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Introduction: This systematic review provides a comprehensive description of different surgical techniques for massive rotator cuff tears (MRCTs) using arthroscopic-assisted latissimus dorsi transfer (A-LDT), reporting clinical outcomes and complications., Sources of Data: We searched the literature on Medline (PubMed), Web of Science and Scopus databases using the combined keywords 'latissimus dorsi', 'tendon', 'transfer', 'rotator cuff tears', 'shoulder' and 'arthroscopy' to identify articles published in English, Spanish, French and Italian. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was followed for the manuscript selection., Areas of Agreement: Ten studies (five retrospective and five prospective investigations), all published between 2014 and 2018, fulfilled our inclusion criteria, dealing with 348 (55.7% male) patients, with a mean age of 61.6 years (range 31-83)., Areas of Controversy: A-LDT is a technical demanding procedure. When compared with the open technique, it does not seem to provide significant subjective and objective clinical outcome improvements., Growing Points: A-LDT seems to yield lesser surgical complications and post-operative stiffness. Sparing the deltoid muscle belly could result in a more effective shoulder post-surgery function., Areas Timely for Developing Research: Further comparative randomized controlled trials with longer follow-up are needed to clarify the potentially promising superiority of A-LDT.

Metaplasticity, defined as the plasticity of synaptic plasticity, could affect learning and memory at different neural levels. It was hypothesized that metaplasticity changes on glutamate receptors may affect memory destabilization, promoting or preventing reconsolidation. We investigated the metaplastic effect of NMDA channel blocker MK-801 on sucrose instrumental memory reconsolidation in a behavioural rat model associated to the assessment of molecular markers of metaplasticity, memory retrieval, destabilization and reconsolidation. Following instrumental conditioning and forced abstinence, rats were intraperitoneally treated with MK-801 or vehicle 24 h before the exposure to memory retrieval or not-retrieval. Separate groups were tested for in-vivo extinction of responding (24 h and 7 d after reactivation) or ex-vivo assessment of transcription factor Zif268 and ribosomal protein rpS6 phosphorylation in nucleus accumbens (NAc) and amygdala (Amy). MK-801 significantly inhibited instrumental responding at extinction test, suggesting reconsolidation blockade of instrumental memory. The decrease of Zif268 and phosphorylated-rpS6 levels in NAc and Amy in MK-801/Retrieval vs. Vehicle/Retrieval group supported the behavioural findings. An increase of GluN2B, GluA1 and mGluR5 in NAc, and GluN2B in Amy, 24 h after MK-801 indicated the trigger of associated metaplastic changes. Our findings show that metaplastic changes induced by NMDA receptors blockade affected sucrose instrumental memory retrieval as shown by both behavioural and molecular changes. We hypothesize that these findings however suggested a switch to extinction rather than a reconsolidation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The drug commonly known as U-47700 is a strong μ-opioid agonist with an approximate potency 7.5 times higher than morphine. It has been available in Europe since 2014, where it is usually sold through the internet or black market as an abuse morphine-like substance. In the case reported here, a Caucasian man was found dead in his apartment. Next to the body, the police seized one transparent plastic bag containing a white powder and two amber glass bottles with nasal spray containing few milliliters of a transparent liquid. During the autopsy, no evidence of natural disease or trauma was found to account for the death. Blood, urine and pubic hair were collected and submitted for toxicological analysis. The content of the seized materials was also submitted to a general screening analysis in order to determine its composition. U-47700 was detected in blood, urine and hair samples using an UHPLC/MS-MS method purposely developed. The blood and urine concentrations were 380 and 10,400 ng/mL, respectively. No other drugs of abuse nor ethanol were found in blood and urine specimens. Pubic hair analysis revealed a frequent past exposure to U-47700. Finally, U-47700 was identified as the main component of the powder and the liquids contained in the nasal spray bottles. The combined circumstantial elements and toxicological results of the case revealed the occurrence of an acute intoxication produced by U-47700 abuse. To the best of our knowledge, this is the first fatal intoxication case reported on the Italian territory involving the synthetic opioid U-47700.

