Combined evaluation of prolactin-induced peptide (PIP) and extracellular signal-regulated kinase (ERK) as new sperm biomarkers of FSH treatment efficacy in normogonadotropic idiopathic infertile men.

PURPOSE: Nearly, 40% of the causes of male infertility remain idiopathic. The only suggested treatment in idiopathic oligo- and/or asthenozoospermia in normogonadotropic patients is the FSH. In the current clinical practice, efficacy is exclusively assessable through semen analysis after 3 months of treatment. No molecular markers of treatment efficacy are appliable in clinical practice. The aim of the present work is to evaluate the combination of extracellular signal regulated kinase (ERK) 1 and 2 and prolactin inducible peptide (PIP) as potential markers of idiopathic infertility and FSH treatment efficacy. METHODS: Western blot and confocal microscopy were performed to analyze the modulation of PIP and ERK1/2 in idiopathic infertile patients (IIP) sperm cells. Taking advantage of mass spectrometry analysis, we identified these proteins unequivocally in sperm cells. RESULTS:  We demonstrated a significant decrease of both PIP protein and of ERK1/2 levels in spermatozoa obtained from IIP in comparison to healthy fertile patients (HFP). Conversely, we reported a significant increase of these markers comparing infertile patients before and after 3 months of FSH treatment. Importantly, this correlated with an increase in total number of sperm and sperm motility after FSH treatment. Finally, we identified of PIP and ERK2 proteins in sperm samples by proteomic analysis. CONCLUSIONS: The combined evaluation of ERK1/2 and PIP proteins might represent a useful molecular marker to tailor FSH treatment in the management of male normogonadotropic idiopathic infertility.

A New Potential Resource in the Fight against Candida auris: the Cinnamomum zeylanicum Essential Oil in Synergy with Antifungal Drug.

Candida auris is a multidrug-resistant fungus known to be a global public health problem. The skin-based transmission, together with the marked resistance to drugs, resulted in its rapid spread to all continents. The aim of this study was to identify an essential oil (EO) active in the fight against C. auris. A total of 15 EOs were tested against 10 clinical strains of C. auris. Cinnamomum zeylanicum EO (CZ-EO) was the most effective (MIC90 and MFC90 equal to 0.06% vol/vol). Three fractions obtained from CZ-EO, and the cinnamaldehyde (CIN), the major chemical compound, were tested to identify the principal compound effectives against C. auris. All CIN-containing samples showed anti-fungal activity. To study the synergy with fluconazole, CZ-EO, its active fraction (FR2), and CIN were tested in checkerboard tests. Results show that CZ-EO and FR2, but not CIN, synergize with fluconazole. Furthermore, only the copresence of CZ-EO or FR2 synergize with fluconazole at therapeutic concentrations of the drug (0.45 ± 0.32 µg/mL and 0.64 ± 0.67 µg/mL, respectively), while CIN only shows additive activity. In vivo studies conducted on Galleria mellonella larvae show the absence of toxicity of CZ-EO up to concentrations of 16% vol/vol, and the ability of CZ-EO to reactivate the efficacy of fluconazole when formulated at synergic concentrations. Finally, biochemical tests were made to study the mechanism of action of CZ-EO. These studies show that in the presence of both fluconazole and CZ-EO, the activity of fungal ATPases decreases and, at the same time, the amount of intracellular drug increases. IMPORTANCE This study highlights how small doses of CZ-EO are able to inhibit the secretion of fluconazole and promote its accumulation in the fungal cell. In this manner, the drug is able to exert its pharmacological effects bypassing the resistance of the yeast. If further studies will confirm this synergy, it will be possible to develop new therapeutic formulations active in the fight against C. auris resistances.

Okur-Chung neurodevelopmental syndrome-linked CK2α variants have reduced kinase activity.

The Okur-Chung neurodevelopmental syndrome, or OCNDS, is a newly discovered rare neurodevelopmental disorder. It is characterized by developmental delay, intellectual disability, behavioral problems (hyperactivity, repetitive movements and social interaction deficits), hypotonia, epilepsy and language/verbalization deficits. OCNDS is linked to de novo mutations in CSNK2A1, that lead to missense or deletion/truncating variants in the encoded protein, the protein kinase CK2α. Eighteen different missense CK2α mutations have been identified to date; however, no biochemical or cell biological studies have yet been performed to clarify the functional impact of such mutations. Here, we show that 15 different missense CK2α mutations lead to varying degrees of loss of kinase activity as recombinant purified proteins and when mutants are ectopically expressed in mammalian cells. We further detect changes in the phosphoproteome of three patient-derived fibroblast lines and show that the subcellular localization of CK2α is altered for some of the OCNDS-linked variants and in patient-derived fibroblasts. Our data argue that reduced kinase activity and abnormal localization of CK2α may underlie the OCNDS phenotype.

[Detecting Large Germline Rearrangements of BRCA1 by Next Generation Tumor Sequencing].

A majority of BRCA1/2 (BRCA) pathogenic variants (PVs) are single nucleotide substitutions or small insertions/deletions. Copy number variations (CNVs), also known as large genomic rearrangements (LGRs), have been identified in BRCA genes. LGRs detection is a mandatory analysis in hereditary breast and ovarian cancer families, if no predisposing PVs are found by sequencing. Next generation sequencing (NGS) may be used to detect structural variation, since quantitative analysis of sequencing reads, when coupled with appropriate bioinformatics tools, is capable of estimating and predicting germline LGRs (gLGRs). However, applying this approach to tumor tissue is challenging, and the pipelines for determination of CNV are yet to be optimized. The aim of this study was to validate the Next Generation Tumor Sequencing (NGTS) technology to detect various gLGRs of BRCA1 locus in surgical tumor tissue samples. In this study, seven different BRCA1 gLGRs, previously found in high-grade serous ovarian cancers (HGSOC) patients, were detected in tumor samples collected from the patients at a time of HGSOC surgery. This study demonstrated that NGS can accurately detect BRCA1 gLGRs in primary tumors, suggesting that gLGR evaluation in BRCA1 locus should be performed in cases when the screening for BRCA alterations starts from tumor instead of blood. NGS sequencing of tumor samples may become the preferred method to detect both somatic and germline gLGRs in BRCA-encoding loci.

The role of procalcitonin outside of the Intensive Care Unit (ICU): a multidisciplinary approach.

OBJECTIVE: Biochemical markers are commonly used in medicine to guide diagnostic investigation or therapy duration and/or monitor treatment efficacy. Due to the emergence and spread of antimicrobial resistance, markers able to prompt a more rational use of antimicrobial therapy are regarded with the greatest attention. Procalcitonin (PCT) certainly stands out among others, yet its role must be better established especially outside of the critical care area. Data about PCT utilization in non-critical patients, optimal negativity cut-offs as well as a protocol for measurement timing are all lacking. MATERIALS AND METHODS: To address these issues, a focus group was set up to propose and endorse shared statements regarding the most beneficial use of PCT in real life as infection marker for non-critical patients, based on the authors' experience and a review of recent literature. RESULTS: A group of nine experts in the fields of Infectious Diseases, Internal Medicine, Microbiology, Clinical Chemistry, Surgery and Medical Economics participated in the discussion of nine pre-specified statements. CONCLUSIONS: The potential role for PCT in differentiating infectious and non-infectious clinical syndromes and guiding antimicrobial therapy discontinuation was acknowledged. Moreover, a shared measurement protocol and desirable cut-offs for the non-critical area were proposed. Finally, observations were made about a reasonable selection of the patient population to be tested.

A false positive case of high-sensitivity cardiac troponin in a patient with acute chest pain: Analytical study of the interference.

We report a case of a heterophile antibodies interference in a new high-sensitivity troponin commercial immunoassay (cTNIH Siemens), observed in a patient with possible acute coronary syndrome (ACS). The analytical interference was investigated with standard laboratories procedures. The false positive result was found with different troponin methods and kits. We also investigated the protein sequence of cTnl and no sequence variants were detected. The discordance between clinical pictures and high concentration of cTnl, together with the collaboration between clinicians and laboratory staff avoided possible erroneous diagnosis and further invasive investigations to the patient.

Clinical effect of corticosteroids in asthma-affected horses: A quantitative synthesis.

BACKGROUND: There are limited findings from low-powered studies based on few number of subjects with equine asthma. Furthermore, no studies have been performed to assess a meaningful clinically detectable impact of corticosteroids in equine asthma. OBJECTIVES: To assess and compare the clinical effect of inhaled and systemic corticosteroids in equine asthma and identify a quantitative clinical score suitable to assess the Minimal Important Difference (MID), expressed as the Minimally Clinically Detectable Difference (MCDD). STUDY DESIGN: Pair-wise and network meta-analysis. METHODS: Literature searches for studies on corticosteroid therapy in equine asthma were performed. The risk of publication bias was assessed by Funnel plots and Egger's test. The effect on changes in clinical scores vs. control was analysed via random-effects models and Bayesian networks. RESULTS: Corticosteroids significantly improved the clinical condition (Standardised Mean Difference: -1.52, 95% CrI -2.07 to -0.98; P<0.001 vs. control). No difference was detected between inhaled and systemic corticosteroids with regard to the changes in clinical scores (Relative Effect: 0.08, 95% CrI -1.45 to 1.32; P = 0.8). An Improved clinically Detectable Equine Asthma Scoring System (IDEASS) indicated that corticosteroids improved the clinical condition of asthmatic horses by 30% compared with controls (IDEASS value: -2.36, 95% CI -3.39 to -1.33; P<0.001). A one-point change in IDEASS represented the MCDD in equine asthma. MAIN LIMITATIONS: Moderate quality of evidence for systemic corticosteroids. CONCLUSIONS: Inhaled corticosteroids are effective in improving the clinical condition of horses with equine asthma and prevent exacerbations. Systemic corticosteroids should be used only in selected cases with symptomatic airway hyperresponsiveness during exacerbation. IDEASS requires further validation but may represent a suitable approach to rank the level of asthma severity and assess the clinical effect of pharmacotherapy in horses with equine asthma.

Pharmacological treatments in asthma-affected horses: A pair-wise and network meta-analysis.

BACKGROUND: Equine asthma is a disease characterised by reversible airflow obstruction, bronchial hyper-responsiveness and airway inflammation following exposure of susceptible horses to specific airborne agents. Although clinical remission can be achieved in a low-airborne dust environment, repeated exacerbations may lead to irreversible airway remodelling. The available data on the pharmacotherapy of equine asthma result from several small studies, and no head-to-head clinical trials have been conducted among the available medications. OBJECTIVES: To assess the impact of the pharmacological interventions in equine asthma and compare the effect of different classes of drugs on lung function. STUDY DESIGN: Pair-wise and network meta-analysis. METHODS: Literature searches for clinical trials on the pharmacotherapy of equine asthma were performed. The risk of publication bias was assessed by funnel plots and Egger's test. Changes in maximum transpulmonary or pleural pressure, pulmonary resistance and dynamic lung compliance vs. control were analysed via random-effects models and Bayesian networks. RESULTS: The results obtained from 319 equine asthma-affected horses were extracted from 32 studies. Bronchodilators, corticosteroids and chromones improved maximum transpulmonary or pleural pressure (range: -8.0 to -21.4 cmH2 O; P<0.001). Bronchodilators, corticosteroids and furosemide reduced pulmonary resistance (range: -1.2 to -1.9 cmH2 O/L/s; P<0.001), and weakly increased dynamic lung compliance. Inhaled ß2 -adrenoreceptor (ß2 -AR) agonists and inhaled corticosteroids had the highest probability of being the best therapies. Long-term treatments were more effective than short-term treatments. MAIN LIMITATIONS: Weak publication bias was detected. CONCLUSIONS: This study demonstrates that long-term treatments with inhaled corticosteroids and long-acting ß2 -AR agonists may represent the first choice for treating equine asthma. Further high quality clinical trials are needed to clarify whether inhaled bronchodilators should be preferred to inhaled corticosteroids or vice versa, and to investigate the potential superiority of combination therapy in equine asthma.

Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.

Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1-4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem-loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding.

MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response.

The p53 inhibitor, MDM4 (MDMX) is a cytoplasmic protein with p53-activating function under DNA damage conditions. Particularly, MDM4 promotes phosphorylation of p53 at Ser46, a modification that precedes different p53 activities. We investigated the mechanism by which MDM4 promotes this p53 modification and its consequences in untransformed mammary epithelial cells and tissues. In response to severe DNA damage, MDM4 stimulates p53Ser46(P) by binding and stabilizing serine-threonine kinase HIPK2. Under these conditions, the p53-inhibitory complex, MDM4/MDM2, dissociates and this allows MDM4 to promote p53/HIPK2 functional interaction. Comparative proteomic analysis of DNA damage-treated cells versus -untreated cells evidenced a diffuse downregulation of proteins with anti-apoptotic activity, some of which were targets of p53Ser46(P)/HIPK2 repressive activity. Importantly, MDM4 depletion abolishes the downregulation of these proteins indicating the requirement of MDM4 to promote p53-mediated transcriptional repression. Consistently, MDM4-mediated HIPK2/p53 activation precedes HIPK2/p53 nuclear translocation and activity. Noteworthy, repression of these proteins was evident also in mammary glands of mice subjected to γ-irradiation and was significantly enhanced in transgenic mice overexpressing MDM4. This study evidences the flexibility of MDM2/MDM4 heterodimer, which allows the development of a positive activity of cytoplasmic MDM4 towards p53-mediated transcriptional function. Noteworthy, this activity uncovers coordinated repression of molecules with shared anti-apoptotic function which precedes active cell apoptosis and that are frequently overexpressed and/or markers of tumour phenotype in human cancer.

Breast cancer stem cells rely on fermentative glycolysis and are sensitive to 2-deoxyglucose treatment.

A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result in tumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential to understand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternative strategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in order to point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cells were compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identify a metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift from mitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactate dehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, we show that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone and shows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis may be a potentially effective strategy to target BCSCs.

Metabolomics signature improves the prediction of cardiovascular events in elderly subjects.

AIMS: Age is one of the most important determinants of cardiovascular health, therefore the management of cardiovascular diseases (CVD) in elderly people entails great challenge. A possible explanation of vascular senescence process is the mitochondrial damage and dysfunction. We hypothesized that metabolomic profiling would identify biomarkers predicting major cardiovascular events (MACEs) in elderly people, improving the clinical standard cardiovascular risk factors. METHODS AND RESULTS: Targeted-mass-spectrometry-based profiling of 49 metabolites was performed in a group of very old participants (n = 67, mean age = 85 ± 3 years) with a high rate of previous CVD (68%). Principal Component Analysis, Random Survival Forest analysis and Cox proportional hazards regression modeling were used to evaluate the relation between the metabolite factors and recurring MACEs. We tested discrimination ability and reclassification of clinical and metabolomic models. At follow-up (median = 3.5 years), 17 MACEs occurred (5 cardiovascular deaths, 1 nonfatal myocardial infarction, 7 nonfatal strokes and 4 peripheral artery surgeries) (incidence = 7.3% person-years). Metabolite factor 1, composed by medium- and long-chain acylcarnitines, and factor 7 (alanine) were independently associated with MACEs, after adjustment for clinical CV covariates [HR = 1.77 (95%CI = 1.11-2.81, p = 0.016) and HR = 2.18 (95%CI = 1.17-4.07, p = 0.014), respectively]. However, only factor 1 significantly increases the prediction accuracy of the Framingham Recurring-Coronary-Heart-Disease-Score, with a significant improvement in discrimination (integrated discrimination improvement = 7%, p = 0.01) and correctly reclassifying 41% of events and 37% of non-events resulting in a cNRI = 0.79 (p = 0.005). CONCLUSIONS: Aging mitochondrial dysfunction evaluated by metabolomic profiling is associated with MACEs, independently of standard predictors.

Proteomics boosts translational and clinical microbiology.

The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient. Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Distinct proteomic profiles characterise non-erosive from erosive reflux disease.

BACKGROUND: Erosive reflux disease (ERD) and non-erosive reflux disease (NERD) are often regarded as part of the spectrum of the same disease. AIM: To elucidate molecular features that characterise NERD and ERD at the protein level. METHODS: A total of 56 consecutive subjects were enrolled: 10 healthy subjects, 24 with NERD and 22 with ERD. Eight specimens were taken from macroscopically normal mucosa at 5 cm of gastro-oesophageal junction. Four were processed for the proteins extraction and four for evaluation using haematoxylin-eosin and immunohistochemistry. We used shotgun proteomics to identify tentative disease molecular features for ERD or NERD. Candidate distinctive proteins were verified using immunohistochemistry. RESULTS: Shotgun proteomics analysis revealed 33 differentially expressed proteins in NERD vs. ERD samples, involved in cellular proliferation, keratinisation, stress responses and sugar metabolism. Based on a gene ontology meta-analysis, seven of them were further analysed using Western blotting (WB) and four also using immunohistochemistry. We identified novel candidate disease molecular features for GERD and few distinctive proteins to discriminate NERD and ERD. In particular, Transitional Endoplasmic Reticulum ATPase (TER ATPase), GAPDH, Alpha 1 Acid Glycoprotein 1, Annexin A1, Calmodulin and 14-3-3 proteins were confirmed at WB analysis. CONCLUSIONS: Non-erosive reflux disease and ERD are distinct disease entities at the protein level. This study proposes an array of candidate biomarkers possibly useful to discriminate between NERD and ERD.

Novel strategies for tracing the exposure of meat cattle to illegal growth-promoters.

Official monitoring of residues in cattle throughout the European Union in 2007 found <0.2% non-compliance for the use of illegal growth-promoters (GPs), including sex steroids, corticosteroids and ß-agonists. There is evidence, however, that these figures may underestimate the real incidence of GP abuse in meat cattle breeding. One source of evidence arises from the introduction of new detection strategies in response to the demand for safe and wholesome food. These strategies are based on the biological effects of the different GP classes in target species, with a focus on identifying reliable and cost effective biomarkers to improve detection methods. This review summarises the published data relating to experimental and field studies performed in meat cattle, emphasising the impact of the 'omic' technologies and bioinformatics to discover suitable biomarkers for residue surveillance. Further research is required before any potential biomarkers can be utilised for large scale high throughput screening tests.

The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity.

The cytochrome cbb3 is an isoenzyme in the family of cytochrome c oxidases. This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (Vmax=100+/-9 mol NO x mol cbb3(-1) x min(-1) and Km=12+/-2.5 microm), which is lost upon denaturation. This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption. As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb3-type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.

Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri.

Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm. This is the first crystallisation report of a (cbb(3))-type oxidase.

Riluzole inhibits the persistent sodium current in mammalian CNS neurons.

The effects of 0.1-100 microM riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch-clamp whole-cell recordings in voltage-clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose-dependently inhibited INa,p up to a complete blocking of the current (EC50 2 microM), showing a significant effect at therapeutic drug concentrations. A comparative dose-effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole.