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Astrocytes provide metabolic support for neurons and modulate their functions by releasing a plethora of neuroactive molecules diffusing to neighboring cells. Here we report that astrocytes also play a role in cortical neurons' vulnerability to Herpes simplex virus type-1 (HSV-1) infection through the release of extracellular ATP. We found that the interaction of HSV-1 with heparan sulfate proteoglycans expressed on the plasma membrane of astrocytes triggered phospholipase C-mediated IP3-dependent intracellular Ca(2+)transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca(2+)concentration that activates the Glycogen Synthase Kinase (GSK)-3 beta, whose action is necessary for HSV-1 entry/replication in these cells. Indeed, in co-cultures of neurons and astrocytes HSV-1-infected neurons were only found in proximity of infected astrocytes releasing ATP, whereas in the presence of fluorocitrate, an inhibitor of astrocyte metabolism, switching-off the HSV-1-induced ATP release, very few neurons were infected. The addition of exogenous ATP, mimicking that released by astrocytes after HSV-1 challenge, restored the ability of HSV-1 to infect neurons co-cultured with metabolically-inhibited astrocytes. The ATP-activated, P2R-mediated, and GSK-3-dependent molecular pathway underlying HSV-1 infection is likely shared by neurons and astrocytes, given that the blockade of either P2Rs or GSK-3 activation inhibited infection of both cell types. These results add a new layer of information to our understanding of the critical role played by astrocytes in regulating neuronal functions and their response to noxious stimuli including microbial agents via Ca2+-dependent release of neuroactive molecules.

Nearly 40–50% of infertility problems are estimated to be of female origin. Previous studies dedicated to the analysis of metabolites in follicular fluid (FF) produced contrasting results, although some valuable indexes capable to discriminate control groups (CTRL) from infertile females (IF) and correlate with outcome measures of assisted reproduction techniques were in some instances found. In this study, we analyzed in blind FF of 35 control subjects (CTRL = patients in which inability to obtain pregnancy was exclusively due to a male factor) and 145 IF (affected by: endometriosis, n = 19; polycystic ovary syndrome, n = 14; age-related reduced ovarian reserve, n = 58; reduced ovarian reserve, n = 29; unexplained infertility, n = 14; genetic infertility, n = 11) to determine concentrations of 55 water- and fat-soluble low molecular weight compounds (antioxidants, oxidative/nitrosative stress-related compounds, purines, pyrimidines, energy-related metabolites, and amino acids). Results evidenced that 27/55 of them had significantly different values in IF with respect to those measured in CTRL. The metabolic pattern of these potential biomarkers of infertility was cumulated (in both CTRL and IF) into a Biomarker Score index (incorporating the metabolic anomalies of FF), that fully discriminated CTRL (mean Biomarker Score value = 4.00 ± 2.30) from IF (mean Biomarker Score value = 14.88 ± 3.09, p < 0.001). The Biomarker Score values were significantly higher than those of CTRL in each of the six subgroups of IF. Posterior probability curves and ROC curve indicated that values of the Biomarker Score clustered CTRL and IF into two distinct groups, based on the individual FF metabolic profile. Furthermore, Biomarker Score values correlated with outcome measures of ovarian stimulation, in vitro fertilization, number and quality of blastocysts, clinical pregnancy, and healthy offspring. These results strongly suggest that the biochemical quality of FF deeply influences not only the effectiveness of IVF procedures but also the following embryonic development up to healthy newborns. The targeted metabolomic analysis of FF (using empowered Redox Energy Test) and the subsequent calculation of the Biomarker Score evidenced a set of 27 low molecular weight infertility biomarkers potentially useful in the laboratory managing of female infertility and to predict the success of assisted reproduction techniques.

Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM–MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM–MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their “real” biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Under physiological conditions, reactive oxygen species (ROS) play pivotal roles in various processes of human spermatozoa. Indeed, semen requires the intervention of ROS to accomplish different stages of its maturation. However, ROS overproduction is a well-documented phenomenon occurring in the semen of infertile males, potentially causing permanent oxidative damages to a vast number of biological molecules (proteins, nucleic acids, polyunsaturated fatty acids of biological membrane lipids), negatively affecting the functionality and vitality of spermatozoa. ROS overproduction may concomitantly occur to the excess generation of reactive nitrogen species (RNS), leading to oxidative/nitrosative stress and frequently encountered in various human pathologies. Under different conditions of male infertility, very frequently accompanied by morpho-functional anomalies in the sperm analysis, several studies have provided evidence for clear biochemical signs of damages to biomolecules caused by oxidative/nitrosative stress. In the last decades, various studies aimed to verify whether antioxidant-based therapies may be beneficial to treat male infertility have been carried out. This review analyzed the results of the studies published during the last ten years on the administration of low-molecular-weight antioxidants to treat male infertility in order to establish whether there is a sufficient number of data to justify antioxidant administration to infertile males. An analysis of the literature showed that only 30 clinical studies tested the effects of the administration of low-molecular-weight antioxidants (administered as a single antioxidant or as a combination of different antioxidants with the addition of vitamins and/or micronutrients) to infertile males. Of these studies, only 33.3% included pregnancy and/or live birth rates as an outcome measure to determine the effects of the therapy. Of these studies, only 4 were case–control studies, and only 2 of them found improvement of the pregnancy rate in the group of antioxidant-treated patients. Additionally, of the 30 studies considered in this review, only 43.3% were case–control studies, 66.7% enrolled a number of patients higher than 40, and 40% carried out the administration of a single antioxidant. Therefore, it appears that further studies are needed to clearly define the usefulness of antioxidant-based therapies to treat male infertility.

