Subcellular distribution and Nrf2/Keap1-interacting properties of Glutathione S-transferase P in hepatocellular carcinoma.

The oncogene and drug metabolism enzyme glutathione S-transferase P (GSTP) is also a GSH-dependent chaperone of signal transduction and transcriptional proteins with key role in liver carcinogenesis. In this study, we explored this role of GSTP in hepatocellular carcinoma (HCC) investigating the possible interaction of this protein with one of its transcription factor and metronome of the cancer cell redox, namely the nuclear factor erythroid 2-related factor 2 (Nrf2). Expression, cellular distribution, and function as glutathionylation factor of GSTP1-1 isoform were investigated in the mouse model of N-nitrosodiethylamine (DEN)-induced HCC and in vitro in human HCC cell lines. The physical and functional interaction of GSTP protein with Nrf2 and Keap1 were investigated by immunoprecipitation and gene manipulation experiments. GSTP protein increased its liver expression, enzymatic activity and nuclear levels during DEN-induced tumor development in mice; protein glutathionylation (PSSG) was increased in the tumor masses. Higher levels and a preferential nuclear localization of GSTP protein were also observed in HepG2 and Huh-7 hepatocarcinoma cells compared to HepaRG non-cancerous cells, along with increased basal and Ebselen-stimulated levels of free GSH and PSSG. GSTP activity inhibition with the GSH analogue EZT induced apoptotic cell death in HCC cells. Hepatic Nrf2 and c-Jun, two transcription factors involved in GSTP expression and GSH biosynthesis, were induced in DEN-HCC compared to control animals; the Nrf2 inhibitory proteins Keap1 and ß-TrCP also increased and oligomeric forms of GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and ß-TrCP expression also increased in HCC cells, and GSTP transfection in HepaRG cells induced Nrf2 activation. In conclusion, GSTP expression and subcellular distribution are modified in HCC cells and apparently contribute to the GSH-dependent reprogramming of the cellular redox in this type of cancer directly influencing the transcriptional system Nrf2/Keap1.

Sex differences in neuromuscular and biological determinants of isometric maximal force.

AIM: Force expression is characterized by an interplay of biological and molecular determinants that are expected to differentiate males and females in terms of maximal performance. These include muscle characteristics (muscle size, fiber type, contractility), neuromuscular regulation (central and peripheral factors of force expression), and individual genetic factors (miRNAs and gene/protein expression). This research aims to comprehensively assess these physiological variables and their role as determinants of maximal force difference between sexes. METHODS: Experimental evaluations include neuromuscular components of isometric contraction, intrinsic muscle characteristics (proteins and fiber type), and some biomarkers associated with muscle function (circulating miRNAs and gut microbiome) in 12 young and healthy males and 12 females. RESULTS: Male strength superiority appears to stem primarily from muscle size while muscle fiber-type distribution plays a crucial role in contractile properties. Moderate-to-strong pooled correlations between these muscle parameters were established with specific circulating miRNAs, as well as muscle and plasma proteins. CONCLUSION: Muscle size is crucial in explaining the differences in maximal voluntary isometric force generation between males and females with similar fiber type distribution. Potential physiological mechanisms are seen from associations between maximal force, skeletal muscle contractile properties, and biological markers.

A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology.

Nerve growth factor (NGF) signalling affects spermatogenesis and mature sperm traits. In this paper, we aimed to evaluate the distribution and the role of NGF and its receptors (p75NTR and TrKA) on the reproductive apparatus (testis and epididymis) and sperm of fertile men (F) and men with different pathologies, namely varicocele (V) and urogenital infections (UGIs). We collected semen samples from 21 individuals (31-40 years old) subdivided as follows: V (n = 7), UGIs (n = 7), and F (n = 7). We submitted the semen samples to bacteriological analysis, leucocyte identification, and analysis of sperm parameters (concentration, motility, morphology, and viability). We determined the seminal plasma levels of NGF, interleukin 1ß (IL-1ß), and F2-isoprostanes (F2-IsoPs), and the gene and protein expression of NGF receptors on sperm. We also used immunofluorescence to examine NGF receptors on ejaculated sperm, testis, and epididymis. As expected, fertile men showed better sperm parameters as well as lower levels of NGF, F2-IsoPs, and IL-1ß compared with men with infertility. Notably, in normal sperm, p75NTR and TrKA were localised throughout the entire tail. TrKA was also found in the post-acrosomal sheath. This localisation appeared different in patients with infertility: in particular, there was a strong p75NTR signal in the midpiece and the cytoplasmic residue or coiled tails of altered ejaculated sperm. In line with these findings, NGF receptors were intensely expressed in the epididymis and interstitial tissue of the testis. These data suggest the distinctive involvement of NGF and its receptors in the physiology of sperm from fertile men and men with infertility, indicating a possible role for new targeted treatment strategies.

Randomised controlled trial combining vitamin E-functionalised chocolate with physical exercise to reduce the risk of protein-energy malnutrition in predementia aged people: study protocol for Choko-Age.

OBJECTIVE: Protein-energy malnutrition and the subsequent muscle wasting (sarcopenia) are common ageing complications. It is knowing to be also associated with dementia. Our programme will test the cytoprotective functions of vitamin E combined with the cortisol-lowering effect of chocolate polyphenols (PP), in combination with muscle anabolic effect of adequate dietary protein intake and physical exercise to prevent the age-dependent decline of muscle mass and its key underpinning mechanisms including mitochondrial function, and nutrient metabolism in muscle in the elderly. METHODS AND ANALYSIS: In 2020, a 6-month double-blind randomised controlled trial in 75 predementia older people was launched to prevent muscle mass loss, in respond to the 'Joint Programming Initiative A healthy diet for a healthy life'. In the run-in phase, participants will be stabilised on a protein-rich diet (0.9-1.0 g protein/kg ideal body weight/day) and physical exercise programme (high-intensity interval training specifically developed for these subjects). Subsequently, they will be randomised into three groups (1:1:1). The study arms will have a similar isocaloric diet and follow a similar physical exercise programme. Control group (n=25) will maintain the baseline diet; intervention groups will consume either 30 g/day of dark chocolate containing 500 mg total PP (corresponding to 60 mg epicatechin) and 100 mg vitamin E (as RRR-alpha-tocopherol) (n=25); or the high polyphenol chocolate without additional vitamin E (n=25). Muscle mass will be the primary endpoint. Other outcomes are neurocognitive status and previously identified biomolecular indices of frailty in predementia patients. Muscle biopsies will be collected to assess myocyte contraction and mitochondrial metabolism. Blood and plasma samples will be analysed for laboratory endpoints including nutrition metabolism and omics. ETHICS AND DISSEMINATION: All the ethical and regulatory approvals have been obtained by the ethical committees of the Azienda Ospedaliera Universitaria Integrata of Verona with respect to scientific content and compliance with applicable research and human subjects' regulation. Given the broader interest of the society toward undernutrition in the elderly, we identify four main target audiences for our research activity: national and local health systems, both internal and external to the project; targeted population (the elderly); general public; and academia. These activities include scientific workshops, public health awareness campaigns, project dedicated website and publication is scientific peer-review journals. TRIAL REGISTRATION NUMBER: NCT05343611.

New Challenges for Anatomists in the Era of Omics.

Anatomic studies have traditionally relied on macroscopic, microscopic, and histological techniques to investigate the structure of tissues and organs. Anatomic studies are essential in many fields, including medicine, biology, and veterinary science. Advances in technology, such as imaging techniques and molecular biology, continue to provide new insights into the anatomy of living organisms. Therefore, anatomy remains an active and important area in the scientific field. The consolidation in recent years of some omics technologies such as genomics, transcriptomics, proteomics, and metabolomics allows for a more complete and detailed understanding of the structure and function of cells, tissues, and organs. These have been joined more recently by "omics" such as radiomics, pathomics, and connectomics, supported by computer-assisted technologies such as neural networks, 3D bioprinting, and artificial intelligence. All these new tools, although some are still in the early stages of development, have the potential to strongly contribute to the macroscopic and microscopic characterization in medicine. For anatomists, it is time to hitch a ride and get on board omics technologies to sail to new frontiers and to explore novel scenarios in anatomy.

In Vitro and Ex Vivo Methodologies for T-Cell Trafficking Through Blood-Brain Barrier After TLR Activation.

This chapter describes ex vivo isolation of human T cells and of naïve splenocytes respectively collected from multiple sclerosis patients and healthy controls and experimental autoimmune encephalomyelitis-affected mice. After the magnetic sorting of naïve and activated T helper lymphocytes, we provide details about the cell cultures to measure the interaction with extracellular matrix proteins using standard cell invasion or hand-made in vitro assays, upon different stimuli, through Toll-like receptor(s) ligands, T-cell activators, and cell adhesion molecules modulators. Finally, we describe the methods to harvest and recover T cells to evaluate the properties associated with their trafficking ability.

The unknown and misunderstood life of Ruggero Oddi, the pioneer of biliary system physiology.

Ruggero Oddi was a talented scientist who initiated the modern era of biliary system physiology, not only with the anatomical discovery of the hepatopancreatic sphincter, but also with the detailed description of its spinal center and nerve regulation. However, his personal and scientific life were determined by an incredible series of unfortunate circumstances. Until now most of these events have been unknown, while the few known have produced biographies distorted by fake interpretations. The purpose of this article is to document Oddi's biography and scientific production in detail, comprehensively framing the scientific environment in which his discoveries occurred. It clears many misinterpretations about events in Oddi's life and academic career, bringing to a new light his figure as scientist in gastroenterological field.

Transcriptomics of natural and synthetic vitamin D in human hepatocyte lipotoxicity.

Vitamin D (VD) has been used to prevent nonalcoholic fatty liver disease (NAFLD), a condition of lipotoxicity associated with a defective metabolism and function of this vitamin. Different forms of VD are available and can be used for this scope, but their effects on liver cell lipotoxicity remain unexplored. In this study we compared a natural formulation rich in VD2 (Shiitake Mushroom extract or SM-VD2) with a synthetic formulation containing pure VD3 (SV-VD3) and the bioactive metabolite 1,25(OH)2-D3. These were investigated in chemoprevention mode in human HepaRG liver cells supplemented with oleic and palmitic acid to induce lipotoxicity. All the different forms of VD showed similar efficacy in reducing the levels of lipotoxicity and the changes that lipotoxicity induced on the cellular transcriptome. However, the three forms of VD generated different gene fingerprints suggesting diverse, even if functionally convergent, cytoprotective mechanisms. Main differences were (1) the number of differentially expressed genes (SV-VD3 > 1,25[OH]2-D3 > SM-VD2), (2) their identity that demonstrated significant gene homology between SM-VD2 and 1,25(OH)2-D3, and (3) the number and type of biological functions identified by ingenuity pathway analysis as relevant to liver metabolism and cytoprotection annotations. Immunoblot confirmed a different response of VDR and other VDR-related proteins to natural and synthetic VD formulations, including FXR, PXR, PPARγ/PGC-1α, and CYP3A4 and CYP24A1. In conclusion, different responses of the cellular transcriptome drive the cytoprotective effect of natural and synthetic formulations of VD in the free fatty acid-induced lipotoxicity of human hepatocytes.

Zinc restores functionality in porcine prepubertal Sertoli cells exposed to subtoxic cadmium concentration via regulating the Nrf2 signaling pathway.

Introduction: Among substances released into the environment by anthropogenic activities, the heavy metal cadmium (Cd) is known to induce severe testicular injury causing male subfertility/infertility. Zinc (Zn) is another heavy metal that, unlike Cd, is physiologically present in the testis, being essential for spermatogenesis. We aimed to examine the possibility that 50 µM ZnCl2 could counteract the toxic effects induced by Cd in an in vitro model of porcine prepubertal Sertoli cells (SCs) exposed to both subtoxic (5 µM) and toxic (10 µM) concentrations of CdCl2 for 48 h. Materials and Methods: Apoptosis, cell cycle, and cell functionality were assessed. The gene expression of Nrf2 and its downstream antioxidant enzymes, ERK1/2, and AKT kinase signaling pathways were evaluated. Materials and Results: We found that Zn, in co-treatment with subtoxic and toxic Cd concentration, increased the number of metabolically active SCs compared to Cd exposure alone but restored SC functionality only in co-treatment with subtoxic Cd concentration with respect to subtoxic Cd alone. Exposure of Cd disrupted cell cycle in SCs, and Zn co-treatment was not able to counteract this effect. Cd alone induced SC death through apoptosis and necrosis in a dose-dependent manner, and co-treatment with Zn increased the pro-apoptotic effect of Cd. Subtoxic and toxic Cd exposures activated the Nrf2 signaling pathway by increasing gene expression of Nrf2 and its downstream genes (SOD, HO-1, and GSHPx). Zn co-treatment with subtoxic Cd attenuated upregulation on the Nrf2 system, while with toxic Cd, the effect was more erratic. Studying ERK1/2 and AKT pathways as a target, we found that the phosphorylation ratio of p-ERK1/2 and p-AKT was upregulated by both subtoxic and toxic Cd exposure alone and in co-treatment with Zn. Discussion: Our results suggest that Zn could counteract Cd effects by increasing the number of metabolically active SCs, fully or partially restoring their functionality by modulating Nrf2, ERK1/2, and AKT pathways. Our SC model could be useful to study the effects of early Cd exposure on immature testis, evaluating the possible protective effects of Zn.

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients.

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles - liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85-99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1ß. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses. These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

Chronic Bedridden Condition Is Reflected by Substantial Changes in Plasma Inflammatory Profile.

Absent or reduced physical activity and spontaneous movement over days, weeks, or even years may lead to problems in almost every major organ/system in the human body. In this study, we investigated whether the dysregulation and alteration of plasma protein inflammatory profiling can stratify chronic bedridden conditions observed in 22 elderly chronic bedridden (CBR) individuals with respect to 11 age-matched active (OLD) controls. By using a combination of immune-assay multiplex techniques, a complex of 27 inflammatory mediators was assessed in the plasma collected from the two groups. A specific plasma protein signature is indeed able to distinguish IPO individuals from age-matched OLD controls; while significantly (p < 0.001) higher protein levels of IL-2, IL-7, and IL-12p70 were measured in the plasma of CBR with respect to OLD individuals, significantly (p < 0.01) higher levels of seven inflammatory mediators, including IL-9, PDGF-b, CCL4 (MIP-1b), CCL5 (RANTES), IL-1Ra, CXCL10 (IP10), and CCL2 (MCP-1), were identified in OLD individuals with respect to CBR individuals. These data suggest that the chronic absence of physical activity may contribute to the dysregulation of a complex molecular pattern occurring with ageing and that specific plasma protein signatures may represent potential biomarkers as well as new potential therapeutic targets for new treatments aimed at improving health expectancy.

Wheat germ oil vitamin E cytoprotective effect and its nutrigenomics signature in human hepatocyte lipotoxicity.

Wheat germ oil (WGO) is rich in α-tocopherol (vitamin E, VE), a vitamin that has long been suggested to exert hepatoprotective effects. In this study, this function of WGO-VE and its transcriptomics fingerprint were investigated in comparison with RRR-α-tocopherol and all-rac-α-tocopherol (nVE and sVE, respectively), in human liver cells treated with oleic acid (OA) to develop steatosis and lipotoxicity. Used in chemoprevention mode, all the VE formulations afforded significant reduction of the OA-induced steatosis and its consequent impact on lipotoxicity indicators, including ROS production and efflux (as H2O2), and apoptotic and necrotic cell death. A trend toward a better control of lipotoxicity was observed for WGO-VE and nVE compared to sVE. Gene microarray data demonstrated that these effects of VE formulations were associated with significantly different responses of the cellular transcriptome to compensate for the modifications of OA treatment, including the downregulation of cellular homeostasis genes and the induction of genes associated with defects of liver cell metabolism, fibrosis and inflammation, liver disease and cancer. Ingenuity Pathway Analysis data showed that WGO-VE modulated genes associated with liver carcinogenesis and steatosis, whereas nVE modulated genes involved in liver cell metabolism and viability biofunctions; sVE did not significantly modulate any gene dataset relevant to such biofunctions. In conclusion, WGO-VE prevents lipotoxicity in human liver cells modulating genes that differ from those affected by the natural or synthetic forms of pure VE. These differences can be captured by precision nutrition tools, reflecting the molecular complexity of this VE-rich extract and its potential in preventing specific cues of hepatocellular lipotoxicity.

Short-Term Effects of Side-Stream Smoke on Nerve Growth Factor and Its Receptors TrKA and p75NTR in a Group of Non-Smokers.

Environmental tobacco smoke remains a major risk factor, for both smokers and non-smokers, able to trigger the initiation and/or the progression of several human diseases. Although in recent times governments have acted with the aim of banning or strongly reducing its impact within public places and common spaces, environmental tobacco smoke remains a major pollutant in private places, such as the home environment or cars. Several inflammatory and long-term biomarkers have been analysed and well-described, but the list of mediators modulated during the early phases of inhalation of environmental tobacco smoke needs to be expanded. The aim of this study was to measure the short-term effects after exposure to side-stream smoke on Nerve Growth Factor and its receptors Tropomyosin-related kinase A and neurotrophin p75, molecules already described in health conditions and respiratory diseases. Twenty-one non-smokers were exposed to a home-standardized level of SS as well as to control smoke-free air. Nerve Growth Factor and inflammatory cytokines levels, as well the expression of Tropomyosin-related kinase A and neurotrophin receptor p75, were analysed in white blood cells. The present study demonstrates that during early phases, side-stream smoke exposure induced increases in the percentage of neurotrophin receptor p75-positive white blood cells, in their mean fluorescent intensity, and in gene expression. In addition, we found a positive correlation between the urine cotinine level and the percentage of neurotrophin receptor-positive white blood cells. For the first time, the evidence that short-term exposure to side-stream smoke is able to increase neurotrophin receptor p75 expression confirms the very early involvement of this receptor, not only among active smokers but also among non-smokers exposed to SS. Furthermore, the correlation between cotinine levels in urine and the increase in neurotrophin receptor p75-positive white blood cells could represent a potential novel molecule to be investigated for the detection of SS exposure at early time points.

Melatonin modulates Nrf2 activity to protect porcine pre-pubertal Sertoli cells from the abnormal H2 O2 generation and reductive stress effects of cadmium.

Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre-pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2 O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione-S-transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK-ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co-administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2 O2 levels, was observed both at 5 and 10 µM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd-induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK-ERK1/2 activity upregulation was associated with these effects at 5 µM Cd, whereas glutathione biosynthesis and efflux were involved at 10 µM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2 O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.

The Effect of Interaction NGF/p75NTR in Sperm Cells: A Rabbit Model.

BACKGROUND: Nerve Growth Factor (NGF) plays an important role in the reproductive system through its receptor's interaction (p75NTR). This paper aims to analyze the impact of NGF p75NTR in epididymal and ejaculated rabbit semen during in vitro sperm storage. METHODS: Semen samples from 10 adult rabbit bucks were collected four times (n = 40) and analyzed. NGF was quantified in seminal plasma, and the basal expression of p75NTR in sperm was established (time 0). Moreover, we evaluated p75NTR, the apoptotic rates, and the main sperm parameters, at times 2-4 and 6 h with or without the administration of exogenous NGF. RESULTS: Based on the level of p75NTR, we defined the threshold value (25.6%), and sperm were divided into High (H) and Normal (N). During sperm storage, p75NTR of H samples significantly modulated some relevant sperm parameters. Specifically, comparing H samples with N ones, we observed a reduction in motility and non-capacitated cell number, together with an increased percentage of dead and apoptotic cells. Notably, the N group showed a reduction in dead and apoptotic cells after NGF treatment. Conversely, the NGF administration on H sperm did not change either the percentage of dead cells or the apoptotic rate. CONCLUSION: The concentration of p75NTR on ejaculated sperm modulates many semen outcomes (motility, apoptosis, viability) through NGF interaction affecting the senescence of sperm.

Endoplasmic reticulum stress and NF-kB activation in SARS-CoV-2 infected cells and their response to antiviral therapy.

Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1α-mediated transcriptional regulation of stress response genes with anti-inflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.

Effect of a UV-C Automatic Last-Generation Mobile Robotic System on Multi-Drug Resistant Pathogens.

BACKGROUND: Healthcare-associated infections caused by multi-drug resistant (MDR) pathogens are associated with increased mortality and morbidity among hospitalized patients. Inanimate surfaces, and in particular high-touch surfaces, have often been described as the source for outbreaks of nosocomial infections. The present work aimed to evaluate the efficacy of a last-generation mobile (robotic) irradiation UV-C light device R2S on MDR microorganisms in inanimate surfaces and its translation to hospital disinfection. METHODS: The efficacy of R2S system was evaluated in environmental high-touch surfaces of two separate outpatient rooms of Perugia Hospital in Italy. The static UV-C irradiation effect was investigated on both the bacterial growth of S. aureus, MRSA, P. aeruginosa, and K. pneumoniae KPC and photoreactivation. The antimicrobial activity was also tested on different surfaces, including glass, steel, and plastic. RESULTS: In the environmental tests, the R2S system decreased the number of bacteria, molds, and yeasts of each high-touch spot surface (HTSs) compared with manual sanitization. UV-C light irradiation significantly inhibits in vitro bacterial growth, also preventing photoreactivation. UV-C light bactericidal activity on MDR microorganisms is affected by the type of materials of inanimate surfaces. CONCLUSIONS: The last-generation mobile R2S system is a more reliable sanitizing procedure compared with its manual counterpart.

SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione.

Viral infections sustain their replication cycle promoting a pro-oxidant environment in the host cell. In this context, specific alterations of the levels and homeostatic function of the tripeptide glutathione have been reported to play a causal role in the pro-oxidant and cytopathic effects (CPE) of the virus. In this study, these aspects were investigated for the first time in SARS-CoV2-infected Vero E6 cells, a reliable and well-characterized in vitro model of this infection. SARS-CoV2 markedly decreased the levels of cellular thiols, essentially lowering the reduced form of glutathione (GSH). Such an important defect occurred early in the CPE process (in the first 24 hpi). Thiol analysis in N-acetyl-Cys (NAC)-treated cells and membrane transporter expression data demonstrated that both a lowered uptake of the GSH biosynthesis precursor Cys and an increased efflux of cellular thiols, could play a role in this context. Increased levels of oxidized glutathione (GSSG) and protein glutathionylation were also observed along with upregulation of the ER stress marker PERK. The antiviral drugs Remdesivir (Rem) and Nelfinavir (Nel) influenced these changes at different levels, essentially confirming the importance or blocking viral replication to prevent GSH depletion in the host cell. Accordingly, Nel, the most potent antiviral in our in vitro study, produced a timely activation of Nrf2 transcription factor and a GSH enhancing response that synergized with NAC to restore GSH levels in the infected cells. Despite poor in vitro antiviral potency and GSH enhancing function, Rem treatment was found to prevent the SARS-CoV2-induced glutathionylation of cellular proteins. In conclusion, SARS-CoV2 infection impairs the metabolism of cellular glutathione. NAC and the antiviral Nel can prevent such defect in vitro.

SARS-CoV-2 Survival on Surfaces and the Effect of UV-C Light.

The aim of this study was to establish the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on inanimate surfaces such as plastic, stainless steel, and glass during UV-C irradiation which is a physical means commonly utilized in sanitization procedures. The viral inactivation rate, virus half-life, and percentage of titer reduction after UV-C irradiation were assessed. Infectivity was maintained on plastic and glass until 120 h and on stainless steel until 72 h. The virus half-life was 5.3, 4.4, and 4.2 h on plastic, stainless steel, and glass, respectively. In all cases, titer decay was >99% after drop drying. UV-C irradiation efficiently reduced virus titer (99.99%), with doses ranging from 10.25 to 23.71 mJ/cm2. Plastic and stainless steel needed higher doses to achieve target reduction. The total inactivation of SARS-CoV-2 on glass was obtained with the lower dose applied. SARS-CoV-2 survival can be long lasting on inanimate surfaces. It is worth recommending efficient disinfection protocols as a measure of prevention of viral spread. UV-C can provide rapid, efficient and sustainable sanitization procedures of different materials and surfaces. The dosages and mode of irradiation are important parameters to consider in their implementation as an important means to fight the SARS-CoV-2 pandemic.

Alpha-Tocopherol Metabolites (the Vitamin E Metabolome) and Their Interindividual Variability during Supplementation.

The metabolism of α-tocopherol (α-TOH, vitamin E) shows marked interindividual variability, which may influence the response to nutritional and therapeutic interventions with this vitamin. Recently, new metabolomics protocols have fostered the possibility to explore such variability for the different metabolites of α-TOH so far identified in human blood, i.e., the "vitamin E metabolome", some of which have been reported to promote important biological functions. Such advances prompt the definition of reference values and degree of interindividual variability for these metabolites at different levels of α-TOH intake. To this end, a one-week oral administration protocol with 800 U RRR-α-TOH/day was performed in 17 healthy volunteers, and α-TOH metabolites were measured in plasma before and at the end of the intervention utilizing a recently validated LC-MS/MS procedure; the expression of two target genes of α-TOH with possible a role in the metabolism and function of this vitamin, namely pregnane X receptor (PXR) and the isoform 4F2 of cytochrome P450 (CYP4F2) was assessed by immunoblot in peripheral blood leukocytes. The levels of enzymatic metabolites showed marked interindividual variability that characteristically increased upon supplementation. With the exception of α-CEHC (carboxy-ethyl-hydroxychroman) and the long-chain metabolites M1 and α-13'OH, such variability was found to interfere with the possibility to utilize them as sensitive indicators of α-TOH intake. On the contrary, the free radical-derived metabolite α-tocopheryl quinone significantly correlated with the post-supplementation levels of α-TOH. The supplementation stimulated PXR, but not CYP4F2, expression of leucocytes, and significant correlations were observed between the baseline levels of α-TOH and both the baseline and post-supplementation levels of PXR. These findings provide original analytical and molecular information regarding the human metabolism of α-TOH and its intrinsic variability, which is worth considering in future nutrigenomics and interventions studies.