Hypoxic-ischemic encephalopathy (HIE) is a major cause of perinatal mortality and subsequent severe neurological sequelae. Mild hypothermia is a standard therapy for HIE, but is used only in selected Reference Centers and in neonates >1800 g. Since neuronal death following HIE occurs by a cascade of events triggered by activation of glutamate receptors, we used in vitro and in vivo models of HIE to examine whether the AMPA/kainate receptor antagonist topiramate and the NMDA receptor antagonist memantine could exert neuroprotective effects, alone or in combination with hypothermia. For the in vitro experiments, rat organotypic hippocampal slices were exposed to a 30 min duration of oxygen-glucose deprivation (OGD): treatment with topiramate (1 μM) and memantine (10-30 μM) or hypothermia (35 °C or 32 °C) significantly attenuated CA1 damage after 24 h. The combination of hypothermia with topiramate and memantine enhanced their protective effect. For the in vivo experiments, we used 7 day-old rat pups subjected to permanent left common carotid artery occlusion followed by 120 min of hypoxia. Administration of topiramate or memantine (i.p., 20 mg/kg) immediately and 2 h after hypoxia or exposure to hypothermia (32 °C for 4 h beginning 1 h after hypoxia) significantly reduced the extent of the resulting infarct. The combination of topiramate or memantine with hypothermia elicited a reduction of the infarct that was greater than that produced by drugs or hypothermia alone. Notably, memantine displayed a higher degree of neuroprotection as compared to topiramate both in vitro and in vivo and, when used alone at 20 mg/kg in vivo, produced a greater reduction in brain damage than observed using topiramate in combination with hypothermia. These results suggest that memantine may be more advantageous than topiramate as a therapeutic agent in neonates with HIE treated with hypothermia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Purpose: Dexpramipexole, a drug recently tested in patients with amyotrophic lateral sclerosis (ALS,) is able to bind F1Fo ATP synthase and increase mitochondrial ATP production. Here, we have investigated its effects on experimental ischaemic brain injury., Experimental Approach: The effects of dexpramipexole on bioenergetics, Ca 2+ fluxes, electrophysiological functions and death were evaluated in primary neural cultures and hippocampal slices exposed to oxygen-glucose deprivation (OGD). Effects on infarct volumes and neurological functions were also evaluated in mice following proximal or distal middle cerebral artery occlusion (MCAo). Distribution of dexpramipexole within the ischaemic brain was evaluated by means of mass spectrometry imaging., Key Results: Dexpramipexole increased mitochondrial ATP production in cultured neurons or glia and reduces energy failure, prevents intracellular Ca 2+ overload and affords cytoprotection when cultures are exposed to OGD. This compound also counteracted ATP depletion, mitochondrial swelling, anoxic depolarization, loss of synaptic activity and neuronal death in hippocampal slices subjected to OGD. Post-ischaemic treatment with dexpramipexole, at doses consistent with those already used in ALS patients, reduced brain infarct size and ameliorated neuroscore in mice subjected to transient or permanent MCAo. Notably, the concentrations of dexpramipexole reached within the ischaemic penumbra equalled those found neuroprotective in vitro., Conclusion and Implications: Dexpramipexole, a compound able to increase mitochondrial F1Fo ATP-synthase activity reduced ischaemic brain injury. These findings, together with the excellent brain penetration and favourable safety profile in humans, make dexpramipexole a drug with realistic translational potential for the treatment of stroke., Linked Articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc., (© 2017 The British Pharmacological Society.)

Background: Synthetic opioids are compounds that were created to act on the opioid receptors. Novel synthetic opioids include various analogs of fentanyl (e.g., acetylfentanyl, acryloylfentanyl, carfentanil, furanylfentanyl, 4-fluorobutyrylfentanyl or ocfentanil) and newly emerging non-fentanyl compounds with different chemical structures, such as AH-7921, MT-45, and U-47700. In the last years, these drugs have rapidly emerged on the recreational drug market, and their abuse has been increasing worldwide. Due to the high potency and the low dose required to produce desired effects, the risk of overdose for these compounds including severe health implications, is quite high. Several fatal intoxication cases related to the abuse of synthetic opioids have recently been reported in the literature., Conclusion: As a consequence, the detection of these compounds in biological samples is crucial in order to get a better understanding of their concentration and distribution in body fluids. We overviewed the analytical approaches for the investigation of synthetic opioids in postmortem samples reported in the literature, with special emphasis given to cases of lethal intoxication., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The determination at low concentrations of common psychotropic drugs is increasingly requested in hair samples for the retrospective investigation of habitual drug abuse and dependence as well as in other toxicological investigations. The dramatic improvements of the instrumentation based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) make the detection of tiny amounts of almost whatever drug is in hair possible, even after single-dose intake. Therefore, LC-MS/MS is gradually replacing gas chromatographic techniques in both screening and confirmation procedures, and is increasingly acknowledged as the technique of choice for hair analysis. We describe a simple procedure for the quantitative determination in hair samples of 15 common drugs of abuse, or metabolites, based on methanol extraction and direct analysis by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

The interferon-gamma (IFN-γ) test measures cell mediated immune response (CMI) during the early stages of tuberculosis infection. Although Bovine Tuberculosis (BT) spread in feral pigs is widely documented in literature, the effectiveness of IFN-γ in this species has been only recently reported. One of the major obstacle of this assay is that whole blood samples should be stimulated with purified protein derivative (PPD) cocktail within 8 h from the blood sampling. This study set up a defined broth culture in which lymphocytes, the cell population predominantly responsible for IFN-γ production, are maintained in a steady-state and their vitality is preserved. The IFN-γ production measured from the samples added with the maintenance medium and stored at 4 °C was similar to the enzyme-linked immunosorbent assay (ELISA) optical density values obtained from the same assay performed within 8 h from sampling., (Copyright © 2017. Published by Elsevier Ltd.)

Many neurological disorders of gluten-related diseases (GRD), not directly referable to the gastrointestinal tract, have been reported in association with celiac disease (CD), including ataxia, neuropathy and epilepsy. In particular, people with epilepsy diagnosed with CD seems to be characterized by intractable seizure. In these patients, gluten restriction diet has resulted in a reduction of both seizure frequency and antiepileptic medication. Many hypotheses have been suggested, however, molecular mechanisms that associates GRD and epileptogenesis are yet unknown. In this study, we examined the effects of the toxic gliadin peptide 31-43 in in vivo and in vitro models of kainate-induced-epilepsy. We observed that p31-43 exacerbates kainate neurotoxicity in epilepsy models, through the involvement of the enzymatic activity of transglutaminases. Moreover, electrophysiological recordings in CA3 pyramidal neurons of organotypic hippocampal slices show that p31-43 increases the inward current induced by kainate, the average sEPSC amplitude and the total number of evoked action potentials when applicated alone, thus suggesting that p31-43 is able to influence CA3-CA1 neurotransmission and can potentiate postsynaptic kainate receptors. Our results suggest a possible mechanism underlying the relationship between GRD and epilepsy through a potentiation of kainate-induced neurotoxicity and links the toxic effects of gluten to epilepsy.

Background: Data on both known and unknown drug use in the electronic dance music (EDM) scene is important to inform prevention and harm reduction. While surveys are the most common method of querying drug use, additional biological data can help validate use and detect unknown/unintentional use of drugs such as new psychoactive substances (NPS). We sought to determine the extent of both known and unknown use of various substances in this high-risk scene., Methods: We hair-tested 90 self-reported past-year ecstasy/MDMA/Molly users attending EDM parties in New York City during the summer of 2016 using UHPLC-MS/MS. Results were compared to self-reported past-year use., Results: Three quarters (74.4%) tested positive for MDMA, a third (33.3%) tested positive for an NPS, and 27.8% tested positive specifically for one or more synthetic cathinones (e.g., butylone, ethylone, pentylone, methylone, alpha-PVP). Half (51.1%) of participants tested positive for a drug not self-reported, with most testing positive for synthetic cathinones (72.0%), methamphetamine (69.0%), other NPS stimulants (e.g., 4-FA, 5/6-APB; 66.7%), or new dissociatives (e.g., methoxetamine, diphenidine; 60.0%). Attending parties every other week or more often, reporting higher-frequency ecstasy pill use, having tested one's ecstasy, and having found out one's ecstasy was adulterated, were risk factors for testing positive for synthetic cathinones and NPS in general., Conclusion: Hair testing appears to be a valuable addition to drug epidemiology studies. Many EDM party attendees-even those who test their ecstasy-are unknowingly using NPS and/or other drugs. Prevention information and harm reduction may help reduce unknown/unintentional use., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Transcriptional deregulation emerges as a key pathogenetic mechanism in ALS pathogenesis, and non-class-specific histone deacetylase (HDACs) inhibitors proved of therapeutic efficacy in preclinical models of ALS. When tested in patients, however, these drugs failed, probably because of a lack of selectivity toward pathogenetic HDACs. Here, we studied the effects of MC1568, an inhibitor of Class-II HDACs which have been reported to contribute to ALS pathogenesis. We focused on transcriptional regulation of glutamate transporter EAAT2, whose reduced expression may contribute to motor neuron degeneration in ALS. We report that MC1568 highly increased EAAT2 transcripts in primary cultures of mouse glia, but these increases did not correlate with increased glutamate uptake capacity. Accordingly, we found that MC1568 augmented protein expression of EAAT2 together with its sumoylation, a post-translational modification typically altering protein function and localization. When tested in SOD1G93A mice, however, MC1568 fully restored the reduced spinal cord expression of EAAT2 and glutamate uptake up to control levels. A prolonged treatment with MC1568 (from onset to end stage) was unable to prolong survival of mice. Data reveal a key role of Class-II HDACs in expression and function of glutamate transporter, further corroborating preclinical and clinical evidence that the sole restoration of glutamate uptake is not of therapeutic relevance to ALS therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Diphenidine is a dissociative drug that shows several psychotropic effects including euphoria, shifts in perception of reality, hallucinations, and transient anterograde amnesia. In this study, a case of acute intoxication occurring after diphenidine intake is reported. A 30-year-old Caucasian male was hospitalized after he was found in a confused and agitated state and unable to communicate. The physical examination displayed tachycardia, miotic pupils, and increased both body temperature and respiratory rate. After a liquid-liquid extraction procedure, GC/MS analysis revealed the presence of diphenidine in plasma and urine at concentrations of 308 and 631 ng/mL, respectively. Methylphenidate and diclazepam were also detected in the plasma. The clinical progress of the patient was favorable, and his symptoms were cured with a symptomatic treatment. The combined circumstantial elements and toxicological results of the case reported revealed the occurrence of an acute intoxication ascribable to the recreational abuse of diphenidine., (© 2017 American Academy of Forensic Sciences.)

Hundreds of new psychoactive substances (NPS) have emerged in the drug market over the last decade. Few drug surveys in the USA, however, ask about use of NPS, so prevalence and correlates of use are largely unknown. A large portion of NPS use is unintentional or unknown as NPS are common adulterants in drugs like ecstasy/Molly, and most NPS are rapidly eliminated from the body, limiting efficacy of urine, blood and saliva testing. We utilized a novel method of examining prevalence of NPS use in a high-risk population utilizing hair-testing. Hair samples from high-risk nightclub and dance music attendees were tested for 82 drugs and metabolites (including NPS) using ultra-high performance liquid chromatography-tandem mass spectrometry. Eighty samples collected from different parts of the body were analyzed, 57 of which detected positive for at least one substance-either a traditional or new drug. Among these, 26 samples tested positive for at least one NPS-the most common being butylone (25 samples). Other new drugs detected include methylone, methoxetamine, 5/6-APB, α-PVP and 4-FA. Hair analysis proved a powerful tool to gain objective biological drug-prevalence information, free from possible biases of unintentional or unknown intake and untruthful reporting of use. Such testing can be used actively or retrospectively to validate survey responses and inform research on consumption patterns, including intentional and unknown use, polydrug-use, occasional NPS intake and frequent or heavy use., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes. In particular, the latter cells provide an opportunity to investigate in parallel the electrophysiological and biochemical modifications that take place when the cells are exposed for various lengths of time to variously aggregated wild-type TTR, a condition that characterizes senile systemic amyloidosis. In this study, we investigated biochemical and electrophysiological modifications in cardiomyocytes exposed to amyloid oligomers or fibrils of wild-type TTR or to its T4-stabilized form, which resists tetramer disassembly, misfolding, and aggregation. Amyloid TTR cytotoxicity results in mitochondrial potential modification, oxidative stress, deregulation of cytoplasmic Ca 2+ levels, and Ca 2+ cycling. The altered intracellular Ca 2+ cycling causes a prolongation of the action potential, as determined by whole-cell recordings of action potentials on isolated mouse ventricular myocytes, which may contribute to the development of cellular arrhythmias and conduction alterations often seen in patients with TTR amyloidosis. Our data add information about the biochemical, functional, and viability alterations that occur in cardiomyocytes exposed to aggregated TTR, and provide clues as to the molecular and physiological basis of heart dysfunction in sporadic senile systemic amyloidosis and familial amyloid cardiomyopathy forms of TTR amyloidoses., (Copyright © 2016. Published by Elsevier Inc.)

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 μM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10-100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Chronic osteomyelitis is a challenging problem, and malignant transformation is a rare occurrence. We report a case of a patient with squamous cell carcinoma arising from an osteomyelitic hotbed and discuss through a literature review the etiopathogenesis, diagnosis, and treatment of this lesion., Case Presentation: A 69-year-old Italian man had sustained an exposed tibial fracture 40 years ago during a road accident, for which he had undergone various surgical osteosynthesis treatments with multiple antibiotic therapies. He presented to our hospital because of recurrence of a fistula at the proximal third of the anterior region of the tibia. For 2 months, we treated the lesion with antibiotics, and local medication with curettage. We saw no evidence of lesion improvement, and we advised the patient to undergo a knee amputation, which he refused. The alternative we chose was a surgical toilet of the osteomyelitic hotbed and used bioglass as a bone substitute. After 2 months of follow-up, we noticed a fulminating, budding formation in the area of the surgical wound that turned out to be a squamous cell carcinoma on biopsy. The patient again refused the amputation and underwent a wide-margin surgical debridement. After 2 months, the carcinoma recurred, and an above-the-knee amputation was performed., Conclusions: Our experience with this case indicates that amputation is the most appropriate treatment for squamous carcinoma occurring in patients with chronic osteomyelitis. To avoid risks of lymphonodular and organ metastasization, this radical surgical procedure should not be delayed. Early diagnosis and timely therapy can prevent amputation only in selected cases. Surgeons who treat osteomyelitis and chronic wounds should be aware of the risk of tumor degeneration. Squamous cell carcinoma associated with chronic osteomyelitis has a low-grade malignancy, but implications of lymphonodular involvement and organ metastasis should not be excluded.

The Technical Document TD2014EAAS was drafted by the World Anti-Doping Agency (WADA) in order to fight the spread of endogenous anabolic androgenic steroids (EAAS) misuse in several sport disciplines. In particular, adoption of the so-called Athlete Biological Passport (ABP) - Steroidal Module allowed control laboratories to identify anomalous EAAS concentrations within the athletes' physiological urinary steroidal profile. Gas chromatography (GC) combined with mass spectrometry (MS), indicated by WADA as an appropriate technique to detect urinary EAAS, was utilized in the present study to develop and fully-validate an analytical method for the determination of all EAAS markers specified in TD2014EAAS, plus two further markers hypothetically useful to reveal microbial degradation of the sample. In particular, testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, dehydroepiandrosterone, 5α-dihydrotestosterone, were included in the analytical method. Afterwards, the multi-parametric feature of ABP profile was exploited to develop a robust approach for the detection of EAAS misuse, based on multivariate statistical analysis. In particular, Principal Component Analysis (PCA) was combined with Hotelling T(2) tests to explore the EAAS data obtained from 60 sequential urine samples collected from six volunteers, in comparison with a reference population of single urine samples collected from 96 volunteers. The new approach proved capable of identifying anomalous results, including (i) the recognition of samples extraneous to each of the individual urine series and (ii) the discrimination of the urine samples collected from individuals to whom "endogenous" steroids had been administrated with respect to the rest of the samples population. The proof-of-concept results presented in this study will need further extension and validation on a population of sport professionals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation., Methods: Rat organotypic hippocampal slice cultures were exposed for 7 days to EtOH (100 to 300 mM) after 2 days (immature) or 10 days (mature) of culture in vitro; EtOH was then removed from the medium, and 24 hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus., Results: EtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre- and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7 days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole-cell patch-clamp recordings showed that incubation with EtOH for 7 days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSC amplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH., Conclusions: These results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus in vitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders., (Copyright © 2016 by the Research Society on Alcoholism.)

The detection of new psychoactive substances (NPS) in hair proved to provide insight into their current diffusion among the population and the social characteristics of these synthetic drugs' users. Therefore, a UHPLC-MS/MS method was developed in order to determine 31 stimulant and psychedelic substituted phenethylamines, and dissociative drugs in hair samples. The method proved to be simple, fast, specific, and sensitive. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method showed optimal linearity in the interval 10-1000 pg/mg, with correlation coefficient values varying between 0.9981 and 0.9997. Quantitation limits ranged from 1.8 pg/mg for 4-methoxyphencyclidine (4-MeO-PCP) up to 35 pg/mg for 6-(2-aminopropyl)benzofuran (6-APB). The method was applied to (i) 23 real samples taken from proven MDMA and ketamine abusers and (ii) 54 real hair samples which had been previously tested negative during regular drug screening in driver's license recovery. Six samples tested positive for at least one target analyte. Methoxetamine (MXE) was found in three cases (range of concentration 7.7-27 pg/mg); mephedrone (4-MMC) was found in two cases (50-59 pg/mg) while one sample tested positive for methylone at 28 pg/mg. Other positive findings included 4-methylethcathinone (4-MEC), alpha-pyrrolidinovalerophenone (α-PVP), 4-fluoroamphetamine (4-FA), 3,4-methylenedioxypyrovalerone (MDPV), and diphenidine. The present study confirms the increasing diffusion of new designer drugs with enhanced stimulant activity among the target population of poly-abuse consumers.

Ischemic tolerance is an endogenous defense program in which exposure to a subtoxic preconditioning insult results in resistance to a subsequent, otherwise lethal, episode of ischemia. Herein, we evaluated the role of histone acetylation/deacetylation in an in vitro model of preconditioning, using rat organotypic hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to CA1 injury 24 h later: tolerance was induced by exposing the slices to preconditioning bouts of NMDA (3 μM for 60 min) 24 h prior to the toxic OGD challenge. Under these conditions, CA1 damage induced by OGD was reduced. The induction of tolerance was prevented by incubating the slices with HDAC inhibitors. NMDA preconditioning was associated with a mild increase in poly(ADP-ribose) polymerase (PARP) activity that was apparently followed, 3 h later, by a mild increase in histone acetylation. Use of PARP and HDAC inhibitors suggests a possible interaction between PARP and HDAC activities in the development of ischemic tolerance. Finally, both PARP and HDAC inhibitors were able to prevent the increase in pERK1/2 induced by NMDA preconditioning. We propose a model in which mild histone acetylation and PARP activity cooperate in producing a neuroprotective response in the development of ischemic tolerance., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

FLAG is an affinity tag widely used for rapid and highly specific one-step protein purification. Native elution of protein from anti-FLAG antibody resins allows the identification of protein and nucleic acid binding partners and functional analysis using biochemical activity assays., (© 2015 Elsevier Inc. All rights reserved.)

A significant number of laboratories observed that poly (ADP-ribose) polymerase (PARP) inhibitors, administered a few hours after ischemic or traumatic brain injury, may drastically reduce the subsequent neurological damage. It has also been shown that PARP inhibitors, administered for 24 hours to rats with permanent middle cerebral artery occlusion (MCAO), may reduce the number of dying neurons for a long period after surgery, thus suggesting that these agents could reduce the delayed brain damage and the neurological and cognitive impairment (dementia) frequently observed a few months after a stroke. In organotypic hippocampal slices exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), an alkylating agent able to activate PARP, a selective and delayed degeneration of the CA1 pyramidal cells which was anatomically similar to that observed after a short period of oxygen and glucose deprivation (OGD) has been described. Biochemical and electrophysiological approaches showed that MNNG exposure caused an increased expression and function of the calcium permeable α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels in the CA1 but not in the CA3 hippocampal region. PARP inhibitors prevented this increase and reduced CA1 cell death. The AMPA receptor antagonist 2,3-dihydroxy-6- nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione or the selective Ca(2+) permeable AMPA channel blocker 1-Naphthyl acetyl spermine (NASPM), also reduced the MNNG-induced CA1 pyramidal cell death. Since activation of PARP-1 facilitate the expression of Ca(2+) permeable channels and the subsequent delayed cell death, PARP inhibitors administered a few hours after a stroke may not only reduce the early post-ischemic brain damage but also the late neuronal death frequently occurring after severe stroke.

This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities., (© 2015 Elsevier Inc. All rights reserved.)

Background: Buprenorphine (BUP) is a psychoactive pharmaceutical drug largely used to treat opiate addiction. Short-term therapeutic monitoring is supported by toxicological analysis of blood and urine samples, whereas long-term monitoring by means of hair analysis is rarely used. Aim of this work was to develop and validate a highly sensitive ultrahigh-performance liquid chromatography tandem mass spectrometry method to detect BUP and norbuprenorphine (NBUP) in head hair., Methods: Interindividual correlation between oral dosage of BUP and head hair concentration was investigated. Furthermore, an intra-individual study by means of segmental analysis was performed on subjects with variable maintenance dosage. Hair samples from a population of 79 patients in treatment for opiate addiction were analyzed., Results: The validated ultrahigh-performance liquid chromatography tandem mass spectrometry protocol allowed to obtain limits of detection and quantification at 0.6 and 2.2 pg/mg for BUP and 5.0 and 17 pg/mg for NBUP, respectively. Validation criteria were satisfied, assuring selective analyte identification, high detection capability, and precise and accurate quantification. Significant positive correlation was found between constant oral BUP dosage (1-32 mg/d) and the summed up head hair concentrations of BUP and NBUP. Nevertheless, substantial interindividual variability limits the chance to predict the oral dosage taken by each subject from the measured concentrations in head hair. In contrast, strong correlation was observed in the results of intra-individual segmental analysis, which proved reliable to detect oral dosage variations during therapy., Conclusions: Remarkably, all hair samples yielded BUP concentrations higher than 10 pg/mg, even when the lowest dosage was administered. Thus, these results support the selection of 10 pg/mg as a cutoff value.

The distribution of mephedrone in the body fluids and tissues of a subject found dead after the concomitant intake of cocaine and mephedrone is reported. Mephedrone (4-methylmethcathinone) is a designer drug of the phenethylamine family that is able to cause central nervous system stimulation, psychoactivity and hallucinations and that is becoming popular among youth as a recreational drug. Mephedrone has been available in Europe since 2007, and it is sold through the internet and by local shops as bath salt or plant food. In the case reported here, a 25-year-old man was found dead in the apartment of a friend after a night spent in several local clubs. A fragment of a blue diamond-shaped pill was found in the pocket of the trousers worn by the decedent. During the autopsy, no evidence of natural disease or trauma was found to account for this death. Blood, urine and gastric content samples were collected and submitted for toxicological analysis. Moreover, bile, brain, lung and hair samples were collected as additional matrices. The content of the pill was submitted to a general screening analysis in order to determine its composition. Mephedrone was detected in the blood, urine, gastric contents and in the additional matrices using an expressly validated GC/MS method. The blood and urine concentrations were 1.33mg/L and 144mg/L, respectively. Contextually, cocaine and cocaethylene were found in the blood and urine specimens. The distribution of mephedrone in the body organs was evaluated by analyzing the brain, bile and lung specimens. Hair analysis revealed a past exposure to mephedrone, ketamine, MDMA and cocaine. Sildenafil was identified as the main component of the blue, diamond-shaped pill. The quantitative determination of mephedrone in several body fluids and tissues provides significant knowledge about the distribution of this new drug of abuse in the human body after massive ingestion., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Activation of poly(ADP-ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper-reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl-dimethylaminomethyl-thieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma-like events to airway damage. Ovalbumin-sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma-like signs, bronchial hyper-reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1-10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen-induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ-treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8-hydroxy-2'-deoxyguanosine and the lung content of pro-inflammatory cytokines (TNF-α, interleukin (IL)-1β, IL-5, IL-6 and IL-18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ-treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

We report a rare case of acute posterior multifocal placoid pigment epitheliopathy (APMPPE) in an elderly patient and a review of the recent scientific literature. A 65-year-old Caucasian man, forty-five days after return from tropical travel he developed right-sided facial palsy with right hemiplegia, dysarthria and a positive Babinski sign. He started prednisone 90 mg/day. Clinical, laboratory and imaging examinations were reported. Five months later there was a complete neurological and ophthalmological recovery. Although APMPPE was described primarily in young adults, we report the first case of APMPPE associated with neurological involvement in an older patient with a complete restitutio ad integrum.

Purpose: To investigate long-term retinal changes after microincision pars plana vitrectomy surgery (MIVS) and internal limiting membrane (ILM) peeling outcome in retinitis pigmentosa (RP) patients affected by vitreomacular traction syndrome (VMT) with higher vitreous surface adhesion or coexisting epiretinal membrane (ERM)., Methods: Eight RP patients suffering from VMT were evaluated by means of best corrected visual acuity (BCVA), anterior and posterior binocular examination, spectral-domain optical coherence tomography (SD-OCT), MP-1 microperimetry (MP-1), and full-field electroretinogram (ERG), before MIVS and ILM peeling and during the 36-month follow-up. Patients were hospitalized for two days after the surgery. Surgical procedure was performed following this schedule: surgical removal of crystalline lens (MICS), MIVS with 23-gauge sutureless system trocars, core vitreous body removal, and balanced-sterile-salin-solution- (BSS-) air-gas (SF6) exchange., Results: All patients presented visual acuity (VA) increase after MIVS. None of the patients developed ocular hypertension or vitreomacular adhesions during the 3-year follow-up. MP-1 bivariate contour ellipse area (BCEA) was reduced in its dimensions and improved in all patients demonstrating a better fixation., Conclusions: MIVS could be the gold standard therapy in RP patients with VMT and higher vitreous surface adhesion or coexisting ERM if medical therapy is not applicable or not effective.

This protocol outlines a procedure for testing whether two proteins interact. A target protein will be immunoprecipitated using an antibody that recognizes it (or a tagged version of the protein). The immunoprecipitated material will be separated by SDS-PAGE and analyzed by Western blotting to assess the presence of a candidate interacting protein(s)., (© 2014 Elsevier Inc. All rights reserved.)

Hair testing has been used in toxicological investigations for the last two decades, but only recently a remarkable extension of hair analysis to a variety of application fields was observed, besides drug abuse recognition. The dramatic improvements of LC-MS/MS instrumentation make the detection of tiny amounts of almost whatever drug in hair possible, even after single-dose intake. The progresses observed during the last 5 years in the detection of psychoactive substances in hair are reviewed herein. The literature is partitioned according to the target compounds, namely traditional drugs of abuse, new psychoactive substances and pharmaceutical psychoactive substances. The LC-MS/MS methods presented are addressed to determine a single class of drugs, with the primary aim of accurate quantitation, or to perform multiclass analysis, for rapid and effective screening protocols.

The increasing role of hair analysis in forensic toxicological investigations principally owes to recent improvements of mass spectrometric instrumentation. Research achievements during the last 6 years in this distinctive application area of analytical toxicology are reviewed. The earlier state of the art of hair analysis was comprehensively covered by a dedicated book (Kintz, 2007a. Analytical and practical aspects of drug testing in hair. Boca Raton: CRC Press and Taylor & Francis, 382 p) that represents key reference of the present overview. Whereas the traditional organization of analytical methods in forensic toxicology divided target substances into quite homogeneous groups of drugs, with similar structures and chemical properties, the current approach often takes advantage of the rapid expansion of multiclass and multiresidue analytical procedures; the latter is made possible by the fast operation and extreme sensitivity of modern mass spectrometers. This change in the strategy of toxicological analysis is reflected in the presentation of the recent literature material, which is mostly based on a fit-for-purpose logic. Thus, general screening of unknown substances is applied in diverse forensic contexts than drugs of abuse testing, and different instrumentation (triple quadrupoles, time-of-flight analyzers, linear and orbital traps) is utilized to optimally cope with the scope. Other key issues of modern toxicology, such as cost reduction and high sample throughput, are discussed with reference to procedural and instrumental alternatives., (Copyright © 2012 Wiley Periodicals, Inc .)

Forensic investigations involving acute or lethal intoxication, drug-facilitated sexual assault, driving or workplace impairment frequently require the analysis of fresh or postmortem blood samples to check out a wide variety of pharmaceutical and illicit drugs, even after single-dose consumption. A sensitive and selective ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) screening method was developed for fast screening of 88 psychoactive drugs and metabolites in blood samples, including the ones most frequently involved in acute intoxications and forensic investigations in Italy. The new method allows short sample processing and analysis time (the whole procedure can be accomplished in less than 30 min) together with the simultaneous monitoring of a large number of pharmaceutical substances. These features represent crucial factors in the approach of acute intoxications, when the patient requires urgent and appropriate therapy. Blood sample treatment was limited to protein precipitation. Two UHPLC-MS/MS runs in positive and negative electrospray ionization modes were performed. The data were acquired at unit mass resolution in the selected reaction monitoring mode. According to international guidelines, linearity range, precision, trueness, detection and quantification limits, recovery, selectivity, specificity, carryover, and matrix effect phenomena were determined. Despite the limited sample purification and the inherent decreased chance of eliminating any potential interference, the present multiresidue screening method proved extremely effective and sensitive, allowing the detection of all tested drugs, even those belonging to structurally different classes of substances. Moreover, the developed method is easily susceptible to further expansion to encompass more drugs, either new or those becoming important for criminal investigation. This protocol was also applied to the analysis of authentic blood samples collected from victims of various crimes in routine casework, whose relevance in forensic investigations is presented in five cases.

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Although treatment of stroke patients with mild hypothermia is a promising therapeutic approach, chemicals inducing prompt and safe reduction of body temperature are an unmet need. We measured the effects of the transient receptor potential vanilloid-1 (TRPV1) agonist rinvanil on thermoregulation and ischemic brain injury in mice. Intraperitoneal or intracerebroventricular injection of rinvanil induces mild hypothermia that is prevented by the receptor antagonist capsazepine. Both intraischemic and postischemic treatments provide permanent neuroprotection in animals subjected to transient middle cerebral artery occlusion (MCAo), an effect lost in mice artificially kept normothermic. Data indicate that TRPV1 receptor agonists are promising candidates for hypothermic treatment of stroke.

In drug-facilitated crimes, victims are subjected to nonconsensual acts while they are incapacitated by the effects of a drug. A specific LC-MS/MