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Abstract Aim of the study Patients with metabolic syndrome (MetS) have an increased risk of cardiovascular disease. Data obtained from muscle biopsies have demonstrated altered insulin signaling (IS) in patients with MetS. The IS regulates critical cell functions including molecular-regulated cellular metabolite fluxes, protein and energetic metabolism, cell proliferation and apoptosis with consequent regulation of cell life including endothelial homeostasis and blood coagulation. However, little is known about blood cell IS in MetS patients. The aim of this study was to develop a method to evaluate IS in peripheral lymphocytes to identify altered intracellular molecules in patients with MetS to use as risk biomarkers of vascular thrombosis. Patients and Methods We investigated 40 patients with MetS and 20 controls. MetS was defined according to guidelines from the US National Cholesterol Education Program Adult Treatment Panel III. Blood samples were taken from all participants. Total mononuclear cells were isolated from peripheral blood using density gradient centrifugation. IS molecules were evaluated using Western blot analysis followed by computer-assisted densitometer evaluation. Results Lymphocytes of MetS patients showed a reduced mTOR expression (the mammalian target of rapamycin) which is a fundamental molecule of IS. Major impairment of IS was confirmed by reduced upstream and downstream mTOR molecules which regulate fundamental cells metabolic functions. Conclusions In patients with MetS, we found a reduction of mTOR and other mTOR-related molecules involved in insulin resistance, cell repair, coagulation and vasculogenesis. A reduced expression of mTOR may reflect an increased risk of vascular thrombosis.

Neuroinflammation is a hallmark of several neurodegenerative disorders that has been extensively studied in recent years. Microglia, the primary immune cells of the central nervous system (CNS), are key players in this physiological process, demonstrating a remarkable adaptability in responding to various stimuli in the eye and the brain. Within the complex network of neuroinflammatory signals, the fatty acid N-ethanolamines, in particular N-arachidonylethanolamine (anandamide, AEA), emerged as crucial regulators of microglial activity under both physiological and pathological states. In this study, we interrogated for the first time the impact of the signaling of these bioactive lipids on microglial cell responses to a sub-lethal acute UVB radiation, a physical stressor responsible of microglia reactivity in either the retina or the brain. To this end, we developed an in vitro model using mouse microglial BV-2 cells. Upon 24 h of UVB exposure, BV-2 cells showed elevated oxidative stress markers and, cyclooxygenase (COX-2) expression, enhanced phagocytic and chemotactic activities, along with an altered immune profiling. Notably, UVB exposure led to a selective increase in expression and activity of fatty acid amide hydrolase (FAAH), the main enzyme responsible for degradation of fatty acid ethanolamides. Pharmacological FAAH inhibition via URB597 counteracted the effects of UVB exposure, decreasing tumor necrosis factor α (TNF-α) and nitric oxide (NO) release and reverting reactive oxidative species (ROS), interleukin-1β (IL-1β), and interleukin-10 (IL-10) levels to the control levels. Our findings support the potential of enhanced fatty acid amide signaling in mitigating UVB-induced cellular damage, paving the way to further exploration of these lipids in light-induced immune responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fasciani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The identification of new therapeutic targets and the development of innovative therapeutic approaches are the most important challenges for osteosarcoma treatment. In fact, despite being relatively rare, recurrence and metastatic potential, particularly to the lungs, make osteosarcoma a deadly form of cancer. In fact, although current treatments, including surgery and chemotherapy, have improved survival rates, the disease's recurrence and metastasis are still unresolved complications. Insights for analyzing the still unclear molecular mechanisms of osteosarcoma development, and for finding new therapeutic targets, may arise from the study of post-translational protein modifications. Indeed, they can influence and alter protein structure, stability and function, and cellular interactions. Among all the post-translational modifications, ubiquitin-like modifications (ubiquitination, deubiquitination, SUMOylation, and NEDDylation), as well as glycosylation, are the most important for regulating protein stability, which is frequently altered in cancers including osteosarcoma. This review summarizes the relevance of ubiquitin-like modifications and glycosylation in osteosarcoma progression, providing an overview of protein stability regulation, as well as highlighting the molecular mediators of these processes in the context of osteosarcoma and their possible targeting for much-needed novel therapy., Competing Interests: The authors declare no conflicts of interest.

Ubiquitination is a post-translational modification that targets specific proteins on their lysine residues. Depending on the type of ubiquitination, this modification ultimately regulates the stability or degradation of the targeted proteins. Ubiquitination is mediated by three different classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes and, most importantly, the E3 ubiquitin ligases. E3 ligases are responsible for the final step of the ubiquitin cascade, interacting directly with the target proteins. E3 ligases can also be involved in DNA repair, cell cycle regulation and response to stress; alteration in their levels can be involved in oncogenic transformation and cancer progression. Of all the six hundred E3 ligases of the human genome, only three of them are specific to the mitochondrion: MARCH5, RNF185 and MUL1. Their alterations (that reflect on the alteration of the mitochondria functions) can be related to cancer progression, as underlined by the increasing research performed in recent years on these three mitochondrial enzymes. This review will focus on the function and mechanisms of the mitochondrial E3 ubiquitin ligases, as well as their important targets, in cancer development and progression, also highlighting their potential use for cancer therapy.

(1) Background: Obesity, a complex metabolic disease resulting from an imbalance between food consumption and energy expenditure, leads to an increase in adipocytes and chronic inflammatory conditions. The aim of this paper was to synthesize a small series of carvacrol derivatives ( CD1-3 ) that are able to reduce both adipogenesis and the inflammatory status often associated with the progression of the obesity disease. (2) Methods: The synthesis of CD1-3 was performed using classical procedures in a solution phase. Biological studies were performed on three cell lines: 3T3-L1, WJ-MSCs, and THP-1. The anti-adipogenic properties of CD1-3 were evaluated using western blotting and densitometric analysis by assessing the expression of obesity-related proteins, such as ChREBP. The anti-inflammatory effect was estimated by measuring the reduction in TNF-α expression in CD1-3 -treated THP-1 cells. (3) Results: CD1-3 -obtained through a direct linkage between the carboxylic moiety of anti-inflammatory drugs (Ibuprofen, Flurbiprofen, and Naproxen) and the hydroxyl group of carvacrol-have an inhibitory effect on the accumulation of lipids in both 3T3-L1 and WJ-MSCs cell cultures and an anti-inflammatory effect by reducing TNF- α levels in THP-1 cells. (4) Conclusions: Considering the physicochemical properties, stability, and biological data, the CD3 derivative-obtained by a direct linkage between carvacrol and naproxen-resulted in the best candidate, displaying anti-obesity and anti-inflammatory effects in vitro.

Opposing dose-dependent effects of curcumin (Cur) have been documented in Retinal Pigment Epithelium (RPE); therefore, to shed the light on the mechanisms of action is crucial for ophthalmic applications. On this basis we explored new insights about the dose-dependent mechanisms triggered by Cur in human retinal pigment epithelial cells (ARPE-19). Three concentrations (0.01 mM; 0.05 mM; 0.1 mM) of Cur were tested, followed by morphological, molecular, and functional analysis of the cells. Cur 0.01 mM promotes a significant increase in cell proliferation, not affecting cell cycle progression and apoptosis; by contrast, Cur 0.05 mM and 0.1 mM block cellular proliferation and trigger S-phase cell cycle arrest without inducing apoptosis. The observation of neuronal-like morphological changes in Cur 0.05 mM and 0.1 mM were not associated with neuronal differentiation, as observed by the quantification of Neurofilament-200 and by the analysis of voltage-dependent currents by patch clamp. Evaluation of autophagic markers LC3BII and p62 revealed significant modulations, suggesting an important activation of autophagy in ARPE-19 cells treated with Cur 0.05 mM and Cur 0.1 mM; conversely, Cur 0.01 mM did not affect autophagy. Altogether, our findings show new dose-dependent mechanisms of action of Cur that suggest a wide therapeutic application in ocular diseases with different pathogenesis (i.e., proliferative vitreoretinopathy or Age-Related Macular Degeneration).

The mammalian target of rapamycin (mTOR) signaling plays a critical role in cell homeostasis, growth and survival. Here, we investigated the localization of the main mTOR signaling proteins in the organ of Corti of normal-hearing and deafened guinea pigs, as well as their possible modulation by exogenously administered brain-derived neurotrophic factor (BDNF) in deafened guinea pigs. Animals were ototoxically deafened by systemic administration of kanamycin and furosemide, and one week later, the right cochleas were treated with gelatin sponge soaked in rhBDNF, while the left cochleas were used as negative controls. Twenty-four hours after treatment, animals were euthanized, and the cochleas were processed for subsequent analysis. Through immunofluorescence, we demonstrated the localization of AKT, pAKT, mTOR, pmTOR and PTEN proteins throughout the cochlea of guinea pigs for the first time, with a higher expression in supporting cells. Moreover, an increase in mTOR immunostaining was observed in BDNF-treated cochleas by means of fluorescence intensity compared to the other groups. Conversely, Western blot analysis showed no significant differences in the protein levels between groups, probably due to dilution of proteins in the neighboring tissues of the organ of Corti. Altogether, our data indicate that mTOR signaling proteins are expressed by the organ of Corti (with a major role for supporting cells) and that the modulation of mTOR may be a protective mechanism triggered by BDNF in the degenerating organ of Corti.

