The analysis by liquid chromatography coupled with electrospray ionization and high-resolution mass spectrometry [LC-(-)ESI/HRMSn] of the methanol extract revealed the occurrence of thirty-six compounds - flavonoids, phenylethanoid glycosides, iridoids, quinic acid derivatives, aliphatic alcohol glycosides, and oligosaccharides - highlighting the substantial presence, as main peaks, of the iridoid melittoside (2) along with flavonoid compounds such as 4'-O-methylisoscutellarein mono-acetyl-diglycoside/chrysoeriol mono-acetyl-diglycoside (24), trimethoxy- (35) and tetramethoxyflavones (36). This extract was tested for its antimicrobial properties against Gram-positive and negative pathogenic strains. The extract was not active against Gram-negative bacteria tested, but it possessed a good dose-dependent antimicrobial activity towards S. aureus (MIC: 1.0 mg/mL) and L. monocytogenes (MIC: 1.0 mg/mL) Gram-(+) strains. Furthermore, this extract has been tested for its possible antioxidant activity in vitro. In particular, it has been shown that these molecules cause a decrease in DPPH, ABTS, and H2O2 radicals. The extract of S. spreitzenhoferi exhibited anti-DPPH activity (IC50: 0.17 mg/mL), anti-H2O2 activity (IC50: 0.125 mg/mL), and promising antiradical effect with an IC50 value of 0.18 mg/mL for anti-ABTS activity. S. spreitzenhoferi extract caused a decrease in ROS (at the concentration of 200 μg/mL) and an increase in the activity of the antioxidant enzymes SOD, CAT, and GPX in OZ-stimulated PMNs. Furthermore, it exhibited antiproliferative activity against acute myeloid leukemia (U937 cell), causing 50% of cell death at the 0.75 mg/mL

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions

Hepatic and extra-hepatic sequelae, and prevalence of viral hepatitis C infection estimated from routine data in at-risk groups

<p>Abstract</p> <p>Background</p> <p>Concerns about the hepatitis C virus (HCV) are due to the high risk of chronic liver disease and poor treatment efficacy. Synthesizing evidence from multiple data sources is becoming widely used to estimate HCV-infection prevalence. This paper aims to estimate the prevalence of HCV infection, and the hepatic and extrahepatic sequelae in at-risk groups, using routinely collected data in the Lazio region, Italy.</p> <p>Methods</p> <p>HCV laboratory surveillance and dialysis, hospital discharge, and drug-user registers were used as information sources to identify at-risk groups and to estimate HCV prevalence and sequelae.</p> <p>Full name and birth date were used as linkage keys for the various health registries. Prevalence was estimated as the percentage of cases within the general population and the at-risk groups, with 95% confidence intervals (95% CI) from 1997 to 2001. The risk of sequelae was estimated through a follow-up of hospital discharges up to December 31, 2004 and calculated as the prevalence ratio in HCV-positive and HCV-negative people, within each at-risk group, with 95% CI.</p> <p>Results</p> <p>There were 65,127 HCV-infected people in the study period; the prevalence was 1.24% (95%CI = 1.23%-1.25%) in the whole population, higher in males and older adults. Drug users (35.1%; 95%CI = 34.6-35.7) and dialysis patients (21.1%; 95%CI = 20.2%-22.0%) showed the highest values. Medical procedures with little exposure to blood resulted in higher estimates, ranging between 1.3% and 3.4%, which was not conclusively attributable to the surgical procedures. Cirrhosis, hepatocellular carcinoma and encephalopathy were the most frequent hepatic sequelae; cryoglobulinaemia and non-Hodgkin's lymphoma were the most frequent extrahepatic sequelae.</p> <p>Conclusions</p> <p>Synthesising data from multiple routine sources improved estimates of HCV prevalence and sequelae in dialysis patients and drug users, although prevalence validity should be assessed in survey and sequelae need a well-defined longitudinal approach.</p

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background:Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.Methodology/Principal Findings:By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.Conclusions/Significance:This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells. © 2009 Mileo et al

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.TelethonAssociazione Serena Talarico per i giovani nel mondo and Fondazione Giuseppe Tomasello O.N.L.U.S.Mitocon OnlusResearch to Prevent BlindnessInternational Foundation for Optic Nerve Diseases (IFOND)Struggling Within Leber'sPoincenot FamilyEierman FoundationNational Eye InstituteUniv Rome, Dept Radiol Oncol & Pathol, Rome, ItalyUniv Bologna, Dept Biomed & NeuroMotor Sci DIBINEM, Bologna, ItalyUniv Bari, Dept Biosci Biotechnol & Biopharmaceut, Bari, ItalyBellaria Hosp, IRCCS Ist Sci Neurol Bologna, I-40139 Bologna, ItalyUSC, Keck Sch Med, Dept Ophthalmol, Los Angeles, CA USAUSC, Keck Sch Med, Dept Neurosurg, Los Angeles, CA USAUniv Trieste, Dept Reprod Sci Dev & Publ Hlth, Trieste, ItalyUniv Trieste, IRCCS Burlo Garofolo Children Hosp, Trieste, ItalyNewcastle Univ, Inst Med Genet, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, EnglandFdn Ist Neurol Carlo Besta IRCCS, Unit Mol Neurogenet, Milan, ItalyMRC Mitochondrial Biol Unit, Cambridge, EnglandFed Univ São Paulo UNIFESP, Dept Ophthalmol, São Paulo, BrazilUniv São Paulo, Inst Psychol, Dept Expt Psychol, São Paulo, BrazilStudio Oculist dAzeglio, Bologna, ItalyOsped San Giovanni Evangelista, Tivoli, ItalyAzienda Osped San Camillo Forlanini, Rome, ItalyUniv Rome, Dipartimento Metodi & Modelli Econ Finanza & Terr, Rome, ItalyUniv Rome, Dept Mol Med, Rome, ItalyFed Univ São Paulo UNIFESP, Dept Ophthalmol, São Paulo, BrazilTelethon: GGP06233Telethon: GGP11182Telethon: GPP10005National Eye Institute: EY03040Web of Scienc

Lopinavir/Ritonavir and Darunavir/Cobicistat in Hospitalized COVID-19 Patients: Findings From the Multicenter Italian CORIST Study

Background: Protease inhibitors have been considered as possible therapeutic agents for COVID-19 patients. Objectives: To describe the association between lopinavir/ritonavir (LPV/r) or darunavir/cobicistat (DRV/c) use and in-hospital mortality in COVID-19 patients. Study Design: Multicenter observational study of COVID-19 patients admitted in 33 Italian hospitals. Medications, preexisting conditions, clinical measures, and outcomes were extracted from medical records. Patients were retrospectively divided in three groups, according to use of LPV/r, DRV/c or none of them. Primary outcome in a time-to event analysis was death. We used Cox proportional-hazards models with inverse probability of treatment weighting by multinomial propensity scores. Results: Out of 3,451 patients, 33.3% LPV/r and 13.9% received DRV/c. Patients receiving LPV/r or DRV/c were more likely younger, men, had higher C-reactive protein levels while less likely had hypertension, cardiovascular, pulmonary or kidney disease. After adjustment for propensity scores, LPV/r use was not associated with mortality (HR = 0.94, 95% CI 0.78 to 1.13), whereas treatment with DRV/c was associated with a higher death risk (HR = 1.89, 1.53 to 2.34, E-value = 2.43). This increased risk was more marked in women, in elderly, in patients with higher severity of COVID-19 and in patients receiving other COVID-19 drugs. Conclusions: In a large cohort of Italian patients hospitalized for COVID-19 in a real-life setting, the use of LPV/r treatment did not change death rate, while DRV/c was associated with increased mortality. Within the limits of an observational study, these data do not support the use of LPV/r or DRV/c in COVID-19 patients