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Summary: Batrachochytrium salamandrivorans (Bsal) poses a major threat to amphibian, and more specifically caudata, diversity. Bsal is currently spreading through Europe, and mitigation measures aimed at stopping its spread and preventing its introduction into naïve environments are urgently needed. Screening for presence of Bsal and diagnosis of Bsal‐induced disease in amphibians are essential core components of effective mitigation plans. Therefore, the aim of this study was to present an overview of all Bsal diagnostic tools together with their limitations and to suggest guidelines to allow uniform interpretation. Here, we investigate the use of different diagnostic tools in post‐mortem detection of Bsal and whether competition between Bd and Bsal occurs in the species‐specific Bd and Bsal duplex real‐time PCR. We also investigate the diagnostic sensitivity, diagnostic specificity and reproducibility of the Bsal real‐time PCR and show the use of immunohistochemistry in diagnosis of Bsal‐induced chytridiomycosis in amphibian samples stored in formaldehyde. Additionally, we have drawn up guidelines for the use and interpretation of the different diagnostic tools for Bsal currently available, to facilitate standardization of execution and interpretation. [ABSTRACT FROM AUTHOR]

Tetrabromobisphenol A (2,2-bis(3,5-dibromo-4-hydroxyphenyl propane-TBBPA) is the most produced brominated flame retardant, detected in the environment and in biological samples. TBBPA shares structural similarities with thyroid hormones (THs), and it has been shown to interfere with different aspects of TH physiology, this raising concern on its possible effects as an endocrine disruptor in humans and wildlife. THs play a major role in lipid metabolism, with the liver representing one of their main target tissues. At the cellular level, THs act through interactions with TH receptors (TRs), as well as through TR-independent mechanisms. Rat hepatoma FaO cells (a liver cell line defective for functional TRs) overloaded with lipids have been utilized as a model to investigate the anti-steatotic effects of THs in the hepatocyte. In this work, the possible effects of TBBPA in steatotic FaO cells were investigated. Exposure to TBBPA for 24h reduced triglyceride (TAG) content and the size of lipid droplets (LDs); similar effects were obtained with equimolar doses (10−6M) of T3 (3,3′,5-1L 1-triiodothyronine). TBBPA and T3 showed common effects on transcription of genes involved in lipid homeostasis. In particular, TBBPA mainly up-regulated mRNA levels for LD-associated oxidative tissue-enriched PAT protein (OXPAT), peroxisome proliferator-activated receptor (PPAR) isoform β/δ, and the mitochondrial uncoupling protein 2 (UCP2). The results demonstrate that TBBPA can decrease lipid accumulation in steatotic cells through stimulation of oxidative pathways. These data identify novel thyromimetic actions of TBBPA at the cellular level. [ABSTRACT FROM AUTHOR]

Introduction: Coastal seawater pollution poses a public health risk due to the potential ingestion of contaminated water during recreational activities. Wastewater-based epidemiology has revealed the abundant presence of SARS-CoV-2 in seawater emitted from wastewater outlets. The objective of this research was to investigate the impact of seawater on SARS-CoV-2 infectivity to assess the safety of recreational activities in seawater., Methods: Wild SARS-CoV-2 was collected from oral swabs of COVID-19 affected patients and incubated for up to 90 min using the following solutions: (a) standard physiological solution (control), (b) reconstructed seawater (3.5% NaCl), and (c) authentic seawater (3.8%). Samples were then exposed to two different host systems: (a) Vero E6 cells expressing the ACE2 SARS-CoV-2 receptor and (b) 3D multi-tissue organoids reconstructing the human intestine. The presence of intracellular virus inside the host systems was determined using plaque assay, quantitative real-time PCR (qPCR), and transmission electron microscopy., Results: Ultrastructural examination of Vero E6 cells revealed the presence of virus particles at the cell surface and in replicative compartments inside cells treated with seawater and/or reconstituted water only for samples incubated up to 2 min. After a 90-min incubation, the presence of the virus and its infectivity in Vero E6 cells was reduced by 90%. Ultrastructural analysis performed in 3D epi-intestinal tissue did not reveal intact viral particles or infection signs, despite the presence of viral nucleic acid detected by qPCR. Indeed, viral genes (Orf1ab and N) were found in the intestinal luminal epithelium but not in the enteric capillaries. These findings suggest that the intestinal tissue is not a preferential entry site for SARS-CoV-2 in the human body. Additionally, the presence of hypertonic saline solution did not increase the susceptibility of the intestinal epithelium to virus penetration; rather, it neutralized its infectivity., Conclusion: Our results indicate that engaging in recreational activities in a seawater environment does not pose a significant risk for COVID-19 infection, despite the possible presence of viral nucleic acid deriving from degraded and fragmented viruses., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Norese, Nicosia, Cortese, Gentili, Rizzo, Rizzo, Grasselli, De Negri Atanasio, Gagliani, Tiso, Zinni, Pulliero and Izzotti.)

