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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.

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.

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.

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.

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.

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.