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5-(3',4'-Dihydroxyphenyl)-γ-valerolactone (VL) is a bioactive metabolite resulting from the gut microbial metabolism of proanthocyanidins and flavonoids, known for its health-promoting effects, including antidiabetic and anti-inflammatory activities. Although VL has been observed in different in vivo studies, its pre-absorption, distribution, metabolism, excretion, toxicity (ADMET) properties have rarely been investigated. This study aims to address this gap by evaluating the pre-ADMET properties of VL for the first time. Also, the understanding of these properties is significant for correlating the encountered activities to this metabolite. In vitro absorption studies revealed that VL is rapidly metabolized and absorbed as its sulfate phase II conjugate (valerolactone sulfate), which enters systemic circulation and mildly activates the Breast Cancer Resistance Protein efflux transporter. In human S9 liver fraction, a mixture of liver enzymes used to simulate in vivo liver metabolism, VL is metabolized into glucuronic phase II conjugates (valerolactone glucuronide 1 [VLG1] and 2 [VLG2]) with a half-life of 8.72 min and an 80% conversion rate. In human liver microsomes, VL is metabolized at a slower rate (half-life of 23.08 min), suggesting that oxidative metabolism is secondary. Additionally, VL did not activate the pregnane X receptor or inhibit Cytochrome P3A4 (CYP3A4) and Cytochrome P1A2 (CYP1A2) enzymes, indicating no risk of herb-drug interactions with coadministered prescription drugs., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Evidence suggests that polyphenol-rich foods like berries may help counteract aging-related disorders such as vascular dysfunction and arterial stiffness. However, few intervention studies have been conducted in older adults. This study aimed to assess whether the consumption of blueberries may improve vascular function in older subjects. A randomized, controlled, crossover trial was conducted in a group of 20 volunteers over 60 years old. Participants consumed either a blueberry mousse (250 g, providing 480 mg of anthocyanins - ACNs) or a control product (250 mL of sugared water), with treatments separated by at least 1-week. Reactive hyperemia index (RHI), augmentation index (AIx), blood pressure, and heart rate were measured at baseline and 2 h post-consumption. Blood samples were collected at baseline and after 1, 1.5, 2 and 4 h from the intake to evaluate ACN bioavailability, metabolic, and vascular markers. Sixteen subjects completed the trial (9 males, 7 females; mean age 69 ± 5 years). Blueberry consumption significantly increased RHI compared to control (mean difference + 0.42, 95 % CI: 0.01-0.082, p < 0.05). Maximum serum ACN concentration was observed at 2 h (20.3 ± 7.4 ng/mL). No association was found between RHI improvement and total serum ACNs, but a significant positive correlation was detected with delphinidin and cyanidin-3-glucoside (p < 0.01). No effects on AIx, blood pressure, or other markers were found. In conclusion, blueberries may improve peripheral vascular function in older adults, potentially due to increased ACN levels. Further studies are needed to corroborate these findings and elucidate the mechanisms involved., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Context: Carnosine and histidine-containing dipeptides (HCDs) are suggested to have anti-inflammatory and antioxidative benefits, but their effects on circulating adipokines and inflammatory and oxidative stress biomarkers remain unclear., Objectives: The aim of the present systematic review and meta-analysis was to determine the impact of HCD supplementation on inflammatory and oxidative stress biomarkers., Data Sources: A systematic search was performed on Medline via Ovid, Scopus, Embase, ISI Web of Science, and the Cochrane Library databases from inception to 25 January 2023., Data Extraction: Using relevant key words, trials investigating the effects of carnosine/HCD supplementation on markers of inflammation and oxidative stress, including C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), adiponectin, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (TAC), and catalase (CAT) were identified. Meta-analyses were conducted using random-effects models to calculate the weighted mean differences (WMDs) and 95% confidence intervals (CIs)., Data Analysis: A total of 9 trials comprising 350 participants were included in the present meta-analysis. Carnosine/HCD supplementation led to a significant reduction in CRP (WMD: -0.97 mg/L; 95% CI: -1.59, -0.36), TNF-α (WMD: -3.60 pg/mL; 95% CI: -7.03, -0.18), and MDA (WMD: -0.34 μmol/L; 95% CI: -0.56, -0.12) and an elevation in CAT (WMD: 4.48 U/mL; 95% CI: 2.43, 6.53) compared with placebo. In contrast, carnosine/HCD supplementation had no effect on IL-6, adiponectin, GSH, SOD, and TAC levels., Conclusion: Carnosine/HCD supplementation may reduce inflammatory and oxidative stress biomarkers, and potentially modulate the cardiometabolic risks associated with chronic low-grade inflammation and lipid peroxidation., Systematic Review Registration: PROSPERO registration no. CRD42017075354., (© The Author(s) 2023. Published by Oxford University Press on behalf of the International Life Sciences Institute.)

Background/objectives: In vitro studies suggest that carnosine reduces inflammation by upregulating anti-inflammatory mediators and downregulating pro-inflammatory cytokines. However, human clinical trials examining the effects of carnosine on inflammatory biomarkers are scant. We conducted a secondary analysis of a double-blind randomised controlled trial (RCT) to examine the effects of carnosine supplementation on inflammatory markers and adipokines in participants with prediabetes or well-controlled type 2 diabetes (T2D)., Methods: Out of 88 participants who were recruited, 49 adults with prediabetes or well-controlled T2D (HbA1c: 6.6 ± 0.7% [mean ± SD]) who were treated with diet and/or metformin were eligible for inclusion. Participants were randomised to receive 2 g/day of carnosine or a matching placebo for 14 weeks. We measured serum concentrations of monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, IL-10, C-reactive protein (CRP), tumour necrosis factor-α (TNF-α), adiponectin, leptin, adipsin, serpin, and resistin levels at baseline and after 14 weeks. The trial was registered at clinicaltrials.gov (NCT02917928)., Results: Forty-one participants (M = 29/F = 12) aged 53 (42.6, 59.3) years [median (IQR)] completed the trial. After 14 weeks of supplementation, changes in pro- and anti-inflammatory cytokine and adipokine levels did not differ between the carnosine and placebo groups ( p > 0.05 for all). The results remained unchanged after adjustment for confounders including age, sex, and anthropometric measures (e.g., body fat percentage and visceral adipose tissue)., Conclusions: In individuals with prediabetes and well-controlled T2D, carnosine supplementation did not result in any significant changes in inflammatory markers. Larger RCTs with longer follow-up durations are needed to evaluate whether carnosine may be beneficial in individuals with poorly controlled T2D.

Integrity of epithelial and endothelial cell barriers is of critical importance for health, barrier disruption is a hallmark of numerous diseases, of which many are driven by carbonyl stressors such as methylglyoxal (MG). Carnosine and anserine exert some MG-quenching activity, but the impact of these and of other histidine containing dipeptides on cell barrier integrity has not been explored in detail. In human proximal tubular (HK-2) and umbilical vein endothelial (HUVEC) cells, exposure to 200 µM MG decreased transepithelial resistance (TER), i.e. increased ionic permeability and permeability for 4-, 10- and 70-kDa dextran, membrane zonula occludens (ZO-1) abundance was reduced, methylglyoxal 5-hydro-5-methylimidazolones (MG-H1) formation was increased. Carnosine, balenine (ß-ala-1methyl-histidine) and anserine (ß-ala-3-methyl-histidine) ameliorated MG-induced reduction of TER in both cell types. Incubation with histidine, 1-/3-methylhistidine, but not with ß-alanine alone, restored TER, although to a lower extent than the corresponding dipeptides. Carnosine and anserine normalized transport and membrane ZO-1 abundance. Aminoguanidine, a well-described MG-quencher, did not mitigate MG-induced loss of TER. Our results show that the effects of the dipeptides on epithelial and endothelial resistance and junction function depend on the methylation status of histidine and are not exclusively explained by their quenching activity., (© 2024. The Author(s).)