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Polyphenolic compounds constitute a diverse group of natural components commonly occurring in various plant species, known for their potential to exert both beneficial and detrimental effects. Additionally, these polyphenols have also been implicated as endocrine-disrupting (ED) chemicals, raising concerns about their widespread use in the cosmetics industry. In this comprehensive review, we focus on the body of literature pertaining to the estrogenic properties of ED chemicals, with a particular emphasis on the interaction of isoflavones with estrogen receptors. Within this review, we aim to elucidate the multifaceted roles and effects of polyphenols on the skin, exploring their potential benefits as well as their capacity to act as ED agents. By delving into this intricate subject matter, we intend to provoke thoughtful consideration, effectively opening a Pandora's box of questions for the reader to ponder. Ultimately, we invite the reader to contemplate whether polyphenols should be regarded as friends or foes in the realm of skincare and endocrine disruption., Competing Interests: Authors SP-d-R and ES-C were employed by the ProtoQSAR SL. Author MP-S was employed by the Moldrug AI Systems S.L. Authors EP, FR, and MZ-R were employed by the Angel Consulting S.a.s 20122 Milano. The funder had the following involvement with the study preparation of the manuscript. Authors SL and JM were employed by the MatTek In Vitro Life Science Laboratories. The remaining 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rispo, De Negri Atanasio, Demori, Costa, Marchese, Perera-del-Rosario, Serrano-Candelas, Palomino-Schätzlein, Perata, Robino, Ferrari, Ferrando, Letasiova, Markus, Zanotti-Russo and Grasselli.)

Considering the global increase in fish consumption, the growing side-streams coming from the fish supply chain (e.g., skin, fins, tail, heads…), also including undersized or "unwanted catches", have been recently proposed as source of high-value bioactive compounds (e.g., peptides and fatty acids). In this case study, hydrolyzed collagen peptides (HCPs) were extracted from different parts of Mugil cephalus L. using environmentally friendly techniques such as ultrasounds and enzymatic treatments. Both a mixed biomass derived from the skin, fins, and tail, and a whole fish, were considered as starting biomass, simulating the unsorted processing side-streams and an undersized/unwanted catch, respectively. The extracted HCPs were purified in fractions (<3 KDa and >3 KDa) whose yields (about 5% and 0.04-0.3%, respectively) demonstrated the efficiency of the hydrolysis process. The extraction protocol proposed allowed us to also isolate the intermediate products, namely the lipids (about 8-10%) and the non-collagenous proteins (NCs, 16-23%), whose exploitation could be considered. Each sample was characterized using Sircol, UltraViolet-Spectra, and hydroxyproline assay, and the viability of their collagen fractions was tested on human endothelial cells. Significant effects were obtained at a fraction of <3 KDa, in particular at a concentration of 0.13 µg/mL. The T-scratch test was also performed, with positive results in all fractions tested.

Crucial information on the pandemic's spread has been gathered by monitoring the trend of SARS-CoV-2 in wastewater. This surveillance has highlighted that the initial concentration is a critical step of the analytical procedure due to the low viral titer that may be present in this matrix. This paper presents the results of the evaluation of two different wastewater concentration protocols to determine the most efficient and cost-effective. The two methods tested were the following: (a) a biphasic separation system with PEG-dextran and (b) a PEG/NaCl precipitation protocol. Other aspects of the detection method were also investigated including the influence of storage temperature on virus recovery and the heat treatment of pasteurization, which aims to make samples safer for operators and the environment. The PEG/NaCl precipitation method was found to perform better than the biphasic separation system, allowing for more sensitive identification of the presence of the virus and the detection of a higher viral titer than that identified with the biphasic separation in all results. Storage of the samples at 4.3±0.2°C for up to 3 weeks did not adversely affect the virus titer and the pasteurization pre-treatment increases operator safety and maintains the identification of the viral concentration., (© 2023. The Author(s).)

This study presents for the first time a scalable process for the extraction of valuable proteins starting from samples of unsorted mixed tuna scraps which were previously dehydrated by an industrial patented process. The aims of this work were both to avoid the onerous sorting step of tuna leftovers, which generally consists of isolating skin and bones for collagen/gelatin extraction, and to improve the logistic of managing highly perishable biomass thanks to the reduction in its volume and to its microbiological stabilization. In view of a zero-waste economy, all the protein fractions (namely, non-collagenous proteins NCs and ALKs, gelatin, and hydrolyzed gelatin peptides, HGPs) isolated in the proposed single cascade flowchart were stabilized and preliminarily characterized. The extraction flowchart proposed allows one to obtain the following most promising compounds: 1.7 g of gelatin, 3.2 g of HGPs, and 14.6 g of NCs per 100 g of dehydrated starting material. A focus on oven-dried gelatin was reported in terms of proximate analysis, amino acid composition, color parameters, FT-IR spectrum, pH, and viscoelastic properties (5 mPa·s of viscosity and 14.3 °C of gelling temperature). All the obtained extracts are intended to be exploited in food supplements, feed, fertilizers/plant bio-stimulants, packaging, and the cosmetic industry.

