Inter- and Intramolecular Interactions in Ether-Functionalized Ionic Liquids.

The intra- and intermolecular interactions in ether-functionalized ionic liquids (ILs) are studied by means of infrared (IR) spectroscopy measurements of N-ethoxyethyl-N-methylpiperidiniumbis(fluorosulfonyl)imide (P1,2O2-FSI) and N-ethoxyethyl-N-methylmorpholiniumbis(fluorosulfonyl)imide (M1,2O2-FSI). The temperature dependence of the spectra in the medium IR range allows the study of the anion conformer distribution and its variation during phase transitions. In particular, it is found that for both ILs the trans conformer of FSI is more stable than the cis conformer, and the enthalpy differences between them are calculated and are found to decrease upon the addition of a Li salt. The results obtained in the far IR range, combined with ab initio calculation of the ionic couple performed using the B3LYP-D functional and considering both empirical dispersion corrections and the presence of a polar solvent, provide evidence for the occurrence of a hydrogen bonding between the O atom of the anion and its closest H atoms directly linked to a C atom of the cation. The comparison with samples having the same cations but with bis(trifluoromethanesulfonyl)imide (TFSI) as an anion, that is, M1,2O2-TFSI and P1,2O2-TFSI, as well as with samples having cations without the ether-functionalization neither in the ring nor in the side chain, such as N-propyl-N-methylpyrrolidinium-FSI (PYR13-FSI) and 1-butyl-1-methylpyrrolidinium-TFSI (PYR14-TFSI), indicates that the occurrence of such highly directional interaction between anion and cation is better observable in the ether-functionalized samples, in particular in those containing FSI as an anion.

Vigilance on use of drugs, herbal products, and food supplements during pregnancy: focus on fosfomycin.

PURPOSE: Urinary tract infection (UTI) is defined as a common bacterial infection that can lead to significant morbidity such as stricture, fistula, abscess formation, bacteremia, sepsis, pyelonephritis, and kidney dysfunction with a mortality rates reported of 1% in men and 3% in women because of development of pyelonephritis. UTIs are more common in women and the 33% of them require antimicrobials treatment for at least one episode by the age of 24 years. UTIs are the most common infections observed during pregnancy and up to 30% of mothers with not treated asymptomatic bacteriuria may develop acute pyelonephritis which consequently can be associated to adverse maternal and fetal outcomes. All bacteriuria in pregnancy should be treated with antimicrobial treatments being safe for both the mother and the fetus. Approximately one every four women receives prescription of antibiotic treatment during pregnancy, nearly 80% of all the prescription medications during gestation. The use of fosfomycin to treat cystitis in pregnancy generally considered safe and effective. Even though use on antibiotics for urinary tract infections is considered generally safe for the fetus and mothers, this opinion is not based on specific studies monitoring the relationship of among urinary infections, consumption of antibiotics, and pregnancy outcomes. MATERIALS AND METHODS: On this basis we decided to analyze data from the database of our multicenter study PHYTOVIGGEST, reporting data from 5362 pregnancies, focusing on use of fosfomycin. Principal outcomes of pregnancy in women treated with fosfomycin were taken into consideration. RESULTS: Women who have been treated with urinary antibiotics during the pregnancy were 183. With respect to the total number of pregnancies of our sample, these women represented the percentage of 3.49% (187/5362). Analysis of different outcomes of pregnancy such as gestational age, neonatal weight, and neonatal Apgar index did not show any significant difference. At the same time, analysis of data of pregnancy complicancies (such as urgent cesarean delivery, use of general anesthesia, need to induce labor) did not show any difference in women taking fosfomycin during pregnancy and those not taking it. CONCLUSIONS: Our data, based on a large number of pregnancies, confirm the safety use of fosfomycin use in pregnancy.

Melatonin treatment suppresses appetite genes and improves adipose tissue plasticity in diet-induced obese zebrafish.

PURPOSE: Overweight and obesity are important risk factors for diabetes, cardiovascular diseases, and premature death in modern society. Recently, numerous natural and synthetic compounds have been tested in diet-induced obese animal models, to counteract obesity. Melatonin is a circadian hormone, produced by pineal gland and extra-pineal sources, involved in processes which have in common a rhythmic expression. In teleost, it can control energy balance by activating or inhibiting appetite-related peptides. The study aims at testing effects of melatonin administration to control-fed and overfed zebrafish, in terms of expression levels of orexigenic (Ghrelin, orexin, NPY) and anorexigenic (leptin, POMC) genes expression and morphometry of visceral and subcutaneous fat depots. METHODS: Adult male zebrafish (n = 56) were divided into four dietary groups: control, overfed, control + melatonin, overfed + melatonin. The treatment lasted 5 weeks and BMI levels of every fish were measured each week. After this period fishes were sacrificed; morphological and morphometric studies have been carried out on histological sections of adipose tissue and adipocytes. Moreover, whole zebrafish brain and intestine were used for qRT-PCR. RESULTS: Our results demonstrate that melatonin supplementation may have an effect in mobilizing fat stores, in increasing basal metabolism and thus in preventing further excess fat accumulation. Melatonin stimulates the anorexigenic and inhibit the orexigenic signals. CONCLUSIONS: It seems that adequate melatonin treatment exerts anti-obesity protective effects, also in a diet-induced obesity zebrafish model, that might be the result of the restoration of many factors: the final endpoint reached is weight loss and stabilization of weight gain.

The effect of ether-functionalisation in ionic liquids analysed by DFT calculation, infrared spectra, and Kamlet-Taft parameters.

The effect of ether-functionalisation on ionic liquids (ILs) is discussed based on Kamlet-Taft parameters and the infrared (IR) spectra of N-ethoxyethyl-N-methylpiperidinium bis(trifluoromethanesulfonyl)imide ([P1,2O2][TFSI]) and N-ethoxyethyl-N-methylmorpholinium bis(trifluoromethanesulfonyl)imide ([M1,2O2][TFSI]). The results are analysed taking into consideration their ion conformers, electronegativity and hardness, and the IR active vibrations obtained by means of DFT calculations. From the evaluation of Kamlet-Taft parameters, the ether-functionalisation in the cationic ring is found to improve the polarity and hydrogen bond acidity of the ILs. This correlates with the computational result which designates that the oxygen atom in the cationic ring increases the electronegativity of the cation. The comparison with the IR spectra, which were obtained experimentally and computationally, revealed that trans-[TFSI] was preferably formed in [M1,2O2][TFSI] compared to [P1,2O2][TFSI]. Although the Kamlet-Taft parameters indicate that [M1,2O2][TFSI] has a higher polarity, this IL preferably adopts trans-[TFSI], which is normally stabilised with the cations having a lower polarity. This may be due to the presence of the oxygen in the cationic ring which delocalises the electron density of the lowest unoccupied molecular orbitals (LUMO) and increases the conformational freedom of the hydrogen bonds between cations and anions. Moreover, the mixtures of pure ILs with a suitable Li-salt were also investigated to analyze the effect of the Li salt on the polarity and the ion conformers.

A high-power and fast charging Li-ion battery with outstanding cycle-life.

Electrochemical energy storage devices based on Li-ion cells currently power almost all electronic devices and power tools. The development of new Li-ion cell configurations by incorporating innovative functional components (electrode materials and electrolyte formulations) will allow to bring this technology beyond mobile electronics and to boost performance largely beyond the state-of-the-art. Here we demonstrate a new full Li-ion cell constituted by a high-potential cathode material, i.e. LiNi0.5Mn1.5O4, a safe nanostructured anode material, i.e. TiO2, and a composite electrolyte made by a mixture of an ionic liquid suitable for high potential applications, i.e. Pyr1,4PF6, a lithium salt, i.e. LiPF6, and standard organic carbonates. The final cell configuration is able to reversibly cycle lithium for thousands of cycles at 1000 mAg-1 and a capacity retention of 65% at cycle 2000.

Tailoring the physical properties of the mixtures of ionic liquids: a microscopic point of view.

A detailed investigation of the phase diagram of the mixtures of the two ionic liquids N-trimethyl-N-propylammonium bis(trifluoromethylsulfonyl)imide (TMPA-TFSI) and N-trimethyl-N-hexylammonium bis(trifluoromethylsulfonyl)imide (TMHA-TFSI) has been performed in the temperature range between 140 and 330 K by means of DSC and infrared spectroscopy measurements. In the low temperature crystalline states, a large concentration of the trans-TFSI conformer is present in TMPA-TFSI, while almost only cis-TFSI is retained in TMHA-TFSI. For the mixtures (TMPA-TFSI)100-x (TMHA-TFSI)x, at concentrations close to the extremes, solid crystalline phases are still present and they show a strong predominance of the trans conformer of the TFSI ion for x < 15 or a large concentration of the cis conformer of TFSI for x > 85. At intermediate concentrations (33 < x < 67) no crystalline phase develops at low temperatures and both conformers of TFSI survive in the whole temperature range investigated here. We suggest that the competition between the two TFSI conformers at low temperatures can be the origin of the lack of crystalline phases for intermediate concentrations and can be exploited as a valid tool to tailor the physical properties of the mixtures of ionic liquids.

Induction of mild enterocolitis in zebrafish Danio rerio via ingestion of Vibrio anguillarum serovar O1.

Vibrio anguillarum is the etiological agent of a fatal hemorrhagic disease known as vibriosis that affects a wide range of fish species, causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of our study was to evaluate the most effective way to induce mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using 2 methods of administration of V. anguillarum serotype O1 to adult zebrafish Danio rerio: via intraperitoneal injection and via ingestion of infected Artemia nauplii. The results showed that the intraperitoneal administration often caused massive fish death due to severe systemic involvement. In our experiments, the effect of intraperitoneal infection was evident 48 h post infection, with cumulative mortality within 7 d post infection with severe histopathological changes in kidney hematopoietic tissue and in the intestine. In contrast, oral infection via Artemia did not show systemic involvement and only a moderate degree of inflammatory influx of the mucosa, a partial recovery at 12 d post infection, and no mortality. For these reasons, oral infection with live food appears to be the most effective method to induce mild enteritis with a local inflammatory response.

Stabilization of different conformers of bis(trifluoromethanesulfonyl)imide anion in ammonium-based ionic liquids at low temperatures.

The infrared absorption spectra of two ionic liquids with bis(trifluoromethanesulfonyl)imide (TFSI) as an anion and ammonium with different alkyl chains as cations are reported as a function of temperature. Using the comparison with ab initio calculations of the infrared-active intramolecular vibrations, the experimental lines were ascribed to the various ions composing the ionic liquids. In the liquid state of the samples, both conformers of the TFSI ion are present. In the solid state, however, the two conformers survive in N-trimethyl-N-propylammonium bis(trifluoromethanesulfonyl)imide (TMPA-TFSI), while only cis-TFSI is retained in N-trimethyl-N-hexylammonium bis(trifluoromethanesulfonyl)imide (TMHA-TFSI). We suggest that the longer alkyl chains of the former compound stabilize the less stable conformer of TFSI by means of stronger interactions between anions and cations.

Adverse reaction after administration of progesterone

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Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer.

Expression and anatomical distribution of TrkB in the encephalon of the adult zebrafish (Danio rerio).

Neurotrophins are a family of growth factor primarily acting in the nervous system, throughout two categories of membrane receptors on the basis of their high (Trk receptors) or low (p75NTR) affinity. Both neurotrophins and Trk receptors are phylogenetically conserved and are expressed not only in the central and peripheral nervous system but also in non-nervous tissues of vertebrates and some invertebrates. The brain-derived neurotrophic factor (BDNF)/TrkB system plays an important role in the development, phenotypic maintenance and plasticity of specific neuronal populations. Considering that this system is poorly characterized in the central nervous system of teleosts, the expression and anatomical distribution of TrkB in the brain of the adult zebrafish using reverse transcriptase-polymerase chain reaction (RT-PCR), Western-blot and immunohistochemistry were analysed. Both the riboprobe and the antibody used were designed to map within the catalytic domain of TrkB. RT-PCR detected specific TrkB mRNA in brain homogenates, while Western-blot identified one unique protein band with an estimated molecular weight of 145kDa, thus corresponding with the TrkB full-length isiform of the receptor. Immunohistochemistry showed specific TrkB immunoreactivity in restricted areas of the encephalon, i.e. the hypothalamus and a specific neuronal subpopulation of the reticular formation. The present results demonstrate, for the first time, that, as in mammals, the encephalon of adult zebrafish expresses TrkB in specific zones related to food intake, behaviour or motor activity.

Cooperation of histone deacetylase inhibitors SAHA and valproic acid in promoting sodium/iodide symporter expression and function in rat Leydig testicular carcinoma cells.

The presence of the sodium/iodide symporter (NIS) is the prerequisite for the use of the radioiodine in the treatment of thyroid cancer. Thus, stimulators of NIS expression and function are currently investigated in cellular models of various human malignancies, also including extrathyroid cancers. In this study, we analyzed the effects of the histone deacetylase inhibitors (HDACi), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), on NIS expression and function in rat Leydig testicular carcinoma cells (LC540). LC540 cells were exposed to SAHA 3 µM and VPA 3 mM (alone and in combination), and cell viability evaluated by MTT assay and cell counting, NIS mRNA and protein levels by using, respectively, real-time RT-PCR and western blotting. NIS function was evaluated by iodide uptake assay. We found that both HDACi were able to stimulate the transcription of NIS gene, but not its protein expression, while the association of SAHA and VPA increased both NIS transcript and protein levels, resulting in significant sixfold enhancement of radioiodine uptake capacity of LC540 cells. These data demonstrate the presence of an epigenetic control of NIS expression in Leydig tumor cells, suggesting the possibility to use the combination of these two HDACi for a radioiodine-based treatment of these malignancies.

Passiflora incarnata L.: ethnopharmacology, clinical application, safety and evaluation of clinical trials.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Passiflora incarnata Linnaeus comprises approximately 520 species belonging to the Passifloraceae family. The majority of these species are vines found in Central or South America, with rare occurrence in North America, Southeast Asia and Australia. The genus Passiflora incarnata has long been used in traditional herbal medicine for the treatment of insomnia and anxiety in Europe, and it has been used as a sedative tea in North America. Furthermore, this plant has been used for analgesic, anti-spasmodic, anti-asthmatic, wormicidal and sedative purposes in Brazil; as a sedative and narcotic in Iraq; and for the treatment of disorders such as dysmenorrhoea, epilepsy, insomnia, neurosis and neuralgia in Turkey. In Poland, this plant has been used to treat hysteria and neurasthenia; in America, it has been used to treat diarrhoea, dysmenorrhoea, neuralgia, burns, haemorrhoids and insomnia. Passiflora incarnata L. has also been used to cure subjects affected by opiate dependence in India. This review aims to provide up-to-date information about the pharmacology, clinical efficacy and clinical safety of Passiflora incarnata L. based on the scientific literature. In particular, the methodological accuracy of clinical trials is analysed in accordance with current consolidated guidelines on reporting the clinical efficacy of herbal medicine, offering new insight into opportunities for future research and development. METHODS: A bibliographic investigation was performed by examining the available data on Passiflora incarnata L. from globally accepted scientific databases and search engines (Pubmed, Scopus and Web of Science, SciFinder and Google Scholar). We selected studies, case reports, and reviews addressing the pharmacology and safety of Passiflora incarnata. RESULTS: Although numerous Passiflora incarnata L. derivative products have been commercialised as alternative anxiolytic and sedative remedies based on their long tradition of use, their supposed efficacy does not appear to be adequately corroborated by the literature, with clinical studies often featuring inadequate methodologies and descriptions of the products under investigation. This medicinal plant has shown a wide spectrum of pharmacological activities in preclinical experiments, including anxiolytic, sedative, antitussive, antiasthmatic, and antidiabetic activities. The plant has a good safety profile. The clinical trials that we included in this review were designed to evaluate and in some cases confirm promising observations of preclinical pharmacological activity, and the methodological limits of these studies are characterised here. CONCLUSION: In conclusion, clinical studies on the effects of products containing herbal preparations based on Passiflora incarnata reveal crucial weaknesses such as poor details regarding the drug extract ratio, limited patient samples, no description of blinding and randomisation procedures, incorrect definition of placebo, and lack of intention to treat analysis. In conclusion, the results of this review suggest that new clinical trials should be conducted using a more rigorous methodology to assess the traditional putative efficacy of Passiflora incarnata L.

Sodium/iodide symporter is expressed in the majority of seminomas and embryonal testicular carcinomas.

Testicular cancer is the most frequent cancer in young men. The large majority of patients have a good prognosis, but in a small group of tumors, the current treatments are not effective. Radioiodine is routinely used in the treatment of thyroid cancer and is currently investigated as a potential therapeutic tool even for extra-thyroid tumors able to concentrate this radioisotope. Expression of Na(+)/I(-) symporter (NIS (SLC5A5)), the glycoprotein responsible for iodide transport, has been demonstrated in normal testicular tissue. In this study, we analyzed NIS expression in a large series of testicular carcinomas. Our retrospective series included 107 patients operated for testicular tumors: 98 typical seminomas, six embryonal carcinomas, one mixed embryonal choriocarcinoma, and two Leydig cells tumors. Expression and regulation of NIS mRNA and protein levels were also investigated in human embryonal testicular carcinoma cells (NTERA) by real-time RT-PCR and western blotting respectively. Immunohistochemical analysis showed the presence of NIS in the large majority of seminomas (90/98) and embryonal carcinomas (5/7) of the testis but not in Leydig cell carcinomas. Expression of NIS protein was significantly associated with lymphovascular invasion. In NTERA cells treated with the histone deacetylase inhibitors SAHA and valproic acid, a significant increase in NIS mRNA (about 60- and 30-fold vs control, P<0.001 and P<0.01 respectively) and protein levels, resulting in enhanced ability to uptake radioiodine, was observed. Finally, NIS expression in testicular tumors with the more aggressive behavior is of interest for the potential use of targeting NIS to deliver radioiodine in malignant cells.

Serotonin involvement in Rhodiola rosea attenuation of nicotine withdrawal signs in rats.

Rhodiola rosea has been used for centuries in the traditional medicine to stimulate nervous system, to enhance physical and mental performance and to treat fatigue. It is known that administration of Rhodiola rosea extract elicits antidepressant activity, but the mechanism of action still remains unclear. Evidence from animal models and human studies show that nicotine reduces symptoms of depression and that nicotine cessation induces depressive-like symptoms. We investigated the effects of Rhodiola rosea on nicotine withdrawal signs. Nicotine dependence was induced by subcutaneous nicotine injection (2 mg/kg, four times daily) for 14 days. Another group of animals treated with nicotine (for 14 days) and successively with Rhodiola rosea extract was co-administered with selective 5-HT receptorial antagonist WAY 100635 (1 mg/kg). After nicotine withdrawal animals were evaluated for behavioural parameters (locomotor activity, abstinence signs, marble burying test), diencephalic serotonin metabolism and serotonin receptor-1A expression. Results show a significant increase of 5-HT content in N treated with R. rosea, with a significant increase of serotonin receptor 1A, suggesting an involvement of serotonin in beneficial effects of R. rosea on suffering produced by nicotine withdrawal.

Ionic liquid-based membranes as electrolytes for advanced lithium polymer batteries.

Gel-type polymer electrolytes are formed by immobilizing a solution of lithium N,N-bis(trifluoromethanesulfonyl)imide (LiTFSI) in N-n-butyl-N-ethylpyrrolidinium N,N-bis(trifluoromethanesulfonyl)imide (Py24TFSI) ionic liquid (IL) with added mixtures of organic solvents, such as ethylene, propylene and dimethyl carbonates (EC, PC, and DMC, respectively), into a poly(vinylidenefluoride-co-hexafluoropropylene) (PVdF-HFP) matrix, and their properties investigated. The addition of the organic solvent mixtures results in an improvement of the ionic conductivity and in the stabilization of the interface with the lithium electrode. Conductivity values in the range of 10⁻³-10⁻²  S cm⁻¹ are obtained in a wide temperature range. These unique properties allow the effective use of these membranes as electrolytes for the development of advanced polymer batteries based on a lithium metal anode and an olivine-type lithium iron phosphate cathode.

Cytotoxic effects of a novel pyrazolopyrimidine derivative entrapped in liposomes in anaplastic thyroid cancer cells in vitro and in xenograft tumors in vivo.

In this study, we evaluated the activity of two novel pyrazolopyrimidine derivatives (Si 34 and Si 35) against ARO cells, a human anaplastic thyroid cancer cell line. ARO cells exposed to different concentrations of the drugs showed a reduced growth rate and an increase of mortality. After 72 h incubation, doses of 5 and 10 microM Si 34 determined a decrease of cell counts by approximately 25% and approximately 75% compared with those of control cells respectively. Similar findings were observed using Si 35. Treatment with both Si 34 and Si 35 at 10 microM increased cell mortality also ( approximately 29% and approximately 18% respectively). At these concentrations, a decrease in cyclin D1 levels was observed. To improve the biopharmaceutical properties, a liposome formulation was prepared. When entrapped in unilamellar liposomes, Si 34 exerted its cytotoxic effects even at lower doses (maximal inhibition at 5 microM) and after shorter incubation time (48 h) either in ARO or other thyroid cancer cell lines. The effects were associated with weak apoptotic death. Inhibition of epidermal growth factor-stimulated src and ERK phosphorylation, as well as reduction of migration properties of ARO cells was also observed. Moreover, the growth of tumor xenografts induced in severe combined immunodeficiency (SCID) mice was inhibited by i.v. administration of 25-50 mg/kg of the drug liposomal formulation. In conclusion, the liposomal preparation of this novel pyrazolopyrimidine derivative appears to be a promising tool for the treatment of anaplasic thyroid cancer.

Growth inhibition of medullary thyroid carcinoma cells by pyrazolo-pyrimidine derivates.

There is no effective treatment for recurrent or metastatic medullary thyroid carcinoma (MTC), a tumor arising from thyroid C-cells commonly presenting an inherited or acquired RET mutation. In this study we examined the sensitivity of two human MTC cell lines to novel pyrazolopyrimidine derivates, able to inhibit src-family tyrosine kinase activity. In TT cells [carrying the multiple endocrine neoplasia (MEN)2A Ret mutation Cys 634Trp] and MZ-CRC-1 cells (carrying the MEN2B RET mutation Met891Thr), one of these compounds, namely Si 34, determined a significant growth inhibitory effect (approximately 90% vs control for TT, 80% vs control for MZ-CRC-1) mainly due to enhanced cell mortality after a 6-day incubation. At concentrations that increased cell mortality, neither biochemical or morphological characteristics of apoptosis were detected in TT and MZCRC- 1 cells treated with Si 34. These results, when confirmed in other in vivo preclinical models, suggest that this novel tyrosine kinase inhibitor may be useful for the treatment of MTC.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro.

BACKGROUND AND PURPOSE: The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro. EXPERIMENTAL APPROACH: The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting. KEY RESULTS: NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.