In this study, we investigated whether cerium oxide nanoparticles (CeO 2 -NPs), a promising antioxidant nanomaterial, may contrast retinal vascular alterations induced by oxidative damage in vitro and in vivo. For the in vivo experiments, the light damage (LD) animal model of Age-Related Macular Degeneration (AMD) was used and the CeO 2 -NPs were intravitreally injected. CeO 2 -NPs significantly decreased vascular endothelial growth factor (VEGF) protein levels, reduced neovascularization in the deep retinal plexus, and inhibited choroidal sprouting into the photoreceptor layer. The in vitro experiments were performed on human retinal pigment epithelial (ARPE-19) cells challenged with H 2 O 2 ; we demonstrated that CeO 2 -NPs reverted H 2 O 2 -induced oxidative stress-dependent effects on this cell model. We further investigated the RPE-endothelial cells interaction under oxidative stress conditions in the presence or absence of CeO 2 -NPs through two experimental paradigms: (i) treatment of human umbilical vein endothelial cells (HUVECs) with conditioned media from ARPE-19 cells, and (ii) coculture of ARPE-19 and HUVECs. In both experimental conditions, CeO 2 -NPs were able to revert the detrimental effect of H 2 O 2 on angiogenesis in vitro by realigning the level of tubule formation to that of the control. Altogether, our results indicate, for the first time, that CeO 2 -NPs can counteract retinal neovascularization and may be a new therapeutic strategy for the treatment of wet AMD.

Metabolic alterations have been observed in many cancer types. The deregulated metabolism has thus become an emerging hallmark of the disease, where the metabolism is frequently rewired to aerobic glycolysis. This has led to the concept of "metabolic reprogramming", which has therefore been extensively studied. Over the years, it has been characterized the enhancement of aerobic glycolysis, where key mutations in some of the enzymes of the TCA cycle, and the increased glucose uptake, are used by cancer cells to achieve a "metabolic phenotype" useful to gain a proliferation advantage. Many studies have highlighted in detail the signaling pathways and the molecular mechanisms responsible for the glycolytic switch. However, glycolysis is not the only metabolic process that cancer cells rely on. Oxidative Phosphorylation (OXPHOS), gluconeogenesis or the beta-oxidation of fatty acids (FAO) may be involved in the development and progression of several tumors. In some cases, these metabolisms are even more crucial than aerobic glycolysis for the tumor survival. This review will focus on the contribution of these alterations of metabolism to the development and survival of cancers. We will also analyze the molecular mechanisms by which the balance between these metabolic processes may be regulated, as well as some of the therapeutical approaches that can derive from their study., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Purpose: Recent studies have shown the presence of SARS-CoV-2 entry factors on the ocular surface, identifying the eye as an additional entry route for the virus. Moreover, the coexpression of angiotensin-converting enzyme 2 (ACE2) with other SARS-CoV-2 entry factors [transmembrane protease serine 2 (TMPRSS2), transmembrane protease serine 4 (TMPRSS4), and dipeptidyl peptidase-4 (DPP4)] facilitates the virus infection. Methods: Here, we performed a study over 10 adult corneal and limbal tissues from human donors, both male and female between 58 and 85 years of age. Some of the main virus entry factors were analyzed and their expression was quantified and correlated with the age and sex of the donors through western blot. The receptors' localization was investigated through immunofluorescence. Results: Immunofluorescence confirmed the localization of ACE2 and TMPRSS2 on the ocular surface and showed, for the first time, the localization of TMPRSS4 and DPP4 in limbal and corneal epithelial superficial cells. The quantitative analysis showed that the expression of SARS-CoV-2 entry factors on corneal and limbal cells is likely to be modulated in an age-dependent manner, in agreement with the increased susceptibility to COVID-19 in the elderly. Moreover, we found a relationship between the expression of TMPRSS proteases with the activation state of limbal cells in 80-year-old donors. Conclusion: This study provides information on the expression of SARS-CoV-2 entry factors on the ocular surface of 10 adult human donors and is a first observation of a possible age-dependent modulation on corneal and limbal tissues. Our data pave the way to further investigate the susceptibility to the infection through the ocular surface in the elderly.

Chronic heart failure (CHF) is a disease with important clinical and socio-economic ramifications. Malnutrition and severe alteration of the protein components of the body (protein disarrangements), common conditions in CHF patients, are independent correlates of heart dysfunction, disease progression, and mortality. Autophagy, a prominent occurrence in the heart of patients with advanced CHF, is a self-digestive process that prolongs myocardial cell lifespan by the removal of cytosolic components, such as aging organelles and proteins, and recycles the constituent elements for new protein synthesis. However, in specific conditions, excessive activation of autophagy can lead to the destruction of molecules and organelles essential to cell survival, ultimately leading to organ failure and patient death. In this review, we aim to describe the experimental and clinical evidence supporting a pathophysiological role of nutrition and autophagy in the progression of CHF. The understanding of the mechanisms underlying the interplay between nutrition and autophagy may have important clinical implications by providing molecular targets for innovative therapeutic strategies in CHF patients.