Traumatic brain injury (TBI) is the leading cause of death and disability in people less than 40 years of age in Western countries. Currently, there are no satisfying pharmacological treatments for TBI patients. In this study, we subjected rats to severe TBI (sTBI), testing the effects of a single subcutaneous administration, 30 min post-impact, of a new low molecular weight dextran sulfate, named ILB&reg, at three different dose levels (1, 5, and 15 mg/kg body weight). A group of control sham-operated animals and one of untreated sTBI rats were used for comparison (each group n = 12). On day 2 or 7 post-sTBI animals were sacrificed and the simultaneous HPLC analysis of energy metabolites, N-acetylaspartate (NAA), oxidized and reduced nicotinic coenzymes, water-soluble antioxidants, and biomarkers of oxidative/nitrosative stress was carried out on deproteinized cerebral homogenates. Compared to untreated sTBI rats, ILB&reg, improved energy metabolism by increasing ATP, ATP/ adenosine diphosphate ratio (ATP/ADP ratio), and triphosphate nucleosides, dose-dependently increased NAA concentrations, protected nicotinic coenzyme levels and their oxidized over reduced ratios, prevented depletion of ascorbate and reduced glutathione (GSH), and decreased oxidative (malondialdehyde formation) and nitrosative stress (nitrite + nitrate production). Although needing further experiments, these data provide the first evidence that a single post-injury injection of a new low molecular weight dextran sulfate (ILB&reg, ) has beneficial effects on sTBI metabolic damages. Due to the absence of adverse effects in humans, ILB&reg, represents a promising therapeutic agent for the treatment of sTBI patients.

Iron plays a major role in multiple processes involved in cell homeostasis such as metabolism, respiration and DNA synthesis. Cancer cells exhibit pronounced iron retention as compared to healthy counterpart. This phenomenon also occurs in multiple myeloma (MM), a hematological malignancy characterized by terminally differentiated plasma cells (PCs), in which serum ferritin levels have been reported as a negative prognostic marker. The aim of current study is to evaluate the potential role of iron metabolism in promoting drug resistance in myeloma cancer cells with particular regard to the interactions between PCs and tumor-associated macrophages (TAMs) as a source of iron. Our data showed that myeloma cell lines are able to intake and accumulate iron and thus, increasing their scavenger antioxidant-related genes and mitochondrial mass. We further demonstrated that PCs pre-treated with ferric ammonium citrate (FAC) decreased bortezomib (BTZ)-induced apoptosis in vitro and successfully engrafted in zebrafish larvae treated with BTZ. Treating human macrophages with FAC, we observed a switch toward a M2-like phenotype associated with an increased expression of anti-inflammatory markers such as ARG1, suggesting the establishment of an iron-mediated immune suppressive tumor microenvironment favouring myeloma growth. Using mfap4:tomato mutant zebrafish larvae, we further confirmed the increase of PCs-monocytes interactions after FAC treatment which favour BTZ-resistance. Taken together our data support the hypothesis that targeting iron trafficking in myeloma microenvironment may represent a promising strategy to counteract a tumor-supporting milieu and drug resistance., Graphical abstract Image 1, Highlights • Myeloma cell lines uptake iron and increase oxidative-redox related genes. • Iron increases mitochondrial biogenesis and fitness in myeloma cell lines. • Iron promotes bortezomib resistance in myeloma cell lines. • Zebrafish xenografts of myeloma cell lines exposed to iron exhibit bortezomib resistance. • Iron exposition promotes monocytes-plasma cells interactions and immunomodulation.

The proteasome inhibitor bortezomib (BTZ) has emerged as an effective drug for the treatment of multiple myeloma even though many patients relapse from BTZ therapy. The present study investigated the metabolic pathways underlying the acquisition of bortezomib resistance in multiple myeloma. We used two different clones of multiple myeloma cell lines exhibiting different sensitivities to BTZ (U266 and U266-R) and compared them in terms of metabolic profile, mitochondrial fitness and redox balance homeostasis capacity. Our results showed that the BTZ-resistant clone (U266-R) presented increased glycosylated UDP-derivatives when compared to BTZ-sensitive cells (U266), thus also suggesting higher activities of the hexosamine biosynthetic pathway (HBP), regulating not only protein O- and N-glycosylation but also mitochondrial functions. Notably, U266-R displayed increased mitochondrial biogenesis and mitochondrial dynamics associated with stronger antioxidant defenses. Furthermore, U266-R maintained a significantly higher concentration of substrates for protein glycosylation when compared to U266, particularly for UDP-GlcNac, thus further suggesting the importance of glycosylation in the BTZ pharmacological response. Moreover, BTZ-treated U266-R showed significantly higher ATP/ADP ratios and levels of ECP and also exhibited increased mitochondrial fitness and antioxidant response. In conclusions, our findings suggest that the HBP may play a major role in mitochondrial fitness, driving BTZ resistance in multiple myeloma and thus representing a possible target for new drug development for BTZ-resistant patients.

Uveal melanoma (UM) is the most common primary intraocular tumour in adults. The most accurate prognostic factor of UM is classification by gene expression profiling. Currently, the role of epigenetics is much less defined compared to genetic mechanisms. We recently showed a strong prognostic role of the expression levels of histone variant macroH2A1 in UM patients. Here, we assessed the mechanistic effects of macroH2A1 on UM progression. UM cell lines were stably knocked down (KD) for macroH2A1, and proliferation and colony formation capacity were evaluated. Mitochondrial function was assayed through qPCR and HPLC analyses. Correlation between mitochondrial gene expression and cancer aggressiveness was studied using a bioinformatics approach. MacroH2A1 loss significantly attenuated UM cells proliferation and aggressiveness. Furthermore, genes involved in oxidative phosphorylation displayed a decreased expression in KD cells. Consistently, macroH2A1 loss resulted also in a significant decrease of mitochondrial transcription factor A (TFAM) expression, suggesting impaired mitochondrial replication. Bioinformatics analyses uncovered that the expression of genes involved in mitochondrial metabolism correlates with macroH2A1 and with cancer aggressiveness in UM patients. Altogether, our results suggest that macroH2A1 controls UM cells progression and it may represent a molecular target to develop new pharmacological strategies for UM treatment.

Human behavior is influenced by both genetic and environmental factors. Monoamine oxidase A (MAOA) is among the most investigated genetic determinants of violent behaviors, while the monoamine oxidase B (MAOB) is explored in Parkinson's disease. We collected twenty-four post-mortem brain tissue datasets of 3871 and 1820 non-demented males and females, respectively, who died from causes not attributable to neurodegenerative diseases. The gene expressions of MAOA and MAOB (MAO genes) were analyzed in these subjects, who were further stratified according to age into eleven groups ranging from late Infancy (5-9 months) to centenarians (>100 years). MAO genes were differently expressed in brains during the entire life span. In particular, maximal and minimal expression levels were found in early life and around the teen years. Females tended to have higher MAO gene levels throughout their lives than those found in age-matched males, even when expressions were separately measured in different brain regions. We demonstrated the existence of age- and sex- related variations in the MAO transcript levels in defined brain regions. More in-depth protein studies are needed to confirm our preliminary results obtained only on messenger RNAs in order to establish the role played by MAO genes in human development.

Mitochondrial dynamics are regulated by a complex system of proteins representing the mitochondrial quality control (MQC). MQC balances antagonistic forces of fusion and fission determining mitochondrial and cell fates. In several neurological disorders, dysfunctional mitochondria show significant changes in gene and protein expression of the MQC and contribute to the pathophysiological mechanisms of cell damage. In this study, we evaluated the main gene and protein expression involved in the MQC in rats receiving traumatic brain injury (TBI) of different severities. At 6, 24, 48 and 120 hours after mild TBI (mTBI) or severe TBI (sTBI), gene and protein expressions of fusion and fission were measured in brain tissue homogenates. Compared to intact brain controls, results showed that genes and proteins inducing fusion or fission were upregulated and downregulated, respectively, in mTBI, but downregulated and upregulated, respectively, in sTBI. In particular, OPA1, regulating inner membrane dynamics, cristae remodelling, oxidative phosphorylation, was post-translationally cleaved generating differential amounts of long and short OPA1 in mTBI and sTBI. Corroborated by data referring to citrate synthase, these results confirm the transitory (mTBI) or permanent (sTBI) mitochondrial dysfunction, enhancing MQC importance to maintain cell functions and indicating in OPA1 an attractive potential therapeutic target for TBI.

Using a closed-head impact acceleration model of mild or severe traumatic brain injury (mTBI or sTBI, respectively) in rats, we evaluated the effects of graded head impacts on the gene and protein expressions of pyruvate dehydrogenase (PDH), as well as major enzymes of mitochondrial tricarboxylic acid cycle (TCA). TBI was induced in anaesthetized rats by dropping 450 g from 1 (mTBI) or 2 m height (sTBI). After 6 h, 12 h, 24 h, 48 h, and 120 h gene expressions of enzymes and subunits of PDH. PDH kinases and phosphatases (PDK1-4 and PDP1-2, respectively), citrate synthase (CS), isocitrate dehydrogenase (IDH), oxoglutarate dehydrogenase (OGDH), succinate dehydrogenase (SDH), succinyl-CoA synthase (SUCLG), and malate dehydrogenase (MDH) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). In the same samples, the high performance liquid chromatographic (HPLC) determination of acetyl-coenzyme A (acetyl-CoA) and free coenzyme A (CoA-SH) was performed. Sham-operated animals (n = 6) were used as controls. After mTBI, the results indicated a general transient decrease, followed by significant increases, in PDH and TCA gene expressions. Conversely, permanent PDH and TCA downregulation occurred following sTBI. The inhibitory conditions of PDH (caused by PDP1-2 downregulations and PDK1-4 overexpression) and SDH appeared to operate only after sTBI. This produced almost no change in acetyl-CoA and free CoA-SH following mTBI and a remarkable depletion of both compounds after sTBI. These results again demonstrated temporary or steady mitochondrial malfunctioning, causing minimal or profound modifications to energy-related metabolites, following mTBI or sTBI, respectively. Additionally, PDH and SDH appeared to be highly sensitive to traumatic insults and are deeply involved in mitochondrial-related energy metabolism imbalance.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-&gamma, ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-&gamma, (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism, (2) the amelioration of the macrophage energy state, (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde, (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat), (5) the increase of the transforming growth factor-&beta, 1 (TGF-&beta, 1) and the down-regulation of the expressions of interleukins 1&beta, and 6 (IL-1&beta, and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM-MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM-MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

Docosahexaenoic acid (DHA) is an omega‑3 polyunsaturated fatty acid, derived mainly from fish oil. It is well known that DHA is present in high concentrations in nervous tissue and plays an important role in brain development and neuroprotection. However, the molecular mechanisms underlying its role remain to be fully elucidated. In this study, to enhance our understanding of the pathophysiological role of DHA, we investigated the possible neuroprotective mechanisms of action of DHA against hydrogen peroxide (H2O2)‑induced oxidative damage in a rat pheochromocytoma cell line (PC12). Specifically, we evaluated the viability, oxidation potential, and the expression and production of antioxidant/cytoprotective enzymes, and eventual apoptosis. We found that pre‑treatment with DHA (24 h) protected the cells from H2O2‑induced oxidative damage. In particular, pre‑treatment with DHA: i) Antagonized the consistent decrease in viability observed following exposure to H2O2 for 24 h; ii) reduced the high levels of intracellular reactive oxygen species (ROS) associated with H2O2‑induced oxidative stress; iii) increased the intracellular levels of enzymatic antioxidants [superoxide dismutase (SOD) and glutathione peroxidase (GSH‑Px)] both under basal conditions and following H2O2 exposure; iv) augmented the intracellular levels of reduced glutathione (GSH) and ascorbic acid, while it reduced the malondialdehyde (MDA) levels under conditions of oxidative stress; v) upregulated the expression of nuclear factor (erythroid‑derived 2)‑like 2 (NFE2L2) and its downstream target protein, heme‑oxygenase‑1 (HO‑1); and vi) induced an anti‑apoptotic effect by decreasing Bax and increasing Bcl2 expression. These findings provide evidence suggesting that DHA is able to prevent H2O2‑induced oxidative damage to PC12 cells, which is attributed to its antioxidant and anti‑apoptotic effects via the regulation NFE2L2/HO‑1 signaling. Therefore, DHA may play protective role in neurodegenerative diseases associated with oxidative stress.

Reactive oxygen species (ROS) are physiologically involved in functions like sperm maturation, capacitation and acrosome reaction, but their excess is involved in male infertility. Antioxidants in seminal plasma (SP) are an important factor balancing physiologic and harmful ROS activities. In this study, we determined and compared the full profiles of the water- and fat-soluble antioxidants in SP and serum of 15 healthy fertile subjects (ranging between the ages of 35 and 42 years). Ejaculates were obtained after 2–5 days of sexual abstinence. After liquefaction and withdrawal of an aliquot for the sperm count, samples were centrifuged to obtain SP. Thirty min after semen donation, a venous blood sample was collected from each subject. Donors with lower SP concentrations of ascorbic acid (n = 5) or α-tocopherol (n = 5) received a 4 week oral administration of either vitamin C (100 mg/day) or vitamin E (30 mg/day). They were then re-assayed to determine the SP and serum levels of ascorbic acid and α-tocopherol. SP and serum samples were properly processed and analyzed by HPLC methods suitable to determine water (ascorbic acid, glutathione (GSH) and uric acid) and fat-soluble (all-trans-retinoic acid, all-trans-retinol, α-tocopherol, carotenoids and coenzyme Q10) antioxidants. Data demonstrate that only ascorbic acid is higher in SP than in serum (SP/serum ratio = 4.97 ± 0.88). The other water-soluble antioxidants are equally distributed in the two fluids (GSH SP/serum ratio = 1.14 ± 0.34, uric acid SP/serum ratio = 0.82 ± 0.12). All fat-soluble antioxidants are about 10 times less concentrated in SP than in serum. In donors treated with vitamin C or vitamin E, ascorbic acid and α-tocopherol significantly increased in both fluids. However, the SP/serum ratio of ascorbic acid was 4.15 ± 0.45 before and 3.27 ± 0.39 after treatment, whilst those of α-tocopherol were 0.11 ± 0.03 before and 0.10 ± 0.02 after treatment. The results of this study, by showing the peculiar composition in water- and fat-soluble antioxidants SP, indicate that it is likely that still-unknown mechanisms allow ascorbic acid accumulation in SP against a concentration gradient. SP mainly relies its defenses on water- rather than fat-soluble antioxidants and on the mechanisms ensuring their transfer from serum.

Carnosine is an endogenous dipeptide composed of &beta, alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2&minus, &bull, ) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-&alpha, and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-&beta, 1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions.

Introduction Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by hematopoietic/stem cell-derived clonal myeloproliferation leading to cytopenia/cytosis, splenomegaly and bone marrow (BM) fibrosis. The alteration of haematopoiesis associted with BM fibrosis is deeply associated with profound modifications of the BM microenvironment, as a consequence of a defective balance between the vascular niche and the endosteal niche, associated to megakaryocytes, endothelial and mesenchymal stromal cells (MSC) dysfunction through the production of a variety of profibrotic, angiogenic, and pro-inflammatory cytokines, including megakaryocyte-derived PDGF, TGF-beta and osteoprotegerin, IL-6, PDGF, RANTSs, BMP-2, and which can trigger auto-immune mechanisms, chronic inflammation and oxidative stress status. It is well-known that oncogenic lesions can switch the bioenergetics of malignant cells from OXPHOS to glycolysis (Warburg effect) with lactate production, a phenomenon clinically relevant, particularly in PMF where the amount of the enzyme lactate dehydrogenase (LDH) is an established biomarker of leucocyte turnover and an independent biomarker of overall and leukemia-free survival. The shuttling of the main LDH metabolite lactate is implicated in the interplay of cancer cells with neighboring stromal cells which become glycolytic and export lactate. In turn, lactate is taken up by cancer cells and used for oxidative metabolism, to drive angiogenesis in endothelial cells and to inhibit T- cell function. Moreover, there are evidences that lactate may be involved in the promotion of the fibrosis increasing the TGF-beta levels. In this work we aimed to investigate the role of lactate in the BM myelofibrosis. Results. From microarray datasets, we selected 34 PMF patients carrying the JAK2V617F mutation, 28 JAK2 wild-type patients and 16 healthy donors (HD). Our analysis showed that SLC16A1 (MCT1), SLC16A3(MCT2) and SLC16A7 (MCT4) genes were upregulated in patients in respect to healthy donors in a JAK2V617F mutation independent manner. Furthermore, we demonstrated a significant increase of lactate concentration in PB sera from PMF patients compared to HD, associated to higher percentage of circulating granulocyte- and monocyte-myeloid derived suppressor cells (Gr- and Mo-MDSCs), and Treg. Moreover, IDO, LAG3, BTLA, PDL-2, TIM-3 and CD152 levels were significantly increased in PMF sera compaterd to HD ones. To demonstrate that lactate could play a role in driving cancer immune evasion in PMF, healthy peripheral blood mononucleated cells (PBMCs) were incubated in presence of lactate. After 3 days we observed a significant increase of the percentage of Mo-MDSCs, Treg, CD4+PD1+ and CD8+PD1+ lymphocytes. The same results were obtained after incubation of PBMCs with sera from PMF patients. No effects were observed using HD sera. Interestingly, the percentages of Treg and Mo-MDSCs increased after exposure to PMF sera were significantly reduced in presence of the inhibitor of lactate transporter AZD3965. To investigate the role of lactate in the PMF microenvironment, we next exposed healthy MSCs to lactate for 48h. Treated MSCs assumed a CAF-like phenotype increasing expression of aSMA, FAP1 and TGF-beta. Moreover, Masson's trichrome staining showed an increase of collagen deposits in BM-MSCs associated to increased release of IL6, TGF-beta, MMP2, MMP9 and RANTES. Also, exposure to PMF sera induced higher collagen deposits in MSCs and this effect was reverted adding AZD3965. As BM fibrosis is frequently accompanied by osteosclerosis, we also investigated the effects of lactate on ostegenic differentiation of BM-MSCs. After 10 days of treatment with lactate, the BM-MSCs showed a morphological change associated to increased osteogenic gene markers such as BMP2, RUNX2 and SPARC (osteonectin), and higher released levels of calcitonin, BMP-2, MCP-1, sRANKL and osteoprotegerin. Conclusion Our results demonstrate that lactate might be involved in the immune impairment and BM fibrosis of PMF. The inhibition of lactate production and shuttles in myelofibrosis may be a strategy not only to inhibit invasive and metastatic behavior of cancer cells, but also to restore the anti-cancer immune response improving the results of therapy in MF patients. Disclosures Romano: Novartis: Honoraria; Takeda: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen, Takeda, Novartis: Honoraria; Celgene: Consultancy, Honoraria; GILEAD, Incyte: Research Funding. Palumbo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

The COVID-19 global pandemic is caused by SARS-CoV-2, and represents an urgent medical and social issue. Unfortunately, there is still not a single proven effective drug available, and therefore, current therapeutic guidelines recommend supportive care including oxygen administration and treatment with antibiotics. Recently, patients have been also treated with off-label therapies which comprise antiretrovirals, anti-inflammatory compounds, antiparasitic agents and plasma from convalescent patients, all with controversial results. The ubiquitin–proteasome system (UPS) is important for the maintenance of cellular homeostasis, and plays a pivotal role in viral replication processes. In this review, we discuss several aspects of the UPS and the effects of its inhibition with particular regard to the life cycle of the coronaviruses (CoVs). In fact, proteasome inhibition by various chemical compounds, such as MG132, epoxomycin and bortezomib, may reduce the virus entry into the eucariotic cell, the synthesis of RNA, and the subsequent protein expression necessary for CoVs. Importantly, since UPS inhibitors reduce the cytokine storm associated with various inflammatory conditions, it is reasonable to assume that they might be repurposed for SARS-CoV-2, thus providing an additional tool to counteract both virus replication as well as its most deleterious consequences triggered by abnormal immunological response.

Due to a multiplicity of causes provoking traumatic brain injury (TBI), TBI is a highly heterogeneous pathology, characterized by high mortality and disability rates. TBI is an acute neurodegenerative event, potentially and unpredictably evolving into sub-chronic and chronic neurodegenerative events, with transient or permanent neurologic, cognitive, and motor deficits, for which no valid standardized therapies are available. A vast body of literature demonstrates that TBI-induced oxidative/nitrosative stress is involved in the development of both acute and chronic neurodegenerative disorders. Cellular defenses against this phenomenon are largely dependent on low molecular weight antioxidants, most of which are consumed with diet or as nutraceutical supplements. A large number of studies have evaluated the efficacy of antioxidant administration to decrease TBI-associated damage in various animal TBI models and in a limited number of clinical trials. Points of weakness of preclinical studies are represented by the large variability in the TBI model adopted, in the antioxidant tested, in the timing, dosages, and routes of administration used, and in the variety of molecular and/or neurocognitive parameters evaluated. The analysis of the very few clinical studies does not allow strong conclusions to be drawn on the real effectiveness of antioxidant administration to TBI patients. Standardizing TBI models and different experimental conditions, as well as testing the efficacy of administration of a cocktail of antioxidants rather than only one, should be mandatory. According to some promising clinical results, it appears that sports-related concussion is probably the best type of TBI to test the benefits of antioxidant administration.

Human amylin is a 37-residue peptide hormone (hA1-37) secreted by &beta, cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer&rsquo, s disease, alone or co-deposited with &beta, amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal &ldquo, cross-talk&rdquo, We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17&ndash, 29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17&ndash, 29 (3 &micro, M) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17&ndash, 29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17&ndash, 29 (20 &micro, M). We found a complete inhibition of hA17&ndash, 29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

Long-term exposure to ultraviolet (UV) radiation is associated with pathological alterations of the retinal pigment epithelium (RPE). It has been indicated that Cortistatin (CST) and somatostatin (SST) are able to inhibit the neurodegeneration of the RPE associated with diabetic retinopathy and retinal ischemia via activation of SST receptors (SSTRs). To the best of our knowledge, the present study indicated for the first time that treatment with UV‑A (30 and 60 min) causes an increase of CST expression, rather than SST, which was linked with the upregulation of STTR3,4,5 subtype receptor gene expression levels. The study revealed that: i) SST and CST mRNA expression were both detected under basal conditions in a human retinal pigment epithelial cell line (Arpe‑19); ii) SST expression remained constant from baseline to 1 h of UV‑A treatment; iii) CST mRNA expression levels were 80 times increased compared with time 0 and after 30 min of exposition to ultraviolet irradiation; iv) SSTR1, SSTR2 mRNA and low levels of SSTR4 were expressed in basal conditions, whereas SSTR3 and SSTR5 mRNA were not detected under the same conditions; and v) only SSTR3, SSTR4 and SSTR5 were overexpressed after UV‑A treatment, although in a different way. In conclusion, the findings provide reasonable evidence to support the pathophysiological role of the CST/SST/SSTRs system in the adaptive response of the RPE exposed to UV‑A radiation.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

STUDY QUESTION Is the determination of antioxidants, oxidative/nitrosative stress-related compounds, purines, pyrimidines and energy-related metabolites in human seminal plasma of utility to evidence biomarkers related to male infertility? SUMMARY ANSWER The determination of 26 metabolites in seminal plasma allowed to evidence that 21/26 of them are biomarkers of male infertility, as well as to calculate a cumulative index, named Biomarker Score, that fully discriminates fertile controls from infertile patients and partially differentiates infertile without from infertile with spermiogram anomalies. WHAT IS KNOWN ALREADY Epidemiological studies indicated that a male factor is involved in ~50% of cases of pregnancy failure, with a significant percentage of infertile males having no alterations in the spermiogram. Further laboratory analyses of male infertility are mainly dedicated only to gross evaluations of oxidative stress or total antioxidant capacity. STUDY DESIGN, SIZE, DURATION Seminal plasma of 48 fertile controls and 96 infertile patients (master group), were collected from September 2016 to February 2018. A second group of 44 infertile patients (validation group) was recruited in a second, independent centre from September 2017 to March 2018. Samples were analysed in blind using a 'Redox Energy Test' to determine various low-molecular weight compounds, with the aim of finding metabolic profiles and biomarkers related to male infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS In all seminal plasma, 26 water- and fat-soluble compounds (related to antioxidant defences, oxidative/nitrosative stress, purine, pyrimidine and energy metabolism) were analysed using high-performance liquid chromatographic methods. According to spermiogram, infertile patients of both groups were also categorized into normozoospermic (N, no anomalies in the spermiogram), or into the subgroup including all patients with anomalies in the spermiogram (asthenoteratooligozoospermic ATO + asthenozoospermic A + teratozoospermic T + oligozoospermic O). MAIN RESULTS AND THE ROLE OF CHANCE In the master group, results indicated that 21/26 compounds assayed in seminal plasma of infertile males were significantly different from corresponding values determined in fertile controls. These 21 compounds constituted the male infertility biomarkers. Similar results were recorded in patients of the validation group. Using an index cumulating the biochemical seminal plasma anomalies (Biomarker Score), we found that fertile controls had mean Biomarker Score values of 2.01 ± 1.42, whilst infertile patients of the master and of the validation group had mean values of 12.27 ± 3.15 and of 11.41 ± 4.09, respectively (P < 0.001 compared to controls). The lack of statistical differences between the master and the validation groups, in both the metabolic profiles and the Biomarker Score values, allowed to pool patients into a single cohort of infertile males. The Biomarker Score values showed that fertile controls and infertile males clustered into two distinct groups. Infertile patients without (N, n = 42) or with (ATO + A + T + O, n = 98) spermiogram anomalies differed in some biomarkers (ascorbic acid, all-trans retinol, α-tocopherol, cytidine, uridine, guanine). These differences were reinforced by distribution frequencies and posterior probability curves of the Biomarker Score in the three groups. LIMITATIONS, REASONS FOR CAUTION Results were obtained in relatively limited number of human seminal plasma samples. Using the 'Redox Energy Test' it was possible to associate specific metabolic profiles and values of the Biomarker Score to fertile controls or infertile males. However, it was not possible to evaluate whether the different anomalies of the spermiogram are associated with specific metabolic profiles and values of the Biomarker Score. WIDER IMPLICATIONS OF THE FINDINGS The 'Redox Energy Test', coupled with the Biomarker Score that cumulates the biochemical characteristics of seminal plasma into a single index, evidenced a set of low-molecular weight biomarkers potentially useful in the laboratory management of male infertility. STUDY FUNDING/COMPETING INTEREST(S) The study was partly funded with research grants from the University of Catania. None of the authors have any conflicting interests to declare.

Traumatic brain injury (TBI) is a leading and rising cause of death and disability worldwide. There is great interest in S100B and Glial Fibrillary Acid Protein (GFAP) as candidate biomarkers of TBI for diagnosis, triage, prognostication and drug development. However, conflicting results especially on S100B hamper their routine application in clinical practice. To try to address this question, we mimicked TBI damage utilizing a well-validated, simplified in vitro model of graded stretch injury induced in rat organotypic hippocampal slice cultures (OHSC). Different severities of trauma, from mild to severe, have been tested by using an equi-biaxial stretch of the OHSCs at a specified Lagrangian strain of 0 (controls), 5, 10, 20 and 50 %. OHSC were analysed at 3, 6, 18, 24, 48 and 96 h post-injury. Cell death, gene expressions and release into the culture medium of S100B and GFAP were determined at each time point. Gene expression and release of S100B slightly increased only in 20 and 50 % stretched OHSC. GFAP over-expression occurred in 10, 20 and 50 % and was inversely correlated with time post-injury. GFAP release significantly increased with time at any level of injury (p < 0.01 with respect to controls). Consequently, the total amount of GFAP released showed a strong linear relationship with the severity of injury (R(2) = 0.7662; p < 0.001). Under these experimental conditions, S100B seems to be useful in diagnosing only moderate to severe TBI-like injuries. Differently, GFAP demonstrates adequate biomarker requisites since its cellular release is affected by all grades of injury severity.

Methionine sulfoxide reductase A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. MsrA has recently turned attention in coupling with the neurodegenerative disorders and in particular with Alzheimer disease. In fact this neurodegenerative disorder depends to a deposit of beta amyloid a peptide with an oxidizable methionine in position 35 which is proved able to modulate the expression to MsrA in neuronal cells. Here, we firstly provided evidence that pretreatment with Resveratrol and Punicalagin (a potent antioxidant extracted from pomegranate), up-regulate the expression and enzymatic activity of MsrA in human neuroblastoma IMR-32 cells with beta amyloid peptides. This effect determines a lowering of oxidative potential of the cells as demonstrated by the ROS measurement and a protective effect on cellular availability. Therefore we hypothesize a possible prevent role for these molecules in Alzheimer and in other neurodegenerative diseases.

Background Adenylosuccinate lyase (ADSL) deficiency is a defect of purine metabolism affecting purinosome assembly and reducing metabolite fluxes through purine de novo synthesis and purine nucleotide recycling pathways. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The most severe form is characterized by neonatal encephalopathy, absence of spontaneous movement, respiratory failure, intractable seizures, and early death within the first weeks of life. More commonly, ADSL presents purely neurologic clinical picture characterized by severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features (type I) or a more slowly progressing form with later onset, and major features including slight to moderate psychomotor retardation, and transient contact disturbances (type II). Diagnostic markers are the presence of succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (SAdo) in extracellular fluids. ADSL is a rare disorder, although its prevalence remains unknown. Of note, the wide range of essentially nonspecific manifestations and lack of awareness of the condition often prevent diagnosis. Case presentation We present here the case of particularly mild, late onset ADSL that has been unsuccessfully investigated until whole exome sequencing (WES) was performed. Conclusions Besides emphasizing the valuable diagnostic value of WES, this report provides new data further documenting the relatively wide clinical manifestation of ADSL. Electronic supplementary material The online version of this article (doi:10.1186/s13052-017-0383-7) contains supplementary material, which is available to authorized users.

In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ‐aminobutyrate (GABA), tyrosine (Tyr), S‐adenosylhomocysteine (SAH), l‐cystathionine (l‐Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N‐acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham‐operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long‐lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l‐Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu‐Gln/GABA cycle between neurons and astrocytes, and of the methyl‐cycle (demonstrated by decrease in Met, and increase in SAH and l‐Cystat), throughout the post‐injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism.

Tau is a microtubule-associated protein exerting several physiological functions in neurons. In Alzheimer's disease (AD) misfolded tau accumulates intraneuronally and leads to axonal degeneration. However, tau has also been found in the extracellular medium. Recent studies indicated that extracellular tau uploaded from neurons causes synaptic dysfunction and contributes to tau pathology propagation. Here we report novel evidence that extracellular tau oligomers are abundantly and rapidly accumulated in astrocytes where they disrupt intracellular Ca2+ signaling and Ca2+-dependent release of gliotransmitters, especially ATP. Consequently, synaptic vesicle release, the expression of pre- and post-synaptic proteins, and mEPSC frequency and amplitude were reduced in neighboring neurons. Notably, we found that tau uploading from astrocytes required the amyloid precursor protein, APP. Collectively, our findings suggests that astrocytes play a critical role in the synaptotoxic effects of tau via reduced gliotransmitter availability, and that astrocytes are major determinants of tau pathology in AD.

Traumatic brain injury (TBI) is the leading cause of death and disability in people younger than 45 in Western countries. Despite many studies, no reliable biomarkers have been found to assess TBI severity and predict recovery. MicroRNA (miRNA) profiling has become widely used to identify biomarkers and therapeutic targets. Through use of the TaqMan Array Human MicroRNA A+B Cards, the expression of 754 miRNAs was analyzed in serum of five mild TBI (mTBI) patients with extra-cranial injury (EC), five severe TBI (sTBI) patients with EC, and five healthy volunteers (HV) at 1 day and 15 days post-injury. The aim was to find candidate biomarkers able to discriminate between mTBI and sTBI. Following this, it was possible to select 10 miRNAs for further study in an enlarged validation cohort of 120 patients by using single TaqMan assays at the following time-points: T0-1 h, T4-12 h, T48-72 h, and 15 days from the injury. Analysis revealed two miRNAs (miR-425-5p and miR-502) that were significantly downregulated (p 0.05) in mTBI at early time-points and are ideal candidates for diagnosis of mTBI, and two miRNAs (miR-21 and miR-335) that were significantly upregulated (p 0.01) and are valid biomarkers for the diagnosis of sTBI. In addition, miR-425-5p was a strong predictor of 6-month outcome at T0-1 h and T4-12 h, while miR-21 was predictive of the outcome at T4-12 h. The panel of selected miRNAs shows promise as biomarkers to discriminate mTBI from sTBI. In addition, the selected miRNAs represent new potential therapeutic targets.

Ozone therapy has been widely used in everyday clinical practice over the last few years, leading to significant clinical results in the treatment of herniated discs and pain management. Nevertheless, further studies have demonstrated its potential efficacy and safety under other clinical and experimental conditions. However, some of these studies showed controversial results regarding the safety and efficacy of ozone therapy, thus mining its potential use in an everyday clinical practice. To this regard, it should be considered that extensive literature review reported the use of ozone in a significant different dose range and with different delivery systems. The aim of the present review is to describe the various pharmacological effects of ozone in different organs and clinical conditions and to provide possible biochemical and molecular insights for ozone biological properties, thus providing a possible explanation for various controversial clinical outcomes described in the scientific literature.

In this study, the metabolic, enzymatic and gene changes causing cerebral glucose dysmetabolism following graded diffuse traumatic brain injury (TBI) were evaluated. TBI was induced in rats by dropping 450g from 1 (mild TBI; mTBI) or 2m height (severe TBI; sTBI). After 6, 12, 24, 48, and 120h gene expressions and enzymatic activities of glycolysis and pentose phosphate pathway (PPP) enzymes, and levels of lactate, ATP, ADP, ATP/ADP (indexing mitochondrial phosphorylating capacity), NADP(+), NADPH and GSH were determined in whole brain extracts (n=9 rats at each time for both TBI levels). Sham-operated animals (n=9) were used as controls. Results demonstrated that mTBI caused a late increase (48-120h post injury) of glycolytic gene expression and enzymatic activities, concomitantly with mitochondrial functional recovery (ATP and ATP/ADP normalization). No changes in lactate and PPP genes and enzymes, were accompanied by transient decrease in GSH, NADP(+), NADPH and NADPH/NADP(+). Animals following sTBI showed early increase (6-24h post injury) of glycolytic gene expression and enzymatic activities, occurring during mitochondrial malfunctioning (50% decrease in ATP and ATP/ADP). Higher lactate and lower GSH, NADP(+), NADPH, NADPH/NADP(+) than controls were recorded at anytime post injury (p

Multiple sclerosis (MS) is a primary inflammatory demyelinating disease associated with a probably secondary progressive neurodegenerative component. Impaired mitochondrial functioning has been hypothesized to drive neurodegeneration and to cause increased anaerobic metabolism in MS. The aim of our multicentre study was to determine whether MS patients had values of circulating lactate different from those of controls. Patients (n=613) were recruited, assessed for disability and clinically classified (relapsing–remitting, secondary progressive, primary progressive) at the Catholic University of Rome, Italy (n=281), at the MS Centre Amsterdam, The Netherlands (n=158) and at the S. Camillo Forlanini Hospital, Rome, Italy (n=174). Serum lactate levels were quantified spectrophotometrically with the analyst being blinded to all clinical information. In patients with MS serum lactate was three times higher (3.04±1.26mmol/l) than that of healthy controls (1.09±0.25mmol/l, p

Neuroglobin is a neuron-specific hexacoordinated globin capable of binding various ligands, including O2, NO, and CO, the biological function of which is still uncertain. Various studies seem to indicate that neuroglobin is a neuroprotective agent when overexpressed, acting as a potent inhibitor of oxidative and nitrosative stress. In this study, we evaluated the pathophysiological response of the neuroglobin gene and protein expression in the cerebral tissue of rats sustaining traumatic brain injury of differing severity, while simultaneously measuring the oxidant/antioxidant balance. Two levels of trauma (mild and severe) were induced in anesthetized animals using the weight-drop model of diffuse axonal injury. Rats were then sacrificed at 6, 12, 24, 48, and 120h after traumatic brain injury, and the gene and protein expression of neuroglobin and the concentrations of malondialdehyde (as a parameter representative of reactive oxygen species-mediated damage), nitrite + nitrate (indicative of NO metabolism), ascorbate, and glutathione (GSH) were determined in the brain tissue. Results indicated that mild traumatic brain injury, although causing a reversible increase in oxidative/nitrosative stress (increase in malondialdehyde and nitrite + nitrate) and an imbalance in antioxidants (decrease in ascorbate and GSH), did not induce any change in neuroglobin. Conversely, severe traumatic brain injury caused an over nine- and a fivefold increase in neuroglobin gene and protein expression, respectively, as well as a remarkable increase in oxidative/nitrosative stress and depletion of antioxidants. The results of this study, showing a lack of effect in mild traumatic brain injury as well as asynchronous time course changes in neuroglobin expression, oxidative/nitrosative stress, and antioxidants in severe traumatic brain injury, do not seem to support the role of neuroglobin as an endogenous neuroprotective antioxidant agent, at least under pathophysiological conditions.

Objective Canavan disease (OMIM 271900 ) is a severe autosomal recessive neurodegenerative disorder characterized by spongy degeneration of the brain and caused by mutations in the gene encoding for aspartoacylase (ASPA). The enzyme is responsible for the catalyses of the brain-specific compound N-acetylaspartate (NAA). Design and methods We report the case of two Egyptian sibling patients suspected of Canavan disease (CD) showing clinical deterioration, white matter degeneration, megalencephaly and severe intellectual impairment. The patients underwent magnetic resonance imaging (MRI) and biochemical analysis of NAA in biological fluid samples (serum and urine). Subsequently, in order to determine the mutation responsible for CD in these two sibs, a molecular biological examination was performed. Results MRI findings and quantification of high NAA excretion (1378.5 and 680.1 μmol NAA/mmol creatinine in urine of 4 months and 4 years old patients, respectively) confirmed the diagnosis of CD and prompted a search for the responsible mutation. The molecular biological analysis revealed homozygosity for the substitution T530C (Ile177Thr) in the exon 4 of the ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. Conclusions The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression.