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Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II–associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4+ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8+ and CD4+ responses were induced with up to 30% of CD3+CD8+ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.

Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π–π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design., AChBP is used as a structurally accessible prototype for studying ligand binding to nicotinic acetylcholine receptors. Stornaiuolo et al. report that a small molecule binds AChBP in an ordered p–p stack of three molecules per binding site, which may lead to new approaches in drug design.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

Several pharmaceutical companies are nowadays considering the use of agri-food waste as alternative raw material for the extraction of bioactive compounds to include in nutraceuticals and food supplements. This recycling activity is encountering the support of authorities, which are alarmed by air, soil and water pollution generated by agricultural waste disposal. Waste reuse has several economic advantages: (i) its low cost; (ii) its abundance; (iii) the high content of bioactive molecule (antioxidants, minerals, fibers, fatty acids); as well as (iv) the financial support received by governments eager to promote eco-compatible and pollution-reducing practices. While nutraceuticals produced from biowaste are becoming popular, products that have been risk-assessed in terms of safety are quite rare. This despite waste biomass, in virtue of its chemical complexity, could, in many cases, mine the overall safety of the final nutraceutical product. In this review, we summarize the scientific results published on genotoxicity risk-assessment of bioactive compounds extracted from agricultural waste. The review depicts a scenario where the risk-assessment of biowaste derived products is still scarcely diffuse, but when available, it confirms the safety of these products, and lets us envisage their future inclusion in the list of botanicals allowed for formulation intended for human consumption.

In this study, a strain of Anabaena flos-aquae UTEX 1444 was cultivated in six different concentrations of iron (III). Cultures were extracted with organic solvents and analyzed using our dereplication strategy, based on the combined use of high-resolution tandem mass spectrometry and molecular networking. The analysis showed the presence of the siderophores’ family, named synechobactins, only in the zero iron (III) treatment culture. Seven unknown synechobactin variants were present in the extract, and their structures have been determined by a careful HRMS/MS analysis. This study unveils the capability of Anabaena flos-aquae UTEX 1444 to produce a large array of siderophores and may be a suitable model organism for a sustainable scale-up exploitation of such bioactive molecules, for the bioremediation of contaminated ecosystems, as well as in drug discovery.

Thyroid hormones (THs) are key metabolic regulators coordinating short- and long-term energy needs. In skeletal muscle, THs modulate energy metabolism in pathophysiological conditions. Indeed, hypo- and hyperthyroidism are leading causes of muscle weakness and strength; however, the metabolic pathways underlying these effects are still poorly understood. Using molecular, biochemical, and isotope-tracing approaches combined with mass spectrometry and denervation experiments, we find that THs regulate glutamine metabolism and anaplerotic fluxes by up-regulating the glutamate pyruvate transaminase 2 (GPT2) gene. In humans, GPT2 autosomal recessive mutations cause a neurological syndrome characterized by intellectual disability, microcephaly, and progressive motor symptoms. Here, we demonstrate a role of the TH/GPT2 axis in skeletal muscle in which it regulates muscle weight and fiber diameter in resting and atrophic conditions and results in protection from muscle loss during atrophy. These results describe an anabolic route by which THs rewire glutamine metabolism toward the maintenance of muscle mass.

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

Cyanobacteria are ubiquitous photosynthetic microorganisms considered as important contributors to the formation of Earth’s atmosphere and to the process of nitrogen fixation. However, they are also frequently associated with toxic blooms, named cyanobacterial harmful algal blooms (cyanoHABs). This paper reports on an unusual out-of-season cyanoHAB and its dynamics during the COVID-19 pandemic, in Lake Avernus, South Italy. Fast detection strategy (FDS) was used to assess this phenomenon, through the integration of satellite imagery and biomolecular investigation of the environmental samples. Data obtained unveiled a widespread Microcystis sp. bloom in February 2020 (i.e., winter season in Italy), which completely disappeared at the end of the following COVID-19 lockdown, when almost all urban activities were suspended. Due to potential harmfulness of cyanoHABs, crude extracts from the “winter bloom” were evaluated for their cytotoxicity in two different human cell lines, namely normal dermal fibroblasts (NHDF) and breast adenocarcinoma cells (MCF-7). The chloroform extract was shown to exert the highest cytotoxic activity, which has been correlated to the presence of cyanotoxins, i.e., microcystins, micropeptins, anabaenopeptins, and aeruginopeptins, detected by molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.

Simple Summary Cancer cells rewire their metabolism to promote growth, survival, proliferation, and long-term maintenance. Aerobic glycolysis is a prominent trait of many cancers; contextually, glutamine addiction, enhanced glucose uptake and aerobic glycolysis sustain the metabolic needs of rapidly proliferating cancer cells. Thyroid hormone (TH) is a positive regulator of tumor progression and metastatic conversion of squamous cell carcinoma (SCC). Accordingly, overexpression of the TH activating enzyme, D2, is associated with metastatic SCC. The aim of our study was to assess the ability of TH and its activating enzyme in promoting key tracts of cancer progression such as angiogenesis, response to hypoxia and metabolic adaptation. By performing in vivo and in vitro studies, we demonstrate that TH induces VEGF-A in cancer cells and fosters aerobic glycolysis inducing pro-glycolytic mediators, thus implying that TH signal attenuation represents a therapeutic tool to contrast tumor angiogenesis and tumor progression. Abstract Cancer angiogenesis is required to support energetic demand and metabolic stress, particularly during conditions of hypoxia. Coupled to neo-vasculogenesis, cancer cells rewire metabolic programs to sustain growth, survival and long-term maintenance. Thyroid hormone (TH) signaling regulates growth and differentiation in a variety of cell types and tissues, thus modulating hyper proliferative processes such as cancer. Herein, we report that TH coordinates a global program of metabolic reprogramming and induces angiogenesis through up-regulation of the VEGF-A gene, which results in the enhanced proliferation of tumor endothelial cells. In vivo conditional depletion of the TH activating enzyme in a mouse model of cutaneous squamous cell carcinoma (SCC) reduces the concentration of TH in the tumoral cells and results in impaired VEGF-A production and attenuated angiogenesis. In addition, we found that TH induces the expression of the glycolytic genes and fosters lactate production, which are key traits of the Warburg effect. Taken together, our results reveal a TH–VEGF-A–HIF1α regulatory axis leading to enhanced angiogenesis and glycolytic flux, which may represent a target for SCC therapy.

Glucose and glutamine play a crucial role in the metabolic reprogramming of cancer cells. Proliferating cells metabolize glucose in the aerobic glycolysis for energy supply, and glucose and glutamine represent the primary sources of carbon atoms for the biosynthesis of nucleotides, amino acids, and lipids. Glutamine is also an important nitrogen donor for the production of nucleotides, amino acids, and nicotinamide. Several membrane receptors strictly control metabolic reprogramming in cancer cells and are considered new potential therapeutic targets. Formyl-peptide receptor 2 (FPR2) belongs to a small family of GPCRs and is implicated in many physiopathological processes. Its stimulation induces, among other things, NADPH oxidase-dependent ROS generation that, in turn, contributes to intracellular signaling. Previously, by phosphoproteomic analysis, we observed that numerous proteins involved in energetic metabolism are uniquely phosphorylated upon FPR2 stimulation. Herein, we investigated the role of FPR2 in cell metabolism, and we observed that the concentrations of several metabolites associated with the pentose phosphate pathway (PPP), tricarboxylic acid cycle, nucleotide synthesis, and glutamine metabolism, were significantly enhanced in FPR2-stimulated cells. In particular, we found that the binding of specific FPR2 agonists: (i) promotes NADPH production; (ii) activates the non-oxidative phase of PPP; (iii) induces the expression of the ASCT2 glutamine transporter; (iv) regulates oxidative phosphorylation; and (v) induces the de novo synthesis of pyrimidine nucleotides, which requires FPR2-dependent ROS generation.

Phytochemical analysis of the Iranian plant Achillea wilhelmsii led to the isolation of 17 pure secondary metabolites belonging to the classes of sesquiterpenoids and phenolics. Two of these compounds, named wilhemsin (7) and wilhelmsolide (9), are new sesquiterpenoids, and the first shows undescribed structural features. Their structures were elucidated through extensive spectroscopic analysis, mainly based on 1D and 2D NMR, and chemical derivatization. Starting from plant traditional use and previous reports on the activity of the plant extracts, all the pure compounds were evaluated on endpoints related to the treatment of metabolic syndrome. The sesquiterpene hanphyllin (8) showed a selective cholesterol-lowering activity (−12.7% at 30 µM), santoflavone (13) stimulated glucose uptake via the GLUT transporter (+16.2% at 30 µM), while the trimethoxylated flavone salvigenin (14) showed a dual activity in decreasing lipid levels (−22.5% palmitic acid biosynthesis at 30 µM) and stimulating mitochondrial functionality (+15.4% at 30 µM). This study further confirms that, in addition to the antioxidants vitexin, isovitexin, and isoschaftoside, A. wilhelmsii extracts contain molecules that can act at different levels on the metabolic syndrome symptoms.

Grapes and apples are the most cultivated fruits in the Mediterranean basin and their agricultural processing is responsible for the production of a large amount of bio-waste. The reuse of this food biomass would increase the volume of recyclable/renewable biomaterial and lower the environmental impact due to the increasing demand for these biological products. To this purpose, agri-food waste from grape and apple processing have become an important source of phytochemicals, and many pharmaceutical industries are using it as starting material to produce dietary supplements, functional foods, and food additives for human consumption. In virtue of the chemical diversity and complexity of agri-food biowaste, developers and producers of nutraceuticals are advised to assess the safety of their final nutraceutical products, in compliance with European Food Safety Authority regulation. Here, we use the Ames test to assess the mutagenicity of three nutraceuticals obtained from agri-food waste biomasses: Taurisolo&reg, from grape pomace of Vitis vinifera L. cv &lsquo, Aglianico&rsquo, AnnurComplex&reg, from Malus pumila M. cv &lsquo, Annurca&rsquo, and Limoncella Apple Extract from Malus domestica B. cv &lsquo, Limoncella&rsquo, The results showed that all three nutraceuticals were non-mutagenic.

The coronavirus infection is constantly diffusing worldwide and the incidence of death is dramatically increasing, representing one of the greatest disasters in human history. Nowadays, no effective therapeutic approaches have been licensed, despite the rising interest of the scientific research in this specific field, and the daily growing number of publications, while the need to find novel strategies is urgent. Evidence in the literature reported the antiviral activity of polyphenols, the largest class of bioactive compounds in nature. Interestingly, a limited number of studies investigated the efficacy of polyphenols from different raw materials, directly against coronaviruses. The present manuscript aimed to report this evidence and provide a viewpoint on the possibility to use it as a start point for the development of novel natural approaches against this viral infection, eventually designing further appropriate researches.

Type 2 deiodinase (D2), the principal activator of thyroid hormone (TH) signaling in target tissues, is expressed in cutaneous squamous cell carcinomas (SCCs) during late tumorigenesis, and its repression attenuates the invasiveness and metastatic spread of SCC. Although D2 plays multiple roles in cancer progression, nothing is known about the mechanisms regulating D2 in cancer. To address this issue, we investigated putative upstream regulators of D2 in keratinocyte carcinomas. We found that the expression of D2 in SCC cells is positively regulated by the NANOG transcription factor, whose expression, besides being causally linked to embryonic stemness, is associated with many human cancers. We also found that NANOG binds to the D2 promoter and enhances D2 transcription. Notably, blockage of D2 activity reduced NANOG-induced cell migration as well as the expression of key genes involved in epithelial&ndash, mesenchymal transition in SCC cells. In conclusion, our study reveals a link among endogenous endocrine regulators of cancer, thyroid hormone and its activating enzyme, and the NANOG regulator of cancer biology. These findings could provide the basis for the development of TH inhibitors as context-dependent anti-tumor agents.

A key aspect that must be supervised during the development of a biotherapeutic is the presence of elemental impurities in the final drug product: they must be quantified as to ensure that their concentrations does not affect patients’ safety. Regulatory guidelines such as ICH Q3D provides Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes (QA) of a biologic may also take place. In order to understand how certain impurities could affect not only the safety of patients, but also the physicochemical properties of biotherapeutics, here we present a study in which we examined how four commonly observed elemental impurities could impact the QAs of a Fc-fusion protein, under normal storage conditions and after six weeks of incubation at +25 °C and +40 °C. The molecule was indeed treated with increasing concentrations of Ni2+, Cu2+, Zn2+ and Fe3+ and the potential changes in conformation, oxidation, aggregation, and fragmentation were monitored. Our data suggest that keeping the levels of these impurities under the safety threshold limits does not guarantee the product quality. While nickel and zinc slightly altered the physicochemical properties of our Fc-fusion protein, iron and copper appeared to be more harmful for the QAs stability. Indeed, these latter elements might cause significant alterations of the product quality such as to potentially alter its efficacy.

Context Perfluoroalkyl-substances (PFAS) are chemical additives considered harmful for humans. We recently showed that accumulation of perfluoro-octanoic acid (PFOA) in human semen of exposed subjects was associated with altered motility parameters of sperm cells, suggesting direct toxicity. Objectives To determine whether direct exposure of human spermatozoa to PFOA was associated to impairment of cell function. Patients and methods Spermatozoa isolated from semen samples of ten normozoospermic healthy donors were exposed up to 2 h to PFOA, at concentrations from 0.1 to 10 ng/mL. Viability and motility parameters were evaluated by Sperm Class Analyser. Cell respiratory function was assessed by both mitochondrial probe JC-1 and respiratory control ratio (RCR) determination. Sperm accumulation of PFOA was quantified by liquid chromatography–mass spectrometry. Expres- sion of organic ion-transporters OATP1 and SLCO1B2 was assessed by immunofluorescence and respective role in PFOA accumulation was evaluated by either blockade with probenecid or membrane scavenging through β-cyclodextrin (β-CD). Plasma membrane fluidity and electrochemical potential (ΔΨp) were evaluated, respectively, with Merocyanine-540 and Di-3-ANEPPDHQ fluorescent probes. Results Compared to untreated controls, a threefold increase of the percentage of non-motile sperms was observed after 2 h of exposure to PFOA regardless of the concentration of PFOA, whilst RCR was significantly reduced. Only scavenging with β-CD was effective in reducing PFOA accumulation, suggesting membrane involvement. Altered membrane fluidity, reduced ΔΨp and sperm motility loss associated with exposure to PFOA were reverted by β-CD treatment. Conclusion PFOA alters human sperm motility through plasma-membrane disruption, an effect recovered by incubation with β-CD.

Background: Thyroid hormones (THs) are key regulators of development, tissue differentiation, and maintenance of metabolic balance in virtually every cell of the body. Accordingly, severe alteration of TH action during fetal life leads to permanent deficits in humans. The skin is among the few adult tissues expressing the oncofetal protein type 3 deiodinase (D3), the TH inactivating enzyme. Here, we demonstrate that D3 is dynamically regulated during epidermal ontogenesis. Methods: To investigate the function of D3 in a postdevelopmental context, we used a mouse model of conditional epidermal-specific D3 depletion. Loss of D3 resulted in tissue hypoplasia and enhanced epidermal differentiation in a cell-autonomous manner. Results: Accordingly, wound healing repair and hair follicle cycle were altered in the D3-depleted epidermis. Further, in vitro ablation of D3 in primary culture of keratinocytes indicated that various markers of stratified epithelial layers were upregulated, thereby confirming the pro-differentiative action of D3 depletion and the consequent increased intracellular triiodothyronine levels. Notably, loss of D3 reduced the clearance of systemic TH in vivo, thereby demonstrating the critical requirement for epidermal D3 in the maintenance of TH homeostasis. Conclusion: In conclusion, our results show that the D3 enzyme is a key TH-signaling component in the skin, thereby providing a striking example of a physiological context for deiodinase-mediated TH metabolism, as well as a rationale for therapeutic manipulation of deiodinases in pathophysiological contexts.

Hair disorders may considerably impact the social and psychological well-being of an individual. Recent advances in the understanding the biology of hair have encouraged the research and development of novel and safer natural hair growth agents. In this context, we have previously demonstrated&mdash, at both preclinical and clinical level&mdash, that an Annurca apple-based dietary supplement (AMS), acting as a nutraceutical, is endowed with an intense hair-inductive activity (trichogenicity), at once increasing hair tropism and keratin content. Herein, in the framework of preclinical investigations, new experiments in primary human models of follicular keratinocytes and dermal papilla cells have been performed to give an insight around AMS biological effects on specific hair keratins expression. As well as confirming the biocompatibility and the antioxidant proprieties of our nutraceutical formulation, we have proven an engagement of trichokeratins production underlying its biological effects on human follicular cells. Annurca apples are particularly rich in oligomeric procyanidins, natural polyphenols belonging to the broader class of bioflavonoids believed to exert many beneficial health effects. To our knowledge, none of the current available remedies for hair loss has hitherto shown to stimulate the production of hair keratins so clearly.

Taurisolo® is a pomace extract from Aglianico Grapes, a wine cultivar native to Campania (Southern Italy). It exhibits a very high polyphenolic content and, consumed as a nutraceutical, is effective in reducing the level of Trimethylamine N-oxide (TMAO), a cardiovascular disease risk factor marker. We here show the effects of Taurisolo® on rat brain microvascular alterations induced by a diminution in cerebral blood flow (CBFD) for 30 min, due to bilateral common carotid artery occlusion, and subsequent blood flow restoration (CBFR) for 60 min. The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed window implanted into the skull bone. The rat pial arterioles were classified according to Strahler’s ordering scheme, from smaller penetrating arterioles up to the larger ones. Western blotting analysis and mass spectrometry (MS)-based metabolomics were used to investigate the expression of endothelial nitric oxide synthase (eNOS) or the presence of peroxidized cardiolipin and several inflammatory mediators, respectively. Radical Oxygen Species (ROS) formation and neuronal loss were assessed. In rats CBFD and CBFR caused a decrease in arteriolar diameter, increase in fluorescent leakage and in adhesion of leukocytes to venular walls, reduction in the length of perfused capillaries and increment of ROS formation with large infarct size. Taurisolo®, intravenously or orally administered, induced pial arteriolar dilation (up to >30% of baseline), prevented fluorescent leakage, adhesion of leukocytes, ROS formation, while facilitated capillary perfusion and significantly reduced infarct size. These effects were accompanied by an increase in eNOS expression. Mass-spectrometry metabolomics analysis detected a marked decrease in the amount of peroxidized cardiolipin and pronounced reduction in pro-inflammatory prostaglandins and thromboxane Txb2. Altogether, these results extend the nutraceutical potential of Taurisolo® and suggest their eligibility for preventing brain damage due to ischemia-reperfusion injury.

A large body of evidence suggests that supplementation of butyric acid exerts beneficial intestinal and extra-intestinal effects. Unfortunately, unpleasant sensorial properties and unfavourable physico-chemical properties strongly limit its use in food supplements and foods for medicinal purposes. N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) is a new butyric acid releaser in solid form with neutral sensorial properties. The aim of this investigation is to provide preliminary information on its pharmacokinetic and toxicological properties through the study of a) in vivo bioavailability of FBA administered by oral gavage to male and female Swiss CD1 mice in comparison with sodium butyrate, b) the influence of digestion on FBA stability through an in vitro simulated oro-gastro-duodenal digestion process, and c) in vitro toxicological profile by means of the Ames Test and Micronucleus Test. The results reveal that FBA is a good butyric acid releaser, being able to increase butyrate serum concentration in a dose and time dependent manner in both male and female mice with a pharmacokinetic profile similar to that obtained from sodium butyrate as such. These data are confirmed by investigating the influence of digestion on FBA, which undergoes extensive hydrolysis following oro-gastro-duodenal digestion, especially in duodenal conditions, with a residual concentration of less than 10% of the initial FBA concentration. Finally, in the Ames and Micronucleus Tests, FBA does not show any in vitro genotoxicity as it is non mutagenic in the Ames Test and results to be unable to induce chromosome breaks in the Micronucleus Test. In conclusion, FBA is a new butyric acid releaser that can overcome the disadvantages of butyric acid while maintaining the same pharmacokinetic properties and safety profile, as shown by the results of the preliminary in vitro toxicological studies performed in this investigation.

Liver diseases affect millions of people worldwide. In most of the cases, severe hepatic dysfunction and liver cancer stem from mild and common clinical signs including hepatic steatosis, insulin resistance, liver inflammation, and oxidative stress, all together referred to as Nonalcoholic Fatty Liver Disease (NAFLD). Nutraceuticals endowed with antioxidant activity have been shown to reduce NAFLD risk factors and exert hepatoprotective effects. Here, we test the protective effect exerted on liver by the antioxidant Taurisolo, a nutraceutical formulation produced by grape pomace and enriched in Resveratrol and Polyphenols. We analyze the effect of Taurisolo on liver cells by profiling the metabolome of in vitro cultured hepatic HuH7 cells and of C57BL-6J mice fed a High Fat Diet and treated with the nutraceutical. Both in vitro and in vivo, we provide evidence that Taurisolo reduces risk factor markers associated with NAFLD. Taurisolo stimulates glucose uptake and reduces hepatic cholesterol and serum triglycerides. Furthermore, we give new insights into the mechanism of action of Taurisolo. The nutraceutical increases mitochondrial activity and promotes respiration and ATP production, fostering catabolic reactions like fatty acid &beta, oxidation and amino acid catabolism. On the contrary, Taurisolo reduces anabolic reactions like biosynthesis of cholesterol, bile acids, and plasma membrane lipids.

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters . ie1 and . p10 and the enhancer element . hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies. © 2012 Elsevier Inc..

Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. ‘Annurca’ and Malus domestica cv ‘Limoncella’; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.