Targeted Metabolomics Highlights Dramatic Antioxidant Depletion, Increased Oxidative/Nitrosative Stress and Altered Purine and Pyrimidine Concentrations in Serum of Primary Myelofibrosis Patients.

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, ß-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.

Ticagrelor Enhances the Cardioprotective Effects of Ischemic Preconditioning in Stable Patients Undergoing Percutaneous Coronary Intervention: the TAPER-S Randomized Study.

BACKGROUND: Ticagrelor improves clinical outcomes in patients with acute coronary syndrome compared with clopidogrel. Ticagrelor also inhibits cell uptake of adenosine and has been associated with cardioprotective effects in animal models. We sought to investigate the potential cardioprotective effects of ticagrelor, as compared with clopidogrel, in stable patients undergoing percutaneous coronary intervention (PCI). METHODS AND RESULTS: This was a Prospective Randomized Open Blinded End-points (PROBE) trial enrolling stable patients with coronary artery disease (CAD) requiring fractional flow reserve (FFR)-guided PCI of intermediate epicardial coronary lesions. ST-segment elevation at intracoronary (IC)-ECG during a two-step sequential coronary balloon inflations in the reference vessel during PCI was used as an indirect marker of cardioprotection induced by ischemic preconditioning. The primary endpoint of the study was the comparison of the delta (Δ) (difference) ST-segment elevation measured by intracoronary-ECG during two-step sequential coronary balloon inflations. RESULTS: Fifty-three patients were randomized to either clopidogrel or ticagrelor. The study was stopped earlier because the primary endpoint was met at a pre-specified interim analysis. ΔST-segment elevation was significantly higher in ticagrelor as compared to clopidogrel arms (p<0.0001). Ticagrelor was associated with lower angina score during coronary balloon inflations. There was no difference in coronary microvascular resistance between groups. Adenosine serum concentrations were increased in patients treated with ticagrelor as compared to those treated with clopidogrel. CONCLUSIONS: Ticagrelor enhances the cardioprotective effects of ischemic preconditioning compared with clopidogrel in stable patients with CAD undergoing PCI. Further studies are warranted to fully elucidate the mechanisms through which ticagrelor may exert cardioprotective effects in humans. Clinical Trial Registration: http://www.clinicaltrials.gov. Unique Identifier: NCT02701140.

Bilirubin Concentration in Follicular Fluid Is Increased in Infertile Females, Correlates with Decreased Antioxidant Levels and Increased Nitric Oxide Metabolites, and Negatively Affects Outcome Measures of In Vitro Fertilization.

In a previous study, we showed that various low-molecular-weight compounds in follicular fluid (FF) samples of control fertile females (CFF) have different concentrations compared to those found in FF of infertile females (IF), before and after their categorization into different subgroups, according to their clinical diagnosis of infertility. Using the same FF samples of this previous study, we here analyzed the FF concentrations of free and bound bilirubin and compared the results obtained in CFF, IF and the different subgroups of IF (endometriosis, EM, polycystic ovary syndrome, PCOS, age-related reduced ovarian reserve, AR-ROR, reduced ovarian reserve, ROR, genetic infertility, GI and unexplained infertility, UI). The results clearly indicated that CFF had lower values of free, bound and total bilirubin compared to the respective values measured in pooled IF. These differences were observed even when IF were categorized into EM, PCOS, AR-ROR, ROR, GI and UI, with EM and PCOS showing the highest values of free, bound and total bilirubin among the six subgroups. Using previous results of ascorbic acid, GSH and nitrite + nitrate measured in the same FF samples of the same FF donors, we found that total bilirubin in FF increased as a function of decreased values of ascorbic acid and GSH, and increased concentrations of nitrite + nitrate. The values of total bilirubin negatively correlated with the clinical parameters of fertilization procedures (number of retrieved oocytes, mature oocytes, fertilized oocytes, blastocysts, high-quality blastocysts) and with clinical pregnancies and birth rates. Bilirubin concentrations in FF were not linked to those found in serum samples of FF donors, thereby strongly suggesting that its over production was due to higher activity of heme oxygenase-1 (HO-1), the key enzyme responsible for bilirubin formation, in granulosa cells, or cumulus cells or oocytes of IF and ultimately leading to bilirubin accumulation in FF. Since increased activity of HO-1 is one of the main enzymatic intracellular mechanisms of defense towards external insults (oxidative/nitrosative stress, inflammation), and since we found correlations among bilirubin and oxidative/nitrosative stress in these FF samples, it may reasonably be supposed that bilirubin increase in FF of IF is the result of protracted exposures to the aforementioned insults evidently playing relevant roles in female infertility.

Traumatic Brain Injury Alters Cerebral Concentrations and Redox States of Coenzymes Q9 and Q10 in the Rat.

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.

Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application.

OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. METHODS: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). RESULTS: The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: -5.6 to 7.4%), are stable in thymol (variation: -8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). CONCLUSIONS: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.

Metabolic Signature of Energy Metabolism Alterations and Excess Nitric Oxide Production in Culture Media Correlate with Low Human Embryo Quality and Unsuccessful Pregnancy.

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART.

Acute restraint stress impairs histamine type 2 receptor ability to increase the excitability of medium spiny neurons in the nucleus accumbens.

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.

The mechanism of action of a novel neuroprotective low molecular weight dextran sulphate: New platform therapy for neurodegenerative diseases like Amyotrophic Lateral Sclerosis.

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.

ILB®, a Low Molecular Weight Dextran Sulphate, Restores Glutamate Homeostasis, Amino Acid Metabolism and Neurocognitive Functions in a Rat Model of Severe Traumatic Brain Injury.

In a previous study, we found that administration of ILB®, a new low molecular weight dextran sulphate, significantly improved mitochondrial functions and energy metabolism, as well as decreased oxidative/nitrosative stress, of brain tissue of rats exposed to severe traumatic brain injury (sTBI), induced by the closed-head weight-drop model of diffused TBI. Using aliquots of deproteinized brain tissue of the same animals of this former study, we here determined the concentrations of 24 amino acids of control rats, untreated sTBI rats (sacrificed at 2 and 7 days post-injury) and sTBI rats receiving a subcutaneous ILB® administration (at the dose levels of 1, 5 and 15 mg/kg b.w.) 30 min post-impact (sacrificed at 2 and 7 days post-injury). Additionally, in a different set of experiments, new groups of control rats, untreated sTBI rats and ILB®-treated rats (administered 30 min after sTBI at the dose levels of 1 or 5 mg/kg b.w.) were studied for their neurocognitive functions (anxiety, locomotor capacities, short- and long-term memory) at 7 days after the induction of sTBI. Compared to untreated sTBI animals, ILB® significantly decreased whole brain glutamate (normalizing the glutamate/glutamine ratio), glycine, serine and γ-aminobutyric acid. Furthermore, ILB® administration restored arginine metabolism (preventing nitrosative stress), levels of amino acids involved in methylation reactions (methionine, L-cystathionine, S-adenosylhomocysteine), and N-acetylaspartate homeostasis. The macroscopic evidences of the beneficial effects on brain metabolism induced by ILB® were the relevant improvement in neurocognitive functions of the group of animals treated with ILB® 5 mg/kg b.w., compared to the marked cognitive decline measured in untreated sTBI animals. These results demonstrate that ILB® administration 30 min after sTBI prevents glutamate excitotoxicity and normalizes levels of amino acids involved in crucial brain metabolic functions. The ameliorations of amino acid metabolism, mitochondrial functions and energy metabolism in ILB®-treated rats exposed to sTBI produced significant improvement in neurocognitive functions, reinforcing the concept that ILB® is a new effective therapeutic tool for the treatment of sTBI, worth being tested in the clinical setting.

Biochemical Discrimination of the Down Syndrome-Related Metabolic and Oxidative/Nitrosative Stress Alterations from the Physiologic Age-Related Changes through the Targeted Metabolomic Analysis of Serum.

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.

A phase II open label clinical study of the safety, tolerability and efficacy of ILB® for Amyotrophic Lateral Sclerosis.

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.

Extracellular tau oligomers affect extracellular glutamate handling by astrocytes through downregulation of GLT-1 expression and impairment of NKA1A2 function.

AIMS: Several studies reported that astrocytes support neuronal communication by the release of gliotransmitters, including ATP and glutamate. Astrocytes also play a fundamental role in buffering extracellular glutamate in the synaptic cleft, thus limiting the risk of excitotoxicity in neurons. We previously demonstrated that extracellular tau oligomers (ex-oTau), by specifically targeting astrocytes, affect glutamate-dependent synaptic transmission via a reduction in gliotransmitter release. The aim of this work was to determine if ex-oTau also impair the ability of astrocytes to uptake extracellular glutamate, thus further contributing to ex-oTau-dependent neuronal dysfunction. METHODS: Primary cultures of astrocytes and organotypic brain slices were exposed to ex-oTau (200 nM) for 1 h. Extracellular glutamate buffering by astrocytes was studied by: Na+ imaging; electrophysiological recordings; high-performance liquid chromatography; Western blot and immunofluorescence. Experimental paradigms avoiding ex-oTau internalisation (i.e. heparin pre-treatment and amyloid precursor protein knockout astrocytes) were used to dissect intracellular vs extracellular effects of oTau. RESULTS: Ex-oTau uploading in astrocytes significantly affected glutamate-transporter-1 expression and function, thus impinging on glutamate buffering activity. Ex-oTau also reduced Na-K-ATPase activity because of pump mislocalisation on the plasma membrane, with no significant changes in expression. This effect was independent of oTau internalisation and it caused Na+ overload and membrane depolarisation in ex-oTau-targeted astrocytes. CONCLUSIONS: Ex-oTau exerted a complex action on astrocytes, at both intracellular and extracellular levels. The net effect was dysregulated glutamate signalling in terms of both release and uptake that relied on reduced expression of glutamate-transporter-1, altered function and localisation of NKA1A1, and NKA1A2. Consequently, Na+ gradients and all Na+ -dependent transports were affected.

The Crosstalk between GPR81/IGFBP6 Promotes Breast Cancer Progression by Modulating Lactate Metabolism and Oxidative Stress.

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.

Altered Follicular Fluid Metabolic Pattern Correlates with Female Infertility and Outcome Measures of In Vitro Fertilization.

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.

ILB® Attenuates Clinical Symptoms and Serum Biomarkers of Oxidative/Nitrosative Stress and Mitochondrial Dysfunction in Patients with Amyotrophic Lateral Sclerosis.

Oxidative/nitrosative stress and mitochondrial dysfunction is a hallmark of amyotrophic lateral sclerosis (ALS), an invariably fatal progressive neurodegenerative disease. Here, as an exploratory arm of a phase II clinical trial (EudraCT Number 2017-005065-47), we used high performance liquid chromatography(HPLC) to investigate changes in the metabolic profiles of serum from ALS patients treated weekly for 4 weeks with a repeated sub-cutaneous dose of 1 mg/kg of a proprietary low molecular weight dextran sulphate, called ILB®. A significant normalization of the serum levels of several key metabolites was observed over the treatment period, including N-acetylaspartate (NAA), oxypurines, biomarkers of oxidative/nitrosative stress and antioxidants. An improved serum metabolic profile was accompanied by significant amelioration of the patients' clinical conditions, indicating a response to ILB® treatment that appears to be mediated by improvement of tissue bioenergetics, decrease of oxidative/nitrosative stress and attenuation of (neuro)inflammatory processes.

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells.

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.

Antioxidant-Based Therapies in Male Infertility: Do We Have Sufficient Evidence Supporting Their Effectiveness?

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.

Ca2+ -dependent release of ATP from astrocytes affects herpes simplex virus type 1 infection of neurons.

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 Ca2+ transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca2+ concentration that activates the Glycogen Synthase Kinase (GSK)-3ß, 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.

Metabolic Reprogramming by Malat1 Depletion in Prostate Cancer.

The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes growth and progression in prostate cancer (PCa); however, little is known about its possible impact in PCa metabolism. The aim of this work has been the assessment of the metabolic reprogramming associated with MALAT1 silencing in human PCa cells and in an ex vivo model of organotypic slice cultures (OSCs). Cultured cells and OSCs derived from primary tumors were transfected with MALAT1 specific gapmers. Cell growth and survival, gene profiling, and evaluation of targeted metabolites and metabolic enzymes were assessed. Computational analysis was made considering expression changes occurring in metabolic markers following MALAT1 targeting in cultured OSCs. MALAT1 silencing reduced expression of some metabolic enzymes, including malic enzyme 3, pyruvate dehydrogenase kinases 1 and 3, and choline kinase A. Consequently, PCa metabolism switched toward a glycolytic phenotype characterized by increased lactate production paralleled by growth arrest and cell death. Conversely, the function of mitochondrial succinate dehydrogenase and the expression of oxidative phosphorylation enzymes were markedly reduced. A similar effect was observed in OSCs. Based on this, a predictive algorithm was developed aimed to predict tumor recurrence in a subset of patients. MALAT1 targeting by gapmer delivery restored normal metabolic energy pathway in PCa cells and OSCs.

Low Molecular Weight Dextran Sulfate (ILB®) Administration Restores Brain Energy Metabolism Following Severe Traumatic Brain Injury in the Rat.

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.