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Abstract N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.

Abstract Glioblastoma (GBM) represents an aggressive brain tumor, characterized by intra- and inter-tumoral heterogeneity and therapy resistance, leading to unfavourable prognosis. An increasing number of studies pays attention on the regulation of ferroptosis, an iron-dependent cell death, as a strategy to reverse drug resistance in cancer. However, the debate on whether this strategy may have important implications for the treatment of GBM is still ongoing. In the present study, we used ferric ammonium citrate and erastin to evaluate ferroptosis induction effects on two human GBM cell lines, U-251 MG, with proneural characteristics, and T98-G, with a mesenchymal profile. The response to ferroptosis induction was markedly different between cell lines, indeed T98-G cells showed an enhanced antioxidant defence, with increased glutathione levels, as compared to U-251 MG cells. Moreover, using bioinformatic approaches and analysing publicly available datasets from patients’ biopsies, we found that GBM with a mesenchymal phenotype showed an up-regulation of several genes involved in antioxidant mechanisms as compared to proneural subtype. Thus, our results suggest that GBM subtypes differently respond to ferroptosis induction, emphasizing the significance of further molecular studies on GBM to better discriminate between various tumor subtypes and progressively move towards personalized therapy.

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is a liver disorder characterized by the ac-cumulation of fat in hepatocytes without alcohol consumption. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress play significant roles in NAFLD pathogenesis. The unfolded protein response in mitochondria (UPRmt) is an adaptive mechanism that aims to restore mitochondrial protein homeostasis and mitigate cellular stress. This study aimed to investigate the effects of ( +)-Lipoic acid (ALA) on UPRmt, inflammation, and oxidative stress in an in vitro model of NAFLD using HepG2 cells treated with palmitic acid and oleic acid to induce steatosis. Results Treatment with palmitic and oleic acids increased UPRmt-related proteins HSP90 and HSP60 (heat shock protein), and decreased CLPP (caseinolytic protease P), indicating ER stress activation. ALA treatment at 1 μM and 5 μM restored UPRmt-related protein levels. PA:OA (palmitic acid:oleic acid)-induced ER stress markers IRE1α (Inositol requiring enzyme-1), CHOP (C/EBP Homologous Protein), BIP (Binding Immunoglobulin Protein), and BAX (Bcl-2-associated X protein) were significantly reduced by ALA treatment. ALA also enhanced ER-mediated protein glycosylation and reduced oxidative stress, as evidenced by decreased GPX1 (Glutathione peroxidase 1), GSTP1 (glutathione S-transferase pi 1), and GSR (glutathione-disulfide reductase) expression and increased GSH (Glutathione) levels, and improved cellular senescence as shown by the markers β-galactosidase, γH2Ax and Klotho-beta. Conclusions In conclusion, ALA ameliorated ER stress, oxidative stress, and inflammation in HepG2 cells treated with palmitic and oleic acids, potentially offering therapeutic benefits for NAFLD providing a possible biochemical mechanism underlying ALA beneficial effects. Graphical Abstract

To date, little is known concerning the circulating levels of biochemically relevant metabolites (antioxidants, oxidative/nitrosative stress biomarkers, purines, and pyrimidines) in patients with primary myelofibrosis (PMF), a rare form of myeloproliferative tumor causing a dramatic decrease in erythropoiesis and angiogenesis. In this study, using a targeted metabolomic approach, serum samples of 22 PMF patients and of 22 control healthy donors were analyzed to quantify the circulating concentrations of hypoxanthine, xanthine, uric acid (as representative purines), uracil, β-pseudouridine, uridine (as representative pyrimidines), reduced glutathione (GSH), ascorbic acid (as two of the main water-soluble antioxidants), malondialdehyde, nitrite, nitrate (as oxidative/nitrosative stress biomarkers) and creatinine, using well-established HPLC method for their determination. Results showed that PMF patients have dramatic depletions of both ascorbic acid and GSH (37.3- and 3.81-times lower circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001), accompanied by significant increases in malondialdehyde (MDA) and nitrite + nitrate (4.73- and 1.66-times higher circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001). Additionally, PMF patients have remarkable alterations of circulating purines, pyrimidines, and creatinine, suggesting potential mitochondrial dysfunctions causing energy metabolism imbalance and consequent increases in these cell energy-related compounds. Overall, these results, besides evidencing previously unknown serum metabolic alterations in PMF patients, suggest that the determination of serum levels of the aforementioned compounds may be useful to evaluate PMF patients on hospital admission for adjunctive therapies aimed at recovering their correct antioxidant status, as well as to monitor patients’ status and potential pharmacological treatments.

Abstract Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) even transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. Here, we report that myeloma MSCs have decreased reliance on mitochondrial metabolism as compared to healthy MSCs and increased tendency to deliver mitochondria to MM cells, suggesting that this intercellular exchange between PCs and stromal cells can be consider part of MSC pro-tumorigenic phenotype. Interestingly, we also showed that PCs promoted expression of connexin 43 (CX43) in MSCs leading to CXCL12 activation and stimulation of its receptor CXCR4 on MM cells favoring protumor mitochondrial transfer. Consistently, we observed that selective inhibition of CXCR4 by plerixafor resulted in a significant reduction of mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. Taken together, our data demonstrated that CXCL12/CXCR4 axis mediates intercellular coupling thus suggesting that the myeloma niche may be exploited as a target to improve and develop therapeutic approaches.

N-acetylaspartate (NAA) is synthesized by the mitochondrial enzyme NAT8L, which uses acetyl-CoA and aspartate as substrates. These metabolites are fundamental for bioenergetics and anabolic requirements of highly proliferating cells, thus, NAT8L modulation may impinge on the metabolic reprogramming of cancer cells. Specifically, aspartate represents a limiting amino acid for nucleotide synthesis in cancer.Here, the expression of the NAT8L enzyme was modulated to verify how it impacts the metabolic adaptations and proliferative capacity of hepatocellular carcinoma. We demonstrated that NAT8L downregulation is associated with increased proliferation of hepatocellular carcinoma cells and immortalized hepatocytes. The overexpression of NAT8L instead decreased cell growth. The pro-tumoral effect of NAT8L silencing depended on glutamine oxidation and the rewiring of glucose metabolism. Mechanistically, NAT8L downregulation triggers aspartate outflow from mitochondria via the exporter SLC25A13 to promote glucose flux into the pentose phosphate pathway, boosting purine biosynthesis. These results were corroborated by the analyses of human and mouse hepatocellular carcinoma samples revealing a decrease in NAT8L expression compared to adjacent non-tumoral tissues. Overall, this work demonstrates that NAT8L expression in liver cells limits the cytosolic availability of aspartate necessary for enhancing the pentose phosphate pathway and purine biosynthesis, counteracting cell proliferation.

Histamine, a monoamine implicated in stress-related arousal states, is synthesized in neurons exclusively located in the hypothalamic tuberomammillary nucleus (TMN) from where they diffusely innervate striatal and mesolimbic networks including the nucleus accumbens (NAc), a vital node in the limbic loop. Since histamine-containing TMN neuron output increases during stress, we hypothesized that exposure of mice to acute restrain stress (ARS) recruits endogenous histamine type 2 receptor (H2R) signaling in the NAc, whose activation increases medium spiny neurons (MSNs) intrinsic excitability via downregulation of A-type K+ currents. We employed an ARS paradigm in which mice were restrained for 120 min, followed by a 20-min recovery period, after which brain slices were prepared for ex vivo electrophysiology. Using whole-cell patch-clamp recordings, we found that pharmacological activation of H2R failed to affect MSN excitability and A-type K+ currents in mice that underwent ARS. Interestingly, in mice treated with H2R-antagonist prior to ARS paradigm, H2R activation increased evoked firing and decreased A-type K+ currents similarly to what observed in control mice. Furthermore, H2R-antagonist treatment ameliorated anxiety-like behavior in ARS mice. Together, our findings indicate that ARS paradigm recruits endogenous H2R signaling in MSNs and suggest the involvement of H2R signaling in stress-related motivational states.

Background: Acute and chronic neurodegenerative diseases represent an immense socioeconomic burden that drives the need for new disease modifying drugs. Common pathogenic mechanisms in these diseases are evident, suggesting that a platform neuroprotective therapy may offer effective treatments. Here we present evidence for the mode of pharmacological action of a novel neuroprotective low molecular weight dextran sulphate drug called ILB®. The working hypothesis was that ILB® acts via the activation of heparin-binding growth factors (HBGF).Methods: Pre-clinical and clinical (healthy people and patients with ALS) in vitro and in vivo studies evaluated the mode of action of ILB®. In vitro binding studies, functional assays and gene expression analyses were followed by the assessment of the drug effects in an animal model of severe traumatic brain injury (sTBI) using gene expression studies followed by functional analysis. Clinical data, to assess the hypothesized mode of action, are also presented from early phase clinical trials.Results: ILB® lengthened APTT time, acted as a competitive inhibitor for HGF-Glypican-3 binding, effected pulse release of heparin-binding growth factors (HBGF) into the circulation and modulated growth factor signaling pathways. Gene expression analysis demonstrated substantial similarities in the functional dysregulation induced by sTBI and various human neurodegenerative conditions and supported a cascading effect of ILB® on growth factor activation, followed by gene expression changes with profound beneficial effect on molecular and cellular functions affected by these diseases. The transcriptional signature of ILB® relevant to cell survival, inflammation, glutamate signaling, metabolism and synaptogenesis, are consistent with the activation of neuroprotective growth factors as was the ability of ILB® to elevate circulating levels of HGF in animal models and humans.Conclusion: ILB® releases, redistributes and modulates the bioactivity of HBGF that target disease compromised nervous tissues to initiate a cascade of transcriptional, metabolic and immunological effects that control glutamate toxicity, normalize tissue bioenergetics, and resolve inflammation to improve tissue function. This unique mechanism of action mobilizes and modulates naturally occurring tissue repair mechanisms to restore cellular homeostasis and function. The identified pharmacological impact of ILB® supports the potential to treat various acute and chronic neurodegenerative disease, including sTBI and ALS.

To date, there is no information on the effect of TBI on the changes in brain CoQ levels and possible variations in its redox state. In this study, we induced graded TBIs (mild TBI, mTBI and severe TBI, sTBI) in male rats, using the weight-drop closed-head impact acceleration model of trauma. At 7 days post-injury, CoQ9, CoQ10 and α-tocopherol were measured by HPLC in brain extracts of the injured rats, as well as in those of a group of control sham-operated rats. In the controls, about the 69% of total CoQ was in the form of CoQ9 and the oxidized/reduced ratios of CoQ9 and CoQ10 were, respectively, 1.05 ± 0.07 and 1.42 ± 0.17. No significant changes in these values were observed in rats experiencing mTBI. Conversely, in the brains of sTBI-injured animals, an increase in reduced and a decrease in oxidized CoQ9 produced an oxidized/reduced ratio of 0.81 ± 0.1 (p < 0.001 compared with both controls and mTBI). A concomitant decrease in both reduced and oxidized CoQ10 generated a corresponding oxidized/reduced ratio of 1.38 ± 0.23 (p < 0.001 compared with both controls and mTBI). An overall decrease in the concentration of the total CoQ pool was also found in sTBI-injured rats (p < 0.001 compared with both controls and mTBI). Concerning α-tocopherol, whilst no differences compared with the controls were found in mTBI animals, a significant decrease was observed in rats experiencing sTBI (p < 0.01 compared with both controls and mTBI). Besides suggesting potentially different functions and intracellular distributions of CoQ9 and CoQ10 in rat brain mitochondria, these results demonstrate, for the first time to the best of knowledge, that sTBI alters the levels and redox states of CoQ9 and CoQ10, thus adding a new explanation to the mitochondrial impairment affecting ETC, OXPHOS, energy supply and antioxidant defenses following sTBI.

Introduction Amyotrophic lateral sclerosis (ALS) is an invariably lethal progressive disease, causing degeneration of neurons and muscle. No current treatment halts or reverses disease advance. This single arm, open label, clinical trial in patients with ALS investigated the safety and tolerability of a novel modified low molecular weight dextran sulphate (LMW-DS, named ILB®) previously proven safe for use in healthy volunteers and shown to exert potent neurotrophic effects in pre-clinical studies. Secondary endpoints relate to efficacy and exploratory biomarkers. Methods Thirteen patients with ALS were treated with 5 weekly subcutaneous injections of ILB®. Safety and efficacy outcome measures were recorded weekly during treatment and at regular intervals for a further 70 days. Functional and laboratory biomarkers were assessed before, during and after treatment. Results No deaths, serious adverse events or participant withdrawals occurred during or after ILB® treatment and no significant drug-related changes in blood safety markers were evident, demonstrating safety and tolerability of the drug in this cohort of patients with ALS. The PK of ILB® in patients with ALS was similar to that seen in healthy controls. The ILB® injection elicited a transient elevation of plasma Hepatocyte Growth Factor, a neurotrophic and myogenic growth factor. Following the ILB® injections patients reported increased vitality, decreased spasticity and increased mobility. The ALSFRS-R rating improved from 36.31 ± 6.66 to 38.77 ± 6.44 and the Norris rating also improved from 70.61 ± 13.91 to 77.85 ± 14.24 by Day 36. The improvement of functions was associated with a decrease in muscle atrophy biomarkers. These therapeutic benefits decreased 3–4 weeks after the last dosage. Conclusions This pilot clinical study demonstrates safety and tolerability of ILB® in patients with ALS. The exploratory biomarker and functional measures must be cautiously interpreted but suggest clinical benefit and have a bearing on the mechanism of action of ILB®. The results support the drug’s potential as the first disease modifying treatment for patients with ALS. Trial registration EudraCT 2017-005065-47.

Down Syndrome (DS) is a neurodevelopmental disorder that is characterized by an accelerated aging process, frequently associated with the development of Alzheimer’s disease (AD). Previous studies evidenced that DS patients have various metabolic anomalies, easily measurable in their serum samples, although values that were found in DS patients were compared with those of age-matched non-DS patients, thus hampering to discriminate the physiologic age-related changes of serum metabolites from those that are truly caused by the pathologic processes associated with DS. In the present study we performed a targeted metabolomic evaluation of serum samples from DS patients without dementia of two age classes (Younger DS Patients, YDSP, aging 20–40 years; Aged DS Patients, ADSP, aging 41–60 years), comparing the results with those that were obtained in two age classes of non-DS patients (Younger non-DS Patients, YnonDSP, aging 30–60 years; Aged-nonDS Patients, AnonDSP, aging 75–90 years). Of the 36 compounds assayed, 30 had significantly different concentrations in Pooled non-DS Patients (PnonDSP), compared to Pooled DS Patients (PDSP). Age categorization revealed that 11/30 compounds were significantly different in AnonDSP, compared to YnonDSP, indicating physiologic, age-related changes of their circulating concentrations. A comparison between YDSP and ADSP showed that 19/30 metabolites had significantly different values from those found in the corresponding classes of non-DS patients, strongly suggesting pathologic, DS-associated alterations of their serum levels. Twelve compounds selectively and specifically discriminated PnonDSP from PDSP, whilst only three discriminated YDSP from ADSP. The results allowed to determine, for the first time and to the best of our knowledge, the true, age-independent alterations of metabolism that are measurable in serum and attributable only to DS. These findings may be of high relevance for better strategies (pharmacological, nutritional) aiming to specifically target the dysmetabolism and decreased antioxidant defenses that are associated with DS.

The combined derangements in mitochondria network, function and dynamics can affect metabolism and ATP production, redox homeostasis and apoptosis triggering, contributing to cancer development in many different complex ways. In hematological malignancies, there is a strong relationship between cellular metabolism, mitochondrial bioenergetics, interconnections with supportive microenvironment and drug resistance. Lymphoma and chronic lymphocytic leukemia cells, e.g., adapt to intrinsic oxidative stress by increasing mitochondrial biogenesis. In other hematological disorders such as myeloma, on the contrary, bioenergetics changes, associated to increased mitochondrial fitness, derive from the adaptive response to drug-induced stress. In the bone marrow niche, a reverse Warburg effect has been recently described, consisting in metabolic changes occurring in stromal cells in the attempt to metabolically support adjacent cancer cells. Moreover, a physiological dynamic, based on mitochondria transfer, between tumor cells and their supporting stromal microenvironment has been described to sustain oxidative stress associated to proteostasis maintenance in multiple myeloma and leukemia. Increased mitochondrial biogenesis of tumor cells associated to acquisition of new mitochondria transferred by mesenchymal stromal cells results in augmented ATP production through increased oxidative phosphorylation (OX-PHOS), higher drug resistance, and resurgence after treatment. Accordingly, targeting mitochondrial biogenesis, electron transfer, mitochondrial DNA replication, or mitochondrial fatty acid transport increases therapy efficacy. In this review, we summarize selected examples of the mitochondrial derangements in hematological malignancies, which provide metabolic adaptation and apoptosis resistance, also supported by the crosstalk with tumor microenvironment. This field promises a rational design to improve target-therapy including the metabolic phenotype.

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.

Breast cancer is the most frequent tumor and the leading cause of cancer deaths in women. In recent years, lactate metabolism and, in particular, its receptor GPR81 have been shown to play a vital role in cancer biology. GPR81 is upregulated in breast cancer and promotes tumor growth by tumor cell-derived lactate. Therefore, the search for possible crosstalk and the involvement of new molecules capable of generating this pathology is always in continuous development. In this study, the relationship between GPR81 and IGFBP6 protein in tumor growth and oxidative stress in the human breast cancer cell line MDA-MB-231 was studied. Cells were treated with lactate or the GPR81 receptor agonist and antagonist 3,5-DHBA and 3-OBA, respectively. In addition, oxidative stress and proliferation were also evaluated in cells challenged with the recombinant IGFBP6 protein. Our data showed that lactate induced cell proliferation and wound healing of the MDA-231 breast cancer cell through the overexpression of both the lactate receptor GPR81 and IGFBP6. The increase in IGFBP6 was able, in turn, to improve the mitochondrial fitness and redox state, as suggested by the reduced levels of mitochondrial ROS production after IGFBP6 treatment, presumably mediated by the increase in the ROS detoxifying genes HMOX1, GSTK1 and NQO1. In conclusion, our data highlight a novel axis between GPR81 and IGFBP6 in MDA-231 cells able to modulate lactate metabolism and oxidative stress. This complex signaling may represent a new therapeutic target for breast cancer.

Abstract 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.

Abstract 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.

The human consumption of food animal products is the main topic of an important debate among professionals in this sector: dietologists, dietitians and nutritional biologists. The red meat provides all the essential amino acids, bioavailable iron, zinc, calcium, lipids and B-group vitamins. A valid alternative to beef could be the buffalo meat. Italy is the largest European producer of buffalo meat and derivatives. The high nutritional characteristics of buffalo meat make it suitable to be included in the Mediterranean diet to customize it in relation to the needs and conditions of the population. Polyunsaturated/saturated fatty acids ratio can be influenced by diet, breed and type of breeding, but muscle tissue fat percentage is the main factor in determining a favorable fatty acid composition. This review focuses on the biochemical and nutritional characteristics of the buffalo meat (content of fats, cholesterol, amino acids, vitamins and minerals), explaining their variability depending on the different breeds, and the favorable implications on the human health. These results suggest that buffalo meat can be a healthier alternative to beef, not only for healthy people in particular physiological conditions (i.e. pregnancy), but also for persons at risk for cardiovascular and cerebrovascular diseases, thus achieving the goal of a personalized nutrition.

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®, 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® 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®) has beneficial effects on sTBI metabolic damages. Due to the absence of adverse effects in humans, ILB® represents a promising therapeutic agent for the treatment of sTBI patients.

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.

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.

Carnosine is an endogenous dipeptide composed of β-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−•) 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-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-β1. 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.

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.