The novel term metabolic dysfunction-associated fatty liver disease (MAFLD), which has been proposed to describe the major cause of hepatic disease, pinpoints the coexistence of multiple metabolic disturbances and liver steatosis, giving rise to different phenotypic manifestations. Within the psychoneuroendocrineimmunological (PNEI) network that regulates body-mind interactions, the stress response plays a pervasive role by affecting metabolic, hormonal, immune, and behavioral balance. In this perspective, we focus on chronic psychosocial stress and high levels of cortisol to highlight their role in MAFLD pathogenesis and worsening. From a PNEI perspective, considering the stress response as a therapeutic target in MAFLD allows for simultaneously influencing multiple pathways in the development of MAFLD, including dysmetabolism, inflammation, feeding behaviors, gut-liver axis, and dysbiosis, with the hope of better outcomes.

Many different amphibian skin peptides have been characterized and proven to exert various biological actions, such as wound-healing, immunomodulatory, anti-oxidant, anti-inflammatory and anti-diabetic effects. In this work, the possible anti-steatotic effect of macrotympanain A1 (MA1) (FLPGLECVW), a skin peptide isolated from the Chinese odorous frog Odorrana macrotympana , was investigated. We used a well-established in vitro model of hepatic steatosis, consisting of lipid-loaded rat hepatoma FaO cells. In this model, a 24 h treatment with 10 µg/mL MA1 exerted a significant anti-steatotic action, being able to reduce intracellular triglyceride content. Accordingly, the number and diameter of cytosolic lipid droplets (LDs) were reduced by peptide treatment. The expression of key genes of hepatic lipid metabolism, such as PPARs and PLINs, was measured by real-time qPCR. MA1 counteracted the fatty acid-induced upregulation of PPARγ expression and increased PLIN3 expression, suggesting a role in promoting lipophagy. The present data demonstrate for the first time a direct anti-steatotic effect of a peptide from amphibian skin secretion and pave the way to further studies on the use of amphibian peptides for beneficial actions against metabolic diseases.

Aluminum is an element found in nature and in cosmetic products. It can interfere with the metabolism of other cations, thus inducing gastrointestinal disorder. In cosmetics, aluminum is used in antiperspirants, lipsticks, and toothpastes. The aim of this work is to investigate aluminum bioavailability after accidental oral ingestion derived from the use of a toothpaste containing a greater amount of aluminum hydroxide than advised by the Scientific Committee on Consumer Safety (SCCS). To simulate in vitro toothpaste accidental ingestion, the INFOGEST model was employed, and the amount of aluminum was measured through the ICP-AES analysis. Tissue barrier integrity was analyzed by measuring transepithelial electric resistance, and the tissue architecture was checked through light microscopy. The margin of safety was also calculated. Overall, our results indicate that the acute exposure to aluminum accidentally ingested from toothpastes is safe for the final user, even in amounts higher than SCCS indications.

Introduction: Ischemia-Reperfusion (I/R) damage is one of the major challenges in cardiothoracic surgeries and in a pathological manner, is identified by exacerbated damage signals resulted from blood supply restriction and subsequent flow restoration and re‑oxygenation. I/R damage includes cellular dysfunction and death, impairing tissue and organ function. Inflammation and oxidative stress are known to underlie either ischemia or reperfusion, leaded by HIF, TNF-α, NF-κB, IL-6 and ROS formation. However, the available approaches to prevent I/R damage has been unsuccessful so far. As agonists of peroxisome-proliferation activation receptor (PPAR) are described as transcription factors related to anti-inflammatory factors, we proposed to observe the effects of novel dual agonist, GQ-11, in I/R-related damage., Methods: Male, Wistar rats, 60 days age and 305 g body weight average were treated with vehicle, pioglitazone or GQ-11 (20 mg/kg) for 7 consecutive days and were submitted to aorta clamping for 30 min followed by 3 h of reperfusion. 18 F-fluorodeoxyglucose ( 18 F-FDG), an analog of glucose associated with inflammation when accumulated, was observed in liver and bowel by positron emission tomography (PET)., Results: GQ-11 decreased 18 F-FDG uptake in liver and bowel when compared to vehicle and pioglitazone. The treatment also modulated inflammatory markers IL-10, TGF-β, IL-6, IL1-β, TNFα, and CCL-2, besides antioxidant enzymes such as catalase, GPx and SOD., Conclusion: Inflammation and oxidative stress showed to be important processes to be regulated in I/R in order to prevent exacerbated responses that leads to cell/tissue dysfunction and death. PPAR agonists - including GQ-11 - might be promising agents in a strategy to avoid tissue dysfunction and death after cardiothoracic surgeries., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nonalcoholic fatty liver disease is a metabolic disorder characterized by lipid overloading in hepatocytes that can progress pathogenically and even end in hepatocellular carcinoma. Nonalcoholic fatty liver disease pharmacological treatment is still limited by unwanted side effects, whereas the use of food components with therapeutic potential is advisable. The culinary use of marine algae is traditional for some populations and reviving worldwide, with promising health outcomes due to the large number of bioactive compounds found in seaweeds. The present review focuses on brown-algae polysaccharides, particularly fucoidan, alginate, and laminarin, and summarizes the experimental evidence of their potential effects against nonalcoholic fatty liver disease onset and progression. In vitro and in vivo studies demonstrate that brown-algae polysaccharides exert beneficial actions on satiety feeling, caloric intake, fat absorption, and modulation of the gut microbiota, which could account for indirect effects on energy and lipid homeostasis, thus diminishing the fat overload in the liver. Specific effects against nonalcoholic fatty liver disease pathogenesis and worsening are also described and sustained by the antioxidant, anti-inflammatory, and antisteatotic properties of brown-algae polysaccharides. Further studies are required to clarify the mechanism of action of brown-algae polysaccharides on liver cells, to determine the composition and bioavailability of brown-algae polysaccharides present in different algal sources and to probe the clinical availability of these compounds in the form of algal foods, food supplements, and regulated therapeutics., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

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.

Fucoidan is a fucose-rich sulfated polysaccharide typically found in the cell wall of marine algae but also recently isolated from terrestrial sources. Due to a variety of biological activities, including antioxidant properties, fucoidan exhibits an attractive therapeutic potential against a wide array of metabolic diseases associated with oxidative stress. We used FTIR, 1 H NMR and 13 C NMR spectroscopy to investigate the structural features of a fucoidan fraction extracted from the brown alga Cystoseira compressa (CYS). The antioxidant potential of CYS was measured by DPPH, ABTS and FRAP assays, which revealed a radical scavenging capacity that was confirmed in in vitro cellular models of hepatic and endothelial cells. The same antioxidant effects were observed for another fucoidan fraction previously identified in the terrestrial tree Eucalyptus globulus (EUC). Moreover, in hepatic cells, CYS and EUC exhibited a significant antisteatotic action, being able to reduce intracellular triglyceride content through the regulation of key genes of hepatic lipid metabolism. EUC exerted stronger antioxidant and antisteatotic effects as compared to CYS, suggesting that both marine and terrestrial sources should be considered for fucoidan extraction and therapeutic applications.

Bisphenol A (BPA) is a widespread environmental contaminant, found in human fluids and tissues. Maternal BPA exposure is associated with alterations in pregnancy outcomes. Because maternal uterine circulation plays a crucial role in normal placenta and fetal growth, we hypothesized that BPA compromises the function of uterine arteries (UAs) and fetoplacental development. Female rats were orally administered with BPA (2.5, 25 and 250 µg/kg/day) or with its vehicle (ethanol) for 30 days before pregnancy and during the first 20 days of pregnancy. To compare the effect of BPA in the reproductive vs. systemic circulation, it was tested on UAs and mesenteric arteries (MAs). Arteries were isolated and examined by pressure myography. Moreover, fetuses and placentas were weighed to provide an index of reproductive performance. In UAs of BPA-treated rats, lumen diameter, acetylcholine-relaxation and expressions of endothelial nitric oxide synthase 3 ( NOS3 ), estrogen receptor α ( ERα ) and peroxisome proliferator-activated receptor ɣ ( PPARɣ ) were reduced. Conversely, no changes were observed in MAs. BPA treatment also reduced placental weights, while fetal weights were increased. For the first time, our results indicate that UAs represent a specific target of BPA during pregnancy and provide insight into the molecular mechanisms that underlie its negative effects on pregnancy outcomes.

Background: The COVID-19 pandemic continues to ravage the human population; therefore, multiple prevention and intervention protocols are being rapidly developed. The aim of our study was to develop a new chemo-prophylactic/-therapeutic strategy that effectively prevents COVID-19 and related complications., Methods: In in vitro studies, COVID-19 infection-sensitive cells were incubated with human oropharyngeal fluids containing high SARS-CoV-2 loads. Levels of infection were determined via intra-cellular virus loads using quantitative PCR (qPCR). Efficacies for infection prevention were determined using several antiviral treatments: lipid-encapsulated ozonized oil (HOO), water-soluble HOO (HOOws), UV, and hydrogen peroxide. In in vivo studies, safety and efficacy of HOO in fighting COVID-19 infection was evaluated in human subjects., Results: HOO in combination with HOOws was the only treatment able to fully neutralize SARS-CoV-2 as well as its capacity to penetrate and reproduce inside sensitive cells. Accordingly, the feasibility of using HOO/HOOws was tested in vivo. Analysis of expired gas in healthy subjects indicates that HOO administration increases oxygen availability in the lung. For our human studies, HOO/HOOws was administered to 52 cancer patients and 21 healthy subjects at high risk for COVID-19 infection, and all of them showed clinical safety. None of them developed COVID-19 infection, although an incidence of at least 11 cases was expected. Efficacy of HOO/HOOws was tested in four COVID-19 patients obtaining recovery and qPCR negativization in less than 10 days., Conclusions: Based on our experience, the HOO/HOOws treatment can be administered at standard doses (three pills per day) for chemo-prophylactic purposes to healthy subjects for COVID-19 prevention and at high doses (up to eight pills per day) for therapeutic purposes to infected patients. This combined prevention strategy can provide a novel protocol to fight the COVID-19 pandemic.

Fucoidan is a fucose-rich sulfated polysaccharide with attractive therapeutic potential due to a variety of biological activities, including antioxidant action. Fucoidan is typically found in the cell wall of marine brown algae, but extra-algal sources have also been discovered. In the present work, for the first time we extracted a water soluble fucoidan fraction from the roots of the terrestrial shrub Ferula hermonis. This fucoidan fraction was termed FUFe, and contained fucose, glucose, sulfate, smaller amounts of monosaccharides such as galactose and mannose, and a minor quantity of proteins. FUFe structural features were investigated by FTIR, 1 H NMR and 13 C NMR spectroscopy. The antioxidant property of FUFe was measured by DPPH, ABTS and FRAP assays, which revealed a high radical scavenging capacity that was confirmed in in vitro cellular models. In hepatic and endothelial cells, 50 μg/mL FUFe could reduce ROS production induced by intracellular lipid accumulation. Moreover, in hepatic cells FUFe exhibited a significant antisteatotic action, being able to reduce intracellular triglyceride content and to regulate the expression of key genes of hepatic lipid metabolism. Altogether, our results candidate FUFe as a possible bioactive compound against fatty liver disease and related vascular damage.

The Ugi four-component reaction employing naturally occurred ferulic acid (FA) is proposed as a convenient method to synthesize feruloyl tertiary amides. Applying this strategy, a peptoid-like derivative of ferulic acid (FEF77) containing 2 additional hydroxy-substituted aryl groups, has been synthesized. The influence of the configuration of the double bond of ferulic acid and feruloyl amide on the antioxidant activity has been investigated thanks to light-mediated isomerization studies. At the cellular level, both FA, trans and cis isomers of FEF77 were able to protect human endothelial cord vein (HECV) cells from the oxidative damage induced by exposure to hydrogen peroxide, as measured by cell viability and ROS production assays. Moreover, in steatotic FaO rat hepatoma cells, an in vitro model resembling non-alcoholic fatty liver disease (NAFLD), the molecules exhibited a lipid-lowering effect, which, along with the antioxidant properties, points to consider feruloyl amides for further investigations in a therapeutic perspective.

Hepatic steatosis is the hallmark of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome and insulin resistance with potential evolution towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Key roles of autophagy and oxidative stress in hepatic lipid accumulation and NAFLD progression are recognized. Here, we employed a rat hepatoma cell model of NAFLD progression made of FaO cells exposed to oleate/palmitate followed or not by TNFα treatment to investigate the molecular mechanisms through which silybin, a lipid-lowering nutraceutical, may improve hepatic lipid dyshomeostasis. The beneficial effect of silybin was found to involve amelioration of the fatty acids profile of lipid droplets, stimulation of the mitochondrial oxidation and upregulation of a microRNA of pivotal relevance in hepatic fat metabolism, miR-122. Silybin was also found to restore the levels of Aquaporin-9 (AQP9) and glycerol permeability while reducing the activation of the oxidative stress-dependent transcription factor NF-κB, and autophagy turnover. In conclusion, silybin was shown to have molecular effects on signaling pathways that were previously unknown and potentially protect the hepatocyte. These actions intersect TG metabolism, fat-induced autophagy and AQP9-mediated glycerol transport in hepatocytes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Overconsumption of fats and sugars is a major cause of development of non‑alcoholic fatty liver disease (NAFLD). The main objectives of the present study were to explore the pathways sustaining the interfering metabolic effects of excess fructose and fatty acids in hepatocytes, and to clarify the mechanisms through which the nutraceutical silybin rescues the functional and metabolic alterations that are associated with the NALFD progression. Cultured hepatocytes were exposed to fructose and fatty acids, alone or in combination, to induce different grades of steatosis in vitro. Cell viability, apoptosis, free radical production, lipid content, lipid peroxidation and activity of lipogenic enzymes were assessed by spectrophotometric assays. Oxygen consumption and mitochondrial respiration parameters were measured using a Seahorse analyzer. Expression of markers for liver steatosis and dysfunction were also evaluated by reverse transcription‑quantitative polymerase chain reaction. The data revealed that fructose and fatty acid combination in vitro had a positive interference on lipogenic pathways, leading to more severe steatosis and liver dysfunction, reduced cell viability, increased apoptosis, oxidative stress and mitochondrial respiration. Hepatic cell abnormalities were almost completely alleviated by silybin treatment. These findings suggest that silybin may serve as a novel and cost‑effective dietary supplement for treatment and/or prevention of hepatosteatosis associated with NAFLD progression.

The chromodomain helicase DNA-binding 4 (CHD4), a member of the nucleosome remodeling and deacetylases (NuRD) complex, has been identified as an oncogene that modulates proliferation and migration of breast cancers (BC). ERBB2 is an oncogenic driver in 20-30% of BC in which its overexpression leads to increased chemoresistance. Here we investigated whether CHD4 depletion affects the ERBB2 cascade and autophagy, which represents a mechanism of resistance against Trastuzumab (Tz), a therapeutic anti-ERBB2 antibody. We show that CHD4 depletion in two ERBB2 + BC cell lines strongly inhibits cell proliferation, induces p27 KIP1 upregulation, Tyr 1248 ERBB2 phosphorylation, ERK1/2 and AKT dephosphorylation, and downregulation of both ERBB2 and PI3K levels. Moreover, CHD4 silencing impairs late stages of autophagy, resulting in increased levels of LC3 II and SQSTM1/p62, lysosomal enlargement and accumulation of autolysosomes (ALs). Importantly, we show that CHD4 depletion and concomitant treatment with Tz prevent cell proliferation in vitro Our results suggest that CHD4 plays a critical role in modulating cell proliferation, ERBB2 signaling cascade and autophagy and provide new insights on CHD4 as a potential target for the treatment of ERBB2 + BC., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Amphibian skin is not to be considered a mere tegument; it has a multitude of functions related to respiration, osmoregulation, and thermoregulation, thus allowing the individuals to survive and thrive in the terrestrial environment. Moreover, amphibian skin secretions are enriched with several peptides, which defend the skin from environmental and pathogenic insults and exert many other biological effects. In this work, the beneficial effects of amphibian skin peptides are reviewed, in particular their role in speeding up wound healing and in protection from oxidative stress and UV irradiation. A better understanding of why some species seem to resist several environmental insults can help to limit the ongoing amphibian decline through the development of appropriate strategies, particularly against pathologies such as viral and fungal infections., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this review.

Purpose: Phenolic compounds (PC) of virgin olive oil exert several biochemical and pharmacological beneficial effects. Some dietary PC seem to prevent/improve obesity and metabolic-related disorders such as non-alcoholic fatty liver disease (NAFLD). We investigated the possible effects of PC extracted from olive pomace (PEOP) and of the main single molecules present in the extract (tyrosol, apigenin, oleuropein, p-coumaric and caffeic acid) in protecting hepatocytes and endothelial cells against triglyceride accumulation and oxidative stress., Methods: Rat hepatoma and human endothelial cells were exposed to a mixture of oleate/palmitate to mimic the condition of NAFLD and atherosclerosis, respectively. Then, cells were incubated for 24 h in the absence or in the presence of PC or PEOP. Different parameters were evaluated, such as lipid accumulation and oxidative stress-related markers., Results: In hepatic cells, expression of peroxisome proliferator-activated receptors (PPARs) and of stearoyl-CoA desaturase 1 (SCD-1) were assessed as index of lipid metabolism. In endothelial cells, expression of intercellular adhesion molecule-1 (ICAM-1), activation of nuclear factor kappa-B (NF-kB), release of nitric oxide (NO), and wound-healing rate were assessed as index of inflammation., Conclusion: PEOP extract ameliorated hepatic lipid accumulation and lipid-dependent oxidative imbalance thus showing potential applications as therapeutic agent tuning down hepatosteatosis and atherosclerosis.

Batrachochytrium salamandrivorans (Bsal) poses a major threat to amphibian, and more specifically caudata, diversity. Bsal is currently spreading through Europe, and mitigation measures aimed at stopping its spread and preventing its introduction into naïve environments are urgently needed. Screening for presence of Bsal and diagnosis of Bsal-induced disease in amphibians are essential core components of effective mitigation plans. Therefore, the aim of this study was to present an overview of all Bsal diagnostic tools together with their limitations and to suggest guidelines to allow uniform interpretation. Here, we investigate the use of different diagnostic tools in post-mortem detection of Bsal and whether competition between Bd and Bsal occurs in the species-specific Bd and Bsal duplex real-time PCR. We also investigate the diagnostic sensitivity, diagnostic specificity and reproducibility of the Bsal real-time PCR and show the use of immunohistochemistry in diagnosis of Bsal-induced chytridiomycosis in amphibian samples stored in formaldehyde. Additionally, we have drawn up guidelines for the use and interpretation of the different diagnostic tools for Bsal currently available, to facilitate standardization of execution and interpretation., (© 2018 Blackwell Verlag GmbH.)

Background: ERBB2 is overexpressed in up to 20-30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2 + ) cells and whether CA might restore Tz sensitivity in Tz-resistant cells., Methods: We have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy., Results: We report that, in ERBB2 + BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21 WAF1 and CDKN1B/p27 KIP1 . Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line., Conclusions: Our data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2 + BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2 + cancers.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and mortality. Oxidative stress and release of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), are major consequences of hepatic lipid overload, which can contribute to progression of NAFLD to non-alcoholic steatohepatitis (NASH). Also, mitochondria are involved in the NAFLD pathogenesis for their role in hepatic lipid metabolism. Definitive treatments for NAFLD/NASH are lacking so far. Silybin, the extract of the milk thistle seeds, has previously shown beneficial effects in NAFLD. Sequential exposure of hepatocytes to high concentrations of fatty acids (FAs) and TNFα resulted in fat overload and oxidative stress, which mimic in vitro the progression of NAFLD from simple steatosis (SS) to steatohepatitis (SH). The exposure to 50 µM silybin for 24 h reduced fat accumulation in the model of NAFLD progression. The in vitro progression of NAFLD from SS to SH resulted in reduced hepatocyte viability, increased apoptosis and oxidative stress, reduction in lipid droplet size, and up-regulation of IκB kinase β-interacting protein and adipose triglyceride lipase expressions. The direct action of silybin on SS or SH cells and the underlying mechanisms were assessed. Beneficial action of silybin was sustained by changes in expression/activity of peroxisome proliferator-activated receptors and enzymes for FA oxidation. Moreover, silybin counteracted the FA-induced mitochondrial damage by acting on complementary pathways: (i) increased the mitochondrial size and improved the mitochondrial cristae organization; (ii) stimulated mitochondrial FA oxidation; (iii) reduced basal and maximal respiration and ATP production in SH cells; (iv) stimulated ATP production in SS cells; and (v) rescued the FA-induced apoptotic signals and oxidative stress in SH cells. We provide new insights about the direct protective effects of the nutraceutic silybin on hepatocytes mimicking in vitro NAFLD progression.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in industrialized countries and is associated with increased risk of cardiovascular, hepatic and metabolic diseases. Molecular mechanisms on the root of the disrupted lipid homeostasis in NAFLD and potential therapeutic strategies can benefit of in vivo and in vitro experimental models of fatty liver. Here, we describe the high fat diet (HFD)-fed rat in vivo model, and two in vitro models, the primary cultured rat fatty hepatocytes or the FaO rat hepatoma fatty cells, mimicking human NAFLD. Liver steatosis was invariably associated with increased number/size of lipid droplets (LDs) and modulation of expression of genes coding for key genes of lipid metabolism such as peroxisome proliferator-activated receptors (Ppars) and perilipins (Plins). In these models, we tested the anti-steatotic effects of 3,5-L-diiodothyronine (T2), a metabolite of thyroid hormones. T2 markedly reduced triglyceride content and LD size acting on mRNA expression of both Ppars and Plins. T2 also stimulated mitochondrial oxidative metabolism of fatty acids. We conclude that in vivo and especially in vitro models of NAFLD are valuable tools to screen a large number of compounds counteracting the deleterious effect of liver steatosis. Because of the high and negative impact of liver steatosis on human health, ongoing experimental studies from our group are unravelling the ultimate translational value of such cellular models of NAFLD.

In vertebrate systems, many endocrine disruptors (EDs) can also interfere with energy and lipid metabolism, thus acting as metabolic disruptors. At the cellular level, these effects are mainly mediated by interactions with nuclear receptors/transcription factors, leading to the modulation of genes involved in lipid homeostasis, as well as by rapid, receptor-independent pathways. Several potential metabolic disruptors are found in aquatic environments. In fish, different EDs have been shown to affect hepatic lipid homeostasis both in vivo and in vitro. However, little information is available in aquatic invertebrates due to our poor knowledge of the regulatory pathways of lipid metabolism. In this work, primary cell cultures from the digestive gland of the bivalve Mytilus galloprovincialis were utilized to investigate the effects of model EDs (bisphenol A (BPA) and perfluorooctane sulphonate (PFOS)) on lipid homeostasis. Both compounds (at 24 and 3h of exposure) increased intracellular lipid and tryglyceride-TAG content, with strongest effects of PFOS at 10 -7 M. Acyl-CoA oxidase activity was unaffected, whereas some changes in the activity of glycolytic, antioxidant/biotransformation enzymes were observed; however, no clear relationship was found with lipid accumulation. Evaluation of mitochondrial membrane potential Δψm and determination of extracellular TAG content indicate that PFOS interferes with mitochondrial function and lipid secretion, whereas BPA mainly affects lipid secretion. Experiments with specific inhibitors showed that activation of PI-3 kinase and extracellularly regulated mitogen-activated protein kinase (ERK MAPK) plays a key role in mediating lipid accumulation. Mussel digestive gland cells represent a simple in vitro model for screening the metabolic effects of EDs in marine invertebrates., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aim: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis., Methods: Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 μmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays., Results: Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-κB). Incubation of steatotic cells with silybin 50 μmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation., Conclusion: We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.

Quartz is a well-known occupational fibrogenic agent able to cause fibrosis and other severe pulmonary diseases such as silicosis and lung cancer. The silicotic pathology owes its severity to the structural and chemo-physical properties of the particles such as shape, size and abundance of surface radicals. In earlier studies, we reported that significant amounts of surface radicals can be generated on crystalline silica by chemical aggression with ascorbic acid (AA), a vitamin naturally abundant in the lung surfactant, and this reaction led to enhanced cytotoxicity and production of inflammatory mediators in a macrophage cell line. However in the lung, other cells acting in the development of silicosis, like fibroblasts and endothelial cells, can come to direct contact with inhaled quartz. We investigated the cytotoxic/pro-inflammatory effects of AA-treated quartz microcrystals (QA) in human primary fibroblasts and endothelial cells as compared to unmodified microcrystals (Q). Our results show that, in fibroblasts, the abundance of surface radicals on quartz microcrystals (Q vs QA) significantly enhanced cell proliferation (with or without co-culture with macrophages), reactive oxygen species (ROS) production, NF-κB nuclear translocation, smooth muscle actin, fibronectin, Bcl-2 and tissue inhibitor of metalloproteinase-1 expression and collagen production. Contrariwise, endothelial cells reacted to the presence of quartz microcrystals independently from the abundance of surface radicals showing similar levels of cytotoxicity, ROS production, cell migration, MCP-1, ICAM-I and fibronectin gene expression when challenged with Q or QA. In conclusion, our in vitro experimental model demonstrates an important and quite unexplored direct contribute of silica surface radicals to fibroblast proliferation and fibrogenic responses., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Excess ethanol consumption and fatty acid intake lead to a cumulative effect on liver steatosis through still unclear mechanisms. This study aimed to characterize the lipid homoeostasis alterations under the exposure of hepatocytes to ethanol alone or combined with fatty acids. FaO hepatoma cells were incubated in the absence (C) or in the presence of 100 mM ethanol (EtOH) or 0.35 mM oleate/palmitate (FFA) alone or in the combination (FFA/EtOH). Content of intra- and extra-cellular triglycerides (TAGs) and of lipid droplets (LDs), expression of lipogenic and lipolytic genes, and oxidative stress-related parameters were evaluated. Exposure to either FFAs or EtOH given separately led to steatosis which was augmented when they were combined. Our results show that FFA/EtOH: (i) increased the LD number, but reduced their size compared to separate treatments; (ii) up-regulated PPARγ and SREBP-1c and down-regulated sirtuin-1 (SIRT1); (iii) impaired FFA oxidation; (iv) did not change lipid secretion and oxidative stress. Our findings indicate that one of the major mechanisms of the metabolic interference between ethanol and fat excess is the impairment of FFA oxidation, in addition to lipogenic pathway stimulation. Interestingly, ethanol combined with FFAs led to a shift from macrovesicular to microvesicular steatosis that represents a more dangerous condition., (Copyright © 2016. Published by Elsevier Ltd.)

Objectives: Oxidative stress seems to be involved in Rett syndrome (RTT). The aim of this study was to assess the antioxidant status in RTT children with MECP2 gene mutations with respect to healthy controls, and to explore novel blood antioxidant markers for RTT severity., Methods: In erythrocytes from RTT females aged 2-14 years (n = 27) and age-matched controls (n = 27), we measured the levels of malonaldehyde and the activity of two antioxidant enzymes, Cu/Zn-superoxide dismutase and catalase, by spectrophotometric assays. In leukocytes, the expression of metallothioneins, the main non-enzymatic antioxidants, was assessed by real-time RT-PCR. In nine selected RTT children, methylome analysis was also performed., Results: Blood of RTT patients showed increased lipid peroxidation and a dysregulated pattern of MT expression, while enzymatic activities did not change significantly with respect to controls. Moreover, we observed no epigenetic dysregulation in CpG-enriched promoter regions of the analysed genes but significant hypomethylation in the random loci., Conclusions: As the haematic level of MT-1A directly correlates with the phenotype severity, this metallothionein can represent a marker for RTT severity. Moreover, the attempt to link the level of blood oxidative stress with MECP2 mutation and specific clinical features led us to draw some interesting conclusions.

Adipose tissue, dietary lipids and de novo lipogenesis are sources of hepatic free fatty acids (FFAs) that are stored in lipid droplets (LDs) as triacylglycerols (TAGs). Destiny of TAGs stored in LDs is determined by LD proteomic equipment. When adipose triglyceride lipase (ATGL) localizes at LD surface the lipid mobilization is stimulated. In this work, an in vitro model of cultured rat hepatocytes mimicking a mild steatosis condition was used to investigate the direct lipid-lowering action of iodothyronines, by focusing, in particular, on LD-associated proteins, FFA oxidation and lipid secretion. Our results demonstrate that in "steatotic" hepatocytes iodothyronines reduced the lipid excess through the recruitment of ATGL on LD surface, and the modulation of the LD-associated proteins Rab18 and TIP47. As an effect of ATGL recruitment, iodothyronines stimulated the lipid mobilization from LDs then followed by the up-regulation of carnitine-palmitoyl-transferase (CPT1) expression and the stimulation of cytochrome-c oxidase (COX) activity that seems to indicate a stimulation of mitochondrial function. The lipid lowering action of iodothyronines did not depend on increased TAG secretion. On the basis of our data, ATGL could be indicated as an early mediator of the lipid-lowering action of iodothyronines able to channel hydrolyzed FFAs toward mitochondrial beta-oxidation rather than secretion.