Replacing cereals with food leftovers could reduce feed-food competition and keep nutrients and energy in the food chain. Former food products (FFPs) are industrial food leftovers no more intended for human but still suitable as alternative and sustainable feedstuffs for monogastric. In this study, omics approaches were applied to evaluate the impact of dietary FFPs on pig liver proteome and plasma peptidome. Thirty-six Swiss Large White male castrated pigs were randomly assigned to three dietary treatments [control (CTR), 30% CTR replaced with salty FFP (SA), 30% CTR replaced with sugary FFP (SU)] from the start of the growing phase (22.4 ± 1.7 kg) until slaughtering (110 ± 3 kg). The low number of differentially regulated proteins in each comparison matrix (SA/SU vs. CTR) and the lack of metabolic interaction indicated a marginal impact on hepatic lipid metabolism. The plasma peptidomics investigation showed low variability between the peptidome of the three dietary groups and identified three possible bioactive peptides in the SA group associated with anti-hypertension and vascular homeostasis regulation. To conclude, the limited modulation of liver proteome and plasma peptidome by the SA and SU diets strenghtened the idea of reusing FFPs as feed ingredients to make pig production more sustainable., (© 2024. The Author(s).)

Vector-borne parasitic diseases (VBPDs) pose a significant threat to public health on a global scale. Collectively, Human African Trypanosomiasis (HAT), Leishmaniasis, and Malaria threaten millions of people, particularly in developing countries. Climate change might alter the transmission and spread of VBPDs, leading to a global burden of these diseases. Thus, novel agents are urgently needed to expand therapeutic options and limit the spread of drug-resistant parasites. Herein, we report the development of broad-spectrum antiparasitic agents by screening a known library of antileishmanial and antimalarial compounds toward Trypanosoma brucei ( T. brucei ) and identifying a 1,3,4-oxadiazole derivative ( 19 ) as anti- T. brucei hit with predicted blood-brain barrier permeability. Subsequently, extensive structure-activity-relationship studies around the lipophilic tail of 19 led to a potent antitrypanosomal and antimalarial compound ( 27 ), with moderate potency also toward Leishmania infantum ( L. infantum ) and Leishmania tropica . In addition, we discovered a pan-active antiparasitic molecule ( 24 ), showing low-micromolar IC 50 s toward T. brucei and Leishmania spp. promastigotes and amastigotes, and nanomolar IC 50 against Plasmodium falciparum , together with high selectivity for the parasites over mammalian cells (THP-1). Early ADME-toxicity assays were used to assess the safety profile of the compounds. Overall, we characterized 24 and 27 , bearing the 1,3,4-oxadiazole privileged scaffold, as broad-spectrum low-toxicity agents for the treatment of VBPDs. An alkyne-substituted chemical probe ( 30 ) was synthesized and will be utilized in proteomics experiments aimed at deconvoluting the mechanism of action in the T. brucei parasite.

Background: The viral main protease (M pro ) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of M pro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract., Methods: The experiments focus on finding MS covalent inhibitors of M pro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site., Results: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known M pro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the M pro pocket observed was also evaluated by computational studies., Conclusions: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind M pro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease associated with obesity and diabetes prevalence. The use of natural compounds has become an attractive approach to prevent NAFLD and its progression. Gamma-oryzanol (Orz) is a natural compound whose beneficial effects on chronic metabolic diseases have been reported. Therefore, we aimed to investigate the preventive effect of Orz on the hepatic proteome in a diet induced NAFLD model. Wistar rats were randomly distributed into three experimental groups (n=6/group) according to the diet received for 30 weeks: Control group, high sugar-fat (HSF) group, and HSF+Orz group. The isolated Orz was added to the chow at the dose of 0.5% (w/w). We evaluated the nutritional profile, characterized the presence of steatosis through histological analysis, triglyceride content in liver tissue and hepatic inflammation. Next, we performed label-free quantitative proteomics of hepatic tissue. Network analysis was performed to describe involved protein pathways. NAFLD induction was characterized by the presence of hepatic steatosis. Orz prevented lipid accumulation. The compound prevented alterations of the hepatic proteome, highlighted by the modulation of lipid metabolism, inflammation, oxidative stress, xenobiotic metabolism, and the sirtuin signaling pathway. It was possible to identify key altered pathways of NAFLD pathophysiology modulated by Orz which may provide insights into NAFLD treatment targets., Competing Interests: Declaration of Competing Interest The authors declare no conflicts., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans -esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity. HPLC-ELSED analysis of the purified mixture from hemp wax revealed C 26 OH and C 28 OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes., Competing Interests: Federica Pellati reports financial support was provided by University of Modena and Reggio Emilia, Department of Life Sciences (FAR2023). The other authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Consumption of diets high in sugar and fat is related to the development of Metabolic dysfunction-associated steatotic liver disease (MASLD). Carnosine (CAR) is a dipeptide with antioxidant and anti-inflammatory action and has been studied for treating diseases. This work aimed to evaluate the effects of CAR on diet-induced MASLD in rats. Male Wistar rats were distributed into 2 groups (17 weeks): normocaloric (Co, n = 12), and hypercaloric diet rich in lipids and simple carbohydrates (MASLD, n = 12). After, the animals were redistributed to begin the treatment with CAR (4 weeks): Co (n = 6), Co + CAR (n = 6), MASLD (n = 6), and MASLD + CAR (n = 6), administered intraperitoneally (250 mg/kg). Evaluations included nutritional, hormonal and metabolic parameters; hepatic steatosis, inflammatory and oxidative markers. MASLD group had a higher adiposity index, systolic blood pressure, glucose, plasma and liver triglycerides and cholesterol, insulin, hepatic steatosis, oxidative markers, and lower PPAR-α (Peroxisome Proliferator-activated receptor α), compared to the Co. CAR attenuated plasma and hepatic triglyceride and cholesterol levels, hepatic steatosis, CD68 + macrophages, and hepatic oxidative markers, in addition to increasing HDL cholesterol levels and PPAR-α, compared to the untreated MASLD group. CAR acts in importants pathophysiological processes of MASLD and may be a therapeutic compound to control the disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Deuterated proanthocyanidin metabolite 5-(3',4'-dihydroxyphenyl)-γ-valerolactone has been successfully produced. This metabolite is responsible for several proanthocyanidin protective effects in the field of cancer chemoprevention, skin wrinkle-prevention, and antimicrobials. The synthetic approach applied employs a short reaction sequence and allows the incorporation of four deuterium atoms on non-exchangeable sites, making it an attractive strategy to produce a stable isotopically labeled internal standard for quantitative mass spectrometry isotope dilution-based methods, as demonstrated by developing an LC-MS/MS method to quantify DHPV in urine samples. Overall, this efficient synthesis provides a valuable analytical tool for the study of the metabolic conversion of proanthocyanidins thus helping to investigate the biological effect and establishing the active dose of the key catabolite 5-(3',4'-dihydroxyphenyl)-γ-valerolactone., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The nonalcoholic fatty liver disease (NAFLD), which is closely related to westernized dietary (WD) patterns, displays a rising epidemiological and economic burden. Since there is no pharmacological therapy approved for this disease, mechanistic studies are warranted. In this work, we investigated the action of carnosine (CAR), a natural dipeptide with several protection roles against oxidative stress in the liver of NAFLD rats. NAFLD was induced by WD-rich sugars and fat, verifying the histological evidence of steatosis. As intraperitoneal administration of CAR reversed liver steatosis, the protein profiles of NAFLD liver and CAR NAFLD liver were evaluated by label-free proteomics approach. A total of 2531 proteins were identified and the 230 and 276 were significantly up- and downregulated, respectively, by CAR treatment of NAFLD rats and involved in fundamental pathways such as oxidative stress and lipid metabolism. Perilipin 2 and apolipoprotein E, components of the plasma membrane of vesicle, resulted in highly downregulated in the CAR-treated NAFLD liver. The advanced bioanalytical approach demonstrated the efficacy of CAR in overcoming the main symptoms of NAFLD, ameliorating the steatosis in the liver., (© 2024 Wiley Periodicals LLC.)

Background: To assess oxidative effects induced by a high-calorie diet on the retina of Wistar rats and test the antioxidative effects of carnosine supplementation., Methods: Wistar rats were randomly divided into the following groups: standard diet (SD), high-calorie diet (HcD), standard diet + carnosine (SD + Car), and high-calorie diet + carnosine (HcD + Car). The body weight, adiposity index, plasma glucose, total lipids, high-density lipoprotein (HDL), low-density lipoprotein (LDL), uric acid, creatinine, and triglycerides of the animals were evaluated. The retinas were analyzed for markers of oxidative stress. Hydrogen peroxide production was assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCF) oxidation. The total glutathione (tGSH), total antioxidant capacity (TAC), protein carbonyl, and sulfhydryl groups of the antioxidant system were analyzed., Results: TAC levels increased in the retinas of the SD + Car group compared to the SD group (p < 0.05) and in the HcD + Car group compared to the HcD group (p < 0.05). The levels of GSH and the GSSH:GSSG ratio were increased in the HcD + Car group compared to the SD + Car group (p < 0.05). An increase in the retinal carbonyl content was observed in the HcD group compared to the SD group (p < 0.05) and in the HcD + Car group compared to the SD + Car group (p < 0.05). A high-calorie diet (HcD) was also associated with a decrease in retinal sulfhydryl-type levels compared to the SD group (p < 0.05)., Conclusion: The results suggest that feeding a high-calorie diet to rats can promote an increase in carbonyl content and a reduction in sulfhydryl groups in their retinas. The administration of carnosine was not effective in attenuating these oxidative markers., Trial Registration: Animal Ethics Committee of Botucatu Medical School - Certificate number 1292/2019., (© 2023. The Author(s).)

Recent evidence indicates that reactive oxygen species play an important causative role in the onset and progression of valvular diseases. Here, we analyzed the oxidative modifications of albumin (HSA) occurring on Cysteine 34 and the antioxidant capacity of the serum in 44 patients with severe aortic stenosis (36 patients underwent aortic valve replacement and 8 underwent a second aortic valve substitution due to a degenerated bioprosthetic valve), and in 10 healthy donors (controls). Before surgical intervention, patients showed an increase in the oxidized form of albumin (HSA-Cys), a decrease in the native reduced form (HSA-SH), and a significant reduction in serum free sulfhydryl groups and in the total serum antioxidant activity. Patients undergoing a second valve replacement showed levels of HSA-Cys, free sulfhydryl groups, and total antioxidant activity similar to those of controls. In vitro incubation of whole blood with aspirin (ASA) significantly increased the free sulfhydryl groups, suggesting that the in vivo treatment with ASA may contribute to reducing oxidative stress. We also found that N-acetylcysteine and its amide derivative were able to regenerate HSA-SH. In conclusion, the systemic oxidative stress reflected by high levels of HSA-Cys is increased in patients with aortic valve stenosis. Thiol-disulfide breaking agents regenerate HSA-SH, thus paving the way to the use these compounds to mitigate the oxidative stress occurring in the disease.

Diet can modulate the different stages of inflammation due to the presence of bioactive compounds such as polyphenols. Apples are a great source of phenolic compounds that show anti-inflammatory and antioxidant properties, and these might be used as a dietary supplement and/or functional element in the treatment of chronic inflammatory illnesses. The aim of our study was to evaluate the anti-inflammatory and antioxidant actions of thinned apple polyphenol (TAP) extracts in a model of paw edema. The experimental model was induced in rats via subplantar injections of 1% λ-Carrageenan (CAR) in the right hind leg, and TAP extract was administered via oral gavage 30 min before and 1 h after the CAR injection at doses of 5 mg/kg and 10 mg/kg, respectively. The inflammatory response is usually quantified by the increase in the size of the paw (edema), which is maximal about 5 h after the injection of CAR. CAR-induced inflammation generates the release of pro-inflammatory mediators and reactive oxygen species (ROS). Furthermore, the inflammatory state induces the pain that involves the peripheral nociceptors, but above all it acts centrally at the level of the spinal cord. Our results showed that the TAP extracts reduced paw histological changes, neutrophil infiltration, mast cell degranulation, and oxidative stress. Additionally, the oral administration of TAP extracts decreased thermal and mechanical hyperalgesia, along with a reduction in spinal microglia and the markers of nociception. In conclusion, we demonstrate that TAP extract is able to modulate inflammatory, oxidative, and painful processes, and is also useful in the treatment of the symptoms associated with paw edema.

The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA., (© 2021 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

The lipid profile of skin is fundamental in the maintenance of the protective barrier against the external environment. Signaling and constitutive lipids of this large organ are involved in inflammation, metabolism, aging, and wound healing, such as phospholipids, triglycerides, FFA, and sphingomyelin. Skin exposure to ultraviolet (UV) radiation results in a photoaging process that is an accelerated form of aging. UV-A radiation deeply penetrates the dermis and promotes damage to DNA, lipids, and proteins by increasing the generation of reactive oxygen species (ROS). Carnosine, an endogenous β-alanyl-L-histidine dipeptide, demonstrated antioxidant properties that prevent photoaging and modification of skin protein profiling, making carnosine a compelling ingredient to consider for use in dermatology. The aim of this research was to investigate the modification of skin lipidome after UV-A treatment in presence or not of topic administration of carnosine. Quantitative analyses based on high-resolution mass spectrometry of nude mice skin-extracted lipids resulted in several modifications of barrier composition after UV-A radiation, with or without carnosine treatment. In total, 328 out of 683 molecules showed significant alteration-262 after UV-A radiation and 126 after UV-A and carnosine treatment versus controls. Importantly, the increased oxidized TGs after UV-A radiation, responsible of dermis photoaging, were completely reverted by carnosine application to prevent the UV-A damage. Network analyses also showed that the production of ROS and the calcium and TNF signaling were modulated by UV-A and carnosine. In conclusion, lipidome analyses attested the carnosine activity to prevent the UV-A damage, reducing the lipid oxidation, the inflammation, and the dysregulation of lipid skin barrier.

Low-grade chronic inflammation in obesity is associated with leptin resistance. In order to alleviate this pathological condition, bioactive compounds capable of attenuating oxidative stress and inflammation have been researched, and bergamot (Citrus bergamia) presents these properties. The aim was to evaluate the effect of bergamot leaves extract on leptin resistance in obese rats. Animals were divided into 2 groups: control diet (C, n = 10) and high sugar-fat diet (HSF, n = 20) for 20 weeks. After detecting hyperleptinemia, animals were divided to begin the treatment with bergamot leaves extract (BLE) for 10 weeks: C + placebo (n = 7), HSF + placebo (n = 7), and HSF + BLE (n = 7) by gavage (50 mg/kg). Evaluations included nutritional, hormonal and metabolic parameters; adipose tissue dysfunction; inflammatory, oxidative markers and hypothalamic leptin pathway. HSF group presented obesity, metabolic syndrome, adipose tissue dysfunction, hyperleptinemia and leptin resistance compared to control group. However, the treated group showed a decrease in caloric consumption and attenuation of insulin resistance. Moreover, dyslipidemia, adipose tissue function, and leptin levels showed an improvement. At the level of the hypothalamus, the treated group showed a reduction of oxidative stress, inflammation and modulation of leptin signaling. In conclusion, BLE properties were able to improve leptin resistance through recovery of the hypothalamic pathway., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ionising radiation (IR) is a cause of lipid peroxidation, and epidemiological data have revealed a correlation between exposure to IR and the development of eye lens cataracts. Cataracts remain the leading cause of blindness around the world. The plasma membranes of lens fibre cells are one of the most cholesterolrich membranes in the human body, forming lipid rafts and contributing to the biophysical properties of lens fibre plasma membrane. Liquid chromatography followed by mass spectrometry was used to analyse bovine eye lens lipid membrane fractions after exposure to 5 and 50 Gy and eye lenses taken from wholebody 2 Gy-irradiated mice. Although cholesterol levels do not change significantly, IR dose-dependant formation of the oxysterols 7β-hydroxycholesterol, 7-ketocholesterol and 5, 6-epoxycholesterol in bovine lens nucleus membrane extracts was observed. Whole-body X-ray exposure (2 Gy) of 12-week old mice resulted in an increase in 7β-hydroxycholesterol and 7-ketocholesterol in their eye lenses. Their increase regressed over 24 h in the living lens cortex after IR exposure. This study also demonstrated that the IR-induced fold increase in oxysterols was greater in the mouse lens cortex than the nucleus. Further work is required to elucidate the mechanistic link(s) between oxysterols and IR-induced cataract, but these data evidence for the first time that IR exposure of mice results in oxysterol formation in their eye lenses., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V.)

The present paper reports a sustainable raw material obtained from the by-products derived from the industrial production of bergamot ( Citrus × Bergamia Risso & Poiteau) essential oils. The procedure to obtain the raw material is designed to maintain as much of the bioactive components as possible and to avoid expensive chemical purification. It consists of spray-drying the fruit juice obtained by squeezing the fruits, which is mixed with the aqueous extract of the pulp, i.e., the solid residue remained after fruit pressing. The resulting powder bergamot juice (PBJ) contains multiple bioactive components, in particular, among others, soluble fibers, polyphenols and amino-acid betaines, such as stachydrine and betonicine. LC-MS analysis identified 86 compounds, with hesperetin, naringenin, apigenin and eridictyol glucosides being the main components. In the second part of the paper, dose-dependent anti-inflammatory activity of PBJ and of stachydrine was found, but neither of the compounds were effective in activating Nrf2. PBJ was then found to be effective in an in vivo model of a metabolic syndrome induced by a high-sugar, high-fat (HSF) diet and evidenced by a significant increase of the values related to a set of parameters: blood glucose, triglycerides, insulin resistance, systolic blood pressure, visceral adipose tissue and adiposity index. PBJ, when given to control rats, did not significantly change these values; in contrast, they were found to be greatly affected in rats receiving an HSF diet. The in vivo effect of PBJ can be ascribed not only to bergamot polyphenols with well-known anti-inflammatory, antioxidant and lipid-regulating effects, but also to the dietary fibers and to the non-phenolic constituents, such as stachydrine. Moreover, since PBJ was found to affect energy homeostasis and to regulate food intake, a mechanism on the regulation of energy homeostasis through leptin networking should also be considered and deserves further investigation.

Metabolic Syndrome (MetS), inflammation and oxidative stress contribute to impairment of skeletal muscle function. Bergamot ( Citrus bergamia ) leaf extract (BLE) has shown protective effects against comorbidities associated with MetS through its anti-inflammatory and antioxidant effects. The aim of this work was to elucidate the antioxidant and anti-inflammatory activity of BLE in skeletal muscles in an experimental model of MetS. Once metabolic syndrome was diagnosed, animals were divided into groups receiving different treatments for 10 weeks, including control diet ( n  = 10), control + BLE ( n  = 10), High Sugar-fat diet (HSF) ( n  = 10), HSF + BLE ( n  = 10). Evaluation included nutritional, metabolic and hormonal analyses, along with measurements of inflammatory status and oxidative stress in soleus and extensor digitorum longus (EDL) muscles. BLE showed positive metabolic effects, with a reduction of plasma triglycerides and insulin resistance and an increase in high-density lipoprotein cholesterol, and protective activity against oxidative stress and inflammation in Soleus and EDL muscles in animals with MetS.

Plant secondary metabolites, known as phytochemicals, have recently gained much attention in light of the "circular economy", to reutilize waste products deriving from agriculture and food industry. Phytochemicals are known for their onco-preventive and chemoprotective effects, among several other beneficial properties. Apple phytochemicals have been extensively studied for their effectiveness in a wide range of diseases, cancer included. This review aims to provide a thorough overview of the main studies reported in the literature concerning apple phytochemicals, mostly polyphenols, in cancer prevention. Although there are many different mechanisms targeted by phytochemicals, the Nrf2 and NF-κB signaling pathways are the ones this review will be focused on, highlighting also the existing crosstalk between these two systems.

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

Very recently, we have developed a new generation of ligands targeting the cannabinoid receptor type 2 (CB2R), namely JR compounds, which combine the pharmacophoric portion of the CB2R positive allosteric modulator (PAM), EC21a , with that of the CB2R selective orthosteric agonist LV62 , both synthesized in our laboratories. The functional examination enabled us to identify JR14a , JR22a , and JR64a as the most promising compounds of the series. In the current study, we focused on the assessment of the bitopic (dualsteric) nature of these three compounds. Experiments in cAMP assays highlighted that only JR22a behaves as a CB2R bitopic (dualsteric) ligand. In parallel, computational studies helped us to clarify the binding mode of these three compounds at CB2R, confirming the bitopic (dualsteric) nature of JR22a . Finally, the potential of JR22a to prevent neuroinflammation was investigated on a human microglial cell inflammatory model.

M pro represents one of the most promising drug targets for SARS-Cov-2, as it plays a crucial role in the maturation of viral polyproteins into functional proteins. HTS methods are currently used to screen M pro inhibitors, and rely on searching chemical databases and compound libraries, meaning that they only consider previously structurally clarified and isolated molecules. A great advancement in the hit identification strategy would be to set-up an approach aimed at exploring un-deconvoluted mixtures of compounds such as plant extracts. Hence, the aim of the present study is to set-up an analytical platform able to fish-out bioactive molecules from complex natural matrices even where there is no knowledge on the constituents. The proposed approach begins with a metabolomic step aimed at annotating the MW of the matrix constituents. A further metabolomic step is based on identifying those natural electrophilic compounds able to form a Michael adduct with thiols, a peculiar chemical feature of many M pro inhibitors that covalently bind the catalytic Cys145 in the active site, thus stabilizing the complex. A final step consists of incubating recombinant M pro with natural extracts and identifying compounds adducted to the residues within the M pro active site by bottom-up proteomic analysis (nano-LC-HRMS). Data analysis is based on two complementary strategies: (i) a targeted search applied by setting the adducted moieties identified as Michael acceptors of Cys as variable modifications; (ii) an untargeted approach aimed at identifying the whole range of adducted peptides containing Cys145 on the basis of the characteristic b and y fragment ions independent of the adduct. The method was set-up and then successfully tested to fish-out bioactive compounds from the crude extract of Scutellaria baicalensis, a Chinese plant containing the catechol-like flavonoid baicalin and its corresponding aglycone baicalein which are well-established inhibitors of M pro . Molecular dynamics (MD) simulations were carried out in order to explore the binding mode of baicalin and baicalein, within the SARS-CoV-2 M pro active site, allowing a better understanding of the role of the nucleophilic residues (i.e. His41, Cys145, His163 and His164) in the protein-ligand recognition process., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giancarlo Aldini reports financial support was provided by Lombardy Region., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A multidisciplinary investigation on Achillea moschata Wulfen (Asteraceae) is outlined herein. This work, part of the European Interreg Italy-Switzerland B-ICE project, originated from an ethnobotanical survey performed in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy) in 2019-2021 which highlighted this species' relevance of use in folk medicine to treat gastrointestinal diseases. In addition, this contribution included analyses of the: (a) phytochemical profile of the aqueous and methanolic extracts of the dried flower heads using LC-MS/MS; (b) morpho-anatomy and histochemistry of the vegetative and reproductive organs through Light, Fluorescence, and Scanning Electron Microscopy; (c) biological activity of the aqueous extract concerning the antioxidant and anti-inflammatory potential through cell-based in vitro models. A total of 31 compounds (5 phenolic acids, 13 flavonols, and 13 flavones) were detected, 28 of which included in both extracts. Covering and secreting trichomes were observed: the biseriate 10-celled glandular trichomes prevailing on the inflorescences represented the main sites of synthesis of the polyphenols and flavonoids detected in the extracts, along with volatile terpenoids. Finally, significant antioxidant and anti-inflammatory activities of the aqueous extract were documented, even at very low concentrations; for the first time, the in vitro tests allowed us to formulate hypotheses about the mechanism of action. This work brings an element of novelty due to the faithful reproduction of the traditional aqueous preparation and the combination of phytochemical and micromorphological research approaches.

Bergamot citrus (Citrus bergamia Risso et Poiteau), have been used as a strategy to prevent or treat comorbidities associated with metabolic syndrome parameters, such as cardiorenal metabolic syndrome (CRMS). The aim was to test the effect of bergamot leaf extract on CRMS and associated pathophysiological factors in rats fed with a high sugar-fat diet. Animals were divided into two experimental groups with control diet (Control, n = 30) and high sugar-fat diet (HSF, n = 30) for 20 weeks. Once CRMS was detected, animals were redivided to begin the treatment with Bergamot Leaf Extract (BLE) by gavage (50 mg/kg) for 10 weeks: control diet + placebo (Control, n = 09), control diet + BLE (Control + BLE, n = 09), HSF diet + placebo (HSF, n = 09), HSF + BLE (n = 09). Evaluation included nutritional, metabolic and hormonal analysis; and renal and cardiac parameters. HSF groups presented obesity, dyslipidemia, hypertension, hyperglycemia, hyperinsulinemia, insulin resistance. BLE showed protection against effects on hypertriglyceridemia, insulin resistance, renal damage, and structural and functional alterations of the heart. Conclusion: Bergamot leaf extract shows potential as a therapeutic to treat CRMS in animals fed with a high sugar-fat diet., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Human serum albumin (HSA) represents the most abundant plasma protein, with relevant antioxidant activity due to the presence of the sulfhydryl group on cysteine at position 34 (Cys34), the latter being one of the major target sites for redox-dependent modifications leading to the formation of mixed disulfide linkages with low molecular weight thiols. Thiolated forms of HSA (Thio-HSA) may be useful as markers of an unbalanced redox state and as a potential therapeutic target. Indeed, we have previously reported that albumin Cys34 can be regenerated in vitro by N -Acetylcysteine (NAC) through a thiol-disulfide breaking mechanism, with a full recovery of the HSA antioxidant and antiplatelet activities. With this case study, we aimed to assess the ability of NAC to regenerate native mercaptoalbumin (HSA-SH) and the plasma antioxidant capacity in subjects with redox unbalance, after oral and intravenous administration. A placebo-controlled crossover study, single-blinded, was performed on six hypertensive subjects, randomized into two groups, on a one-to-one basis with NAC (600 mg/die) or a placebo, orally and intravenously administered. Albumin isoforms, HSA-SH, Thio-HSA, and glutathione levels were evaluated by means of mass spectrometry. The plasma antioxidant activity was assessed by a fluorimetric assay. NAC, orally administered, significantly decreased the Thio-HSA levels in comparison with the pre-treatment conditions (T0), reaching the maximal effect after 60 min (-24.7 ± 8%). The Thio-HSA reduction was accompanied by a concomitant increase in the native HSA-SH levels (+6.4 ± 2%). After intravenous administration of NAC, a significant decrease of the Thio-HSA with respect to the pre-treatment conditions (T0) was observed, with a maximal effect after 30 min (-68.9 ± 10.6%) and remaining significant even after 6 h. Conversely, no effect on the albumin isoforms was detected with either the orally or the intravenously administered placebo treatments. Furthermore, the total antioxidant activity of the plasma significantly increased after NAC infusion with respect to the placebo ( p = 0.0089). Interestingly, we did not observe any difference in terms of total glutathione corrected for hemoglobin, ruling out any effect of NAC on the intracellular glutathione and supporting its role as a disulfide-breaking agent. This case study confirms the in vitro experiments and demonstrates for the first time that NAC is able to regenerate mercaptoalbumin in vivo, allowing us to hypothesize that the recovery of Cys34 content can modulate in vivo oxidative stress and, hopefully, have an effect in oxidative-based diseases.

Purpose: To detect carnosine, anserine and homocarnosine in vivo with chemical exchange saturation transfer (CEST) at 17.2 T., Methods: CEST MR acquisitions were performed using a CEST-linescan sequence developed in-house and optimized for carnosine detection. In vivo CEST data were collected from three different regions of interest (the lower leg muscle, the olfactory bulb and the neocortex) of eight rats., Results: The CEST effect for carnosine, anserine and homocarnosine was characterized in phantoms, demonstrating the possibility to separate individual contributions by employing high spectral resolution (0.005 ppm) and low CEST saturation power (0.15 μ $$ \mu $$ T). The CEST signature of these peptides was evidenced, in vivo, in the rat brain and skeletal muscle. The presence of carnosine and anserine in the muscle was corroborated by in vivo localized spectroscopy (MRS). However, the sensitivity of MRS was insufficient for carnosine and homocarnosine detection in the brain. The absolute amounts of carnosine and derivatives in the investigated tissues were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry using isotopic dilution standard methods and were in agreement with the CEST results., Conclusion: The robustness of the CEST-linescan approach and the favorable conditions for CEST at ultra-high magnetic field allowed the in vivo CEST MR detection of carnosine and related peptides. This approach could be useful to investigate noninvasively the (patho)-physiological roles of these molecules., (© 2022 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

The qualitative profile of thinned apple polyphenols (TAP) fraction (≈24% of polyphenols) obtained by purification through absorbent resin was fully investigated by LC-HRMS in positive and negative ion mode and using ESI source. A total of 68 polyphenols were identified belonging to six different classes: flavanols, flavonols, dihydrochalchones, flavanones, flavones and organic and phenolic acids. The antioxidant and anti-inflammatory activities were then investigated in cell models with gene reporter for NRF2 and NF-κB and by quantitative proteomic (label-free and SILAC) approaches. TAP dose-dependently activated NRF2 and in the same concentration range (10-250 µg/mL) inhibited NF-κB nuclear translocation induced by TNF-α and IL-1α as pro-inflammatory promoters. Proteomic studies elucidated the molecular pathways evoked by TAP treatment: activation of the NRF2 signaling pathway, which in turn up-regulates protective oxidoreductases and their nucleophilic substrates such as GSH and NADPH, the latter resulting from the up-regulation of the pentose phosphate pathway. The increase in the enzymatic antioxidant cellular activity together with the up-regulation of the heme-oxygenase would explain the anti-inflammatory effect of TAP. The results suggest that thinned apples can be considered as a valuable source of apple polyphenols to be used in health care products to prevent/treat oxidative and inflammatory chronic conditions.

Advanced quantitative bioanalytical approaches in combination with network analyses allow us to answer complex biological questions, such as the description of changes in protein profiles under disease conditions or upon treatment with drugs. In the present work, three quantitative proteomic approaches-either based on labelling or not-in combination with network analyses were applied to a new in vitro cellular model of nonalcoholic fatty liver disease (NAFLD) for the first time. This disease is characterized by the accumulation of lipids, inflammation, fibrosis, and insulin resistance. Hepatic G2 cells were used as model, and NAFLD was induced by a complex of oleic acid and bovine albumin. The development of the disease was verified by lipid vesicle staining and by the increase in the expression of perilipin-2-a protein constitutively present in the vesicles during NAFLD. The nLC-MS/MS analyses of peptide samples obtained from three different proteomic approaches resulted in accurate and reproducible quantitative data of protein fold-change expressed in NAFLD versus control cells. The differentially regulated proteins were used to evaluate the involved and statistically enriched pathways. Network analyses highlighted several functional and disease modules affected by NAFLD, such as inflammation, oxidative stress defense, cell proliferation, and ferroptosis. Each quantitative approach allowed the identification of similar modulated pathways. The combination of the three approaches improved the power of statistical network analyses by increasing the number of involved proteins and their fold-change. In conclusion, the application of advanced bioanalytical approaches in combination with pathway analyses allows the in-depth and accurate description of the protein profile of an in vitro cellular model of NAFLD by using high-resolution quantitative mass spectrometry data. This model could be extremely useful in the discovery of new drugs to modulate the equilibrium NAFLD health state.

Different drug classes such as antineoplastic drugs (anthracyclines, cyclophosphamide, 5-fluorouracil, taxanes, tyrosine kinase inhibitors), antiretroviral drugs, antipsychotic, and immunosuppressant drugs are known to induce cardiotoxic and neurotoxic effects. Recent studies have demonstrated that the impairment of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is a primary event in the pathophysiology of drug-induced cardiotoxicity and neurotoxicity. The Nrf2 pathway regulates the expression of different genes whose products are involved in antioxidant and inflammatory responses and the detoxification of toxic species. Cardiotoxic drugs, such as the anthracycline doxorubicin, or neurotoxic drugs, such as paclitaxel, suppress or impair the Nrf2 pathway, whereas the rescue of this pathway counteracts both the oxidative stress and inflammation that are related to drug-induced cardiotoxicity and neurotoxicity. Therefore Nrf2 represents a novel pharmacological target to develop new antidotes in the field of clinical toxicology. Interestingly, carnosine (β-alanyl-l-histidine), an endogenous dipeptide that is characterized by strong antioxidant, anti-inflammatory, and neuroprotective properties is able to rescue/activate the Nrf2 pathway, as demonstrated by different preclinical studies and preliminary clinical evidence. Starting from these new data, in the present review, we examined the evidence on the therapeutic potential of carnosine as an endogenous antidote that is able to rescue the Nrf2 pathway and then counteract drug-induced cardiotoxicity and neurotoxicity.

Enocianina is an anthocyanin-rich extract obtained from grape pomace. It is widely used as a colorant in the food industry and, in addition to anthocyanins, it also contains a variety of polyphenols. To understand whether enocianina, besides its coloring effect, may offer potential health benefit applications, we aimed to fully characterize the profile of four commercial enocianinas and assess their radical scavenging, enzymatic, antioxidant, and anti-inflammatory activities. LC-ESI-MS/MS analysis identified 90 phytochemicals. The relative content of each anthocyanin was assessed by a semi-quantitative analysis, with malvidin derivatives being the most abundant. UV-VIS spectroscopy detected total amounts of polyphenols and anthocyanins of 23% and 3.24%, respectively, indicating that anthocyanins represent a minor fraction of total polyphenols. Multiple linear regression analysis indicated that the radical scavenging activity is related to the total polyphenol content and not to anthocyanins. All four enocianinas dose-dependently activate Nrf2, and such activity was correlated with catechol-containing polyphenol content. Finally, all enocianinas showed dose-dependent anti-inflammatory activity, which at the highest concentrations tested was closely related to the total polyphenol content and was explained by radical scavenging, Nrf2 activation, and other mechanisms related to the polyphenolic components.

Cyclo(His-Pro) (CHP) is a cyclic dipeptide which is endowed with favorable pharmacokinetic properties combined with a variety of biological activities. CHP is found in a number of protein-rich foods and dietary supplements. While being stable at physiological pH, CHP can open yielding two symmetric dipeptides (His-Pro, Pro-His), the formation of which might be particularly relevant from dietary CHP due to the gastric acidic environment. The antioxidant and protective CHP properties were repeatedly reported although the non-enzymatic mechanisms were scantly investigated. The CHP detoxifying activity towards α,β unsaturated carbonyls was never investigated in detail, although its open dipeptides might be effective as already observed for histidine containing dipeptides. Hence, this study investigated the scavenging properties of TRH, CHP and its open derivatives towards 4-hydroxy-2-nonenal. The obtained results revealed that Pro-His possesses a marked activity and is more reactive than l-carnosine. As investigated by DFT calculations, the enhanced reactivity can be ascribed to the greater electrophilicity of the involved iminium intermediate. These findings emphasize that the primary amine (as seen in l-carnosine) can be replaced by secondary amines with beneficial effects on the quenching mechanisms. Serum stability of the tested peptides was also evaluated, showing that Pro-His is characterized by a greater stability than l-carnosine. Docking simulations suggested that its hydrolysis can be catalyzed by serum carnosinase. Altogether, the reported results evidence that the antioxidant CHP properties can be also due to the detoxifying activity of its open dipeptides, which might be thus responsible for the beneficial effects induced by CHP containing food.

Background: Recent studies suggest that acute-combined carnosine and anserine supplementation has the potential to improve the performance of certain cycling protocols. Yet, data on optimal dose, timing of ingestion, effective exercise range, and mode of action are lacking. Three studies were conducted to establish dosing and timing guidelines concerning carnosine and anserine intake and to unravel the mechanism underlying the ergogenic effects., Methods: First, a dose response study A was conducted in which 11 men randomly received placebo, 10, 20, or 30 mg.kg -1 of both carnosine and anserine. They performed 3x maximal voluntary isometric contractions (MVC), followed by a 5 x 6 s repeated cycling sprint ability test (RSA), once before the supplement and 30 and 60 minutes after. In a second study, 15 men performed 3x MVCs with femoral nerve electrical stimulation, followed by an RSA test, once before 30 mg.kg -1 carnosine and anserine and 60 minutes after. Finally, in study C, eight men performed a high intensity cycling training after randomly ingesting 30 mg.kg -1 of carnosine and anserine, a placebo or antihistamines (reduce post-exercise blood flow) to investigate effects on muscle perfusion., Results: Study A showed a 3% peak power (p = 0.0005; 95% CI = 0.07 to 0.27; ES = 0.91) and 4.5% peak torque (p = 0.0006; 95% CI = 0.12 to 0.50; ES = 0.87) improvement on RSA and MVC, with 30 mg.kg -1 carnosine + anserine ingestion 60 minutes before the performance yielding the best results. Study B found no performance improvement on group level; however, a negative correlation (r = -0.54; p = 0.0053; 95% CI = -0.77 to -0.19) was found between carnosinase enzyme activity (responsible for carnosine and anserine breakdown) and performance improvement. No effect of the supplement on neuromuscular function nor on muscle perfusion was found., Conclusions: These studies reveal that acute ingestion of 30 mg.kg -1 of both carnosine and anserine, 60 minutes before a high intensity exercise, can potentially improve performance, such as short cycling sprints or maximal muscle contractions. Subjects with lower carnosinase activity, and thus a slower breakdown of circulating dipeptides, appear to benefit more from this ergogenic effect. Finally, neither the involvement of a direct effect on neuromuscular function, nor an indirect effect on recovery through increased muscle perfusion could be confirmed as potential mechanism of action. The ergogenic mechanism therefore remains elusive., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

N -acetylcysteine (NAC) is able to break down protein disulfides, generating free thiols. This mechanism occurs on mixed disulfides of albumin (HSA) to form mercaptoalbumin (HMA), the main antioxidant species in the plasma. Circulating HSA exists in two main forms: the reduced form (HMA), and the oxidized forms, whose predominant modification is cystenylation (HSA-Cys). Increased levels of oxidized HSA have been detected in several diseases associated with oxidative stress. This study showed that NAC inhibits platelet aggregation by restoring HMA. In addition, the regeneration of HMA by NAC inhibits platelet functions such as intracellular calcium mobilization, reactive oxygen species generation, arachidonic acid metabolites synthesis, and adhesion to the collagen matrix. In our conditions, the exposure of platelets to NAC did not increase GSH levels. However, the inhibition of platelet aggregation was also detected following treatment of platelet-rich plasma with GSH, which, similarly to NAC, reduced HSA-Cys levels. Furthermore, this study showed that cysteine, another compound able to restore HMA by reducing the HSA-Cys content, inhibited platelet aggregation to a similar extent as NAC. The results obtained in this study suggest a new mechanism by which NAC can modulate platelet activation and suggest its possible use as an antiplatelet drug in conditions associated with oxidative stress.

Carnosine is an endogenous β-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Allergic reactions to antibiotics are a major concern in the clinic. ß -lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α -enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α -enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α -enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α -enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α -enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 González-Morena, Sánchez-Gómez, Vida, Pérez-Inestrosa, Salas, Montañez, Altomare, Aldini, Pajares and Pérez-Sala.)

Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13 C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: The receptor for AGEs (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface receptors expressed in many organs, among them, the kidneys. When activated, RAGE leads to a sequence of signaling that results in inflammation and oxidative stress, both involved in kidney disease pathogenesis. Gamma-oryzanol (γOz) comprises a mixture of ferulic acid (FA) esters and phytosterols (sterols and triterpene alcohols) mainly found in rice, with antioxidant and anti-inflammatory activities., Aim: To evaluate the effect of γOz to reduce renal inflammation and oxidative stress by modulating AGEs/RAGE axis in animals submitted to a high sugar-fat diet., Methods: Male Wistar rats (±187g) were randomly divided into two experimental groups: control (n = 7 animals) and high sugar-fat diet (HSF, n = 14 animals) for 20 weeks. After this period, when the presence of renal disease risk factors was detected in the HSF group (insulin resistance, dyslipidemia, increased systolic blood pressure and obesity), the HSF animals were divided to begin the treatment with γOz or continue receiving only HSF for 10 more weeks., Results: No effect of γOz on obesity and metabolic parameters was observed. However, kidney inflammation and oxidative stress decreased as soon as RAGE levels were reduced in HSF + γOz., Conclusion: It is possible to conclude that the gamma- oryzanol was effective in reducing inflammation and oxidative stress in the kidney by modulating the AGEs/RAGE axis.

A fully-detailed LC-MS qualitative profiling of red grape skin, extracted with a mixture of ethanol and water (70:30 v : v ) has permitted the identification of 65 compounds which can be classified into the following chemical classes: organic and phenolic acids (14 compounds), stilbenoids (1 compound), flavanols (21 compounds), flavonols (15 compounds) and anthocyanins (14 compounds). The extraction yield obtained with water at different temperatures (100 °C, 70 °C, room temperature) was then evaluated and the overall polyphenol content indicates that EtOH:H 2 O solvent is the most efficient and selective for polyphenol extraction. However, by analyzing the recovery yield of each single polyphenol, we found that water extraction under heating conditions is effective (extraction yield similar or even better in respect to the binary solvent) for some polyphenolic classes, such as hydrophilic procyanidins, phenolic acids, flavonol glucosides and stilbenoids. However, according to their lipophilic character, a poor yield was found for the most lipophilic components, such as flavonol aglycones, and in general for anthocyanins. The radical scavenging activity was in accordance with the polyphenol content, and hence, much higher for the extract obtained with the binary solvent in respect to water extraction. All the tested extracts were found to have an anti-inflammatory activity in the R3/1 cell line with NF-kb reporter challenged with 0.01 µg/mL of IL-1α, in a 1 to 250 µg/mL concentration range. An intriguing result was that the EtOH:H 2 O extract was found to be superimposable with that obtained using water at 100 °C despite the lower polyphenol content. Taken together, the results show the bioactive potentialities of grape skin extracts and the possibility to exploit this rich industrial waste. Water extraction carried out by heating is an easy, low-cost and environmentally friendly extraction method for some polyphenol classes and may have great potential for extracts with anti-inflammatory activities.

The skin is an important barrier against external attacks from bacteria, radicals, or radiations. UV-A radiations cause significant impairment of this barrier, inducing inflammation, oxidative stress, and wrinkle formation, thereby promoting photoaging. Previous studies reported that carnosine, a potent antioxidant, and carbonyl scavenger agent, may prevent photoaging features in the skin of hairless mice exposed to UV-A radiations. In the present study, we used a quantitative proteomic approach to analyze the changes evoked by carnosine in the skin proteome of hairless mice exposed to UV-A. This approach allowed to quantify more than 2480 proteins, among them consistent differences were observed for 89 proteins in UV-A exposed vs control unexposed skins, and 252 proteins in UV-A-exposed skin preventively treated by carnosine (UVAC) vs UV-A. Several functional pathways were altered in the skins of UV-A exposed hairless mice, including the integrin-linked kinase, calcium signaling, fibrogenesis, cell migration and filament formation. An impairment of mitochondrial function and metabolism was observed, with an up-regulation of cytochrome C oxidase 6B1 and NADH: ubiquinone oxidoreductase S8. Skins pre-treated by carnosine were prevented from UV-A induced proteome alterations. In conclusion, our study emphasizes the potency of a proteomic approach to identify the consequences of UV radiations in the skins, and points out the capacity of carnosine to prevent the alterations of skin proteome evoked by UV-A., (Copyright © 2021. Published by Elsevier Inc.)

Hyaluronic acid (HA) is a glycosaminoglycan very common in commercial products from pharmaceuticals to cosmetics due to its widespread distribution in humans and its diversified physico-chemical proprieties. Despite its extended use and preliminary evidence showing even also opposite activities to the native form, the precise cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear. The 'omics sciences currently in development offer a new and combined perspective on the cellular and organismal environment. This work aims to integrate lipidomics analyses to our previous quantitative proteomics one for a multi-omics vision of intra- and extra-cellular impact of different concentrations (0.125, 0.25, and 0.50%) of LMW-HA (20-50 kDa) on normal human dermal fibroblasts by LC-high resolution mass spectrometry (LC-HRMS). Untargeted lipidomics allowed us to identify 903 unique lipids mostly represented by triacylglycerols, ceramides, and phosphatidylcholines. According to proteomics analyses, LMW-HA 0.50% was the most effective concentration also in the lipidome rearrangement especially stimulating the synthesis of ceramides involved in skin hydration and reparation, cell signaling, and energy balance. Finally, integrative analyses showed 25 nodes covering several intra- and extra-cellular functions. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here pointed out will be useful to further exploit its features and improve current formulations even though further studies on lipids biosynthesis and degradation are necessary.

Candida spp. are pathobionts, as they can switch from commensals to pathogens, responsible for a variety of pathological processes. Adhesion to surfaces, morphological switch and biofilm-forming ability are the recognized virulence factors promoting yeast virulence. Sessile lifestyle also favors fungal persistence and antifungal tolerance. In this study, we investigated, in vitro, the efficacy of two urinary cranberry metabolites, 5-(3',4'-dihydroxy phenyl)-γ-valerolactone (VAL) and 4-hydroxybenzoic acid (4-HBA), in inhibiting C. albicans adhesion and biofilm formation. Both the reference strain SC5314 and clinical isolates were used. We evaluated biomass reduction, by confocal microscopy and crystal violet assay, and the possible mechanisms mediating their inhibitory effects. Both VAL and 4-HBA were able to interfere with the yeast adhesion, by modulating the expression of key genes, HWP1 and ALS3 . A significant dose-dependent reduction in biofilm biomass and metabolic activity was also recorded. Our data showed that the two cranberry metabolites VAL and 4-HBA could pave the way for drug development, for targeting the very early phases of biofilm formation and for preventing genitourinary Candida infections.

Xerostomia, the subjective complaint of dry mouth, is caused by therapeutic interventions or diseases. Nowadays, radiotherapy (RT) in patients with head and neck cancer (HNC) stands out as one of the most important causes of xerostomia. Currently available therapies for the treatment of xerostomia are still less than optimal and xerostomia still represents an unmet clinical need. In this article, we present the results of a prospective clinical study with a new product, Aqualief TM , in patients treated with curative RT with or without chemotherapy for HNC. Aqualief TM is based on two main ingredients, carnosine and karkadé, which have acid buffering and antioxidant properties. The study was performed on 30 patients, with 4 of the patients being lost during the study period. Each patient received randomly one of the two treatments, Aqualief TM or placebo, for 8 days. After a 10-day wash-out period, each patient received the other treatment for a further 8 days. The results show that Aqualief TM stimulated salivation in these patients and reduced the pH drop that was observed in an equivalent placebo-treated population of patients. Moreover, no serious, treatment-related adverse events were observed. Aqualief TM has shown positive results, although with limitations due to unsuccessful trial accrual. Therefore, it may be further investigated as a tool for the treatment of RT-related xerostomia.

Background: Diseases of the oral cavity (OC) with an infectious trigger such as caries and periodontal disease are extremely common in the general population and can also have effects at the cardiovascular level. The oral salivary flow, with its buffering capacity, is able to regulate the pH of the OC and, therefore, significantly contribute to the ecological balance of the microenvironment in which the oral microbiome (OM) develops. On the other side, when the quality/quantity of salivary flow is altered it is supposed the disruption of this balance with the potential increase in oral pathogens and triggered diseases. Among the endogenous substances able to exert a significant effect on the salivary flow and its characteristics, carnosine (Car), a dipeptide originally isolated in skeletal muscle, represents, thanks to the known buffering properties, a promising principle., Methods: We aimed this protocol to evaluate the quantitative/qualitative characteristics of the salivary flow in healthy volunteer subjects (n = 20) and in subjects suffering from common OC pathologies (n = 40), before and after 7 days of supplementation with SaliflussTM (Metis Healthcare srl, Milan, Italy), a Class I medical device on the market as 400 mg mucoadhesive oral tablets that has Car as the main ingredient., Discussion: Combining the characteristics of saliva with the OM and comparing them with OC pathologies, we expect to clarify their reciprocal relationship and, using quantitative proteomics techniques, to help clarify the mechanism of action of Car., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Nonalcoholic steatohepatitis (NASH) is a pathological manifestation with a progressive incidence in response to the epidemic of hepatic steatosis caused primarily by excessive energy intake. The present study unravels affected biological processes and functions by the presence of NASH in rats using a label-free quantitative proteomic strategy. NASH was induced by a Western high-sugar and high-fat diet for 20 weeks. The liver tissue was collected for histology and for a mass spectrometry-based proteomic protocol. The NASH group showed severe lipidosis, hepatocyte ballooning, and the presence of collagen deposition. Among upregulated proteins in NASH perilipin-2 (Plin-2; F6QBA3; difference [diff]: 2.29), ferritin heavy (Fth1; Q66HI5; diff: 2.19) and light (Ftl1; P02793; diff: 1.75) chains, macrophage migration inhibitory factor 1 (Mif; P30904; diff: 1.69), and fibronectin (Fn1; F1LST1; diff: 0.35) were observed, whereas among downregulated proteins, plectin (Q6S399; diff: -3.34), some Cyp2 family proteins of the cytochrome P450 complex, glutathione S-transferases, flavin-containing monooxygenase 1 (Fmo1; P36365; diff: -2.08), acetyl-CoA acetyltransferase 2 (Acat2; Q5XI22; diff: -2.25), acyl-CoA oxidase 2 (Acox2; F1LNW3; diff: -1.59), and acyl-CoA oxidase 3 (Acox3; F1M9A7; diff: -2.41) were observed. Also, biological processes and functions such as LPS/IL-1 inhibition of RXR, fatty acid metabolism, Nrf2-mediated oxidative stress response, xenobiotic metabolism, and PXR/RXR and CAR/RXR activations were predicted to be affected. In conclusion, the liver of rats with NASH induced by Western diet shows a decreased capacity of metabolizing lipids, fatty acids, and xenobiotic compounds that predispose fibrosis development., (© 2021 Wiley Periodicals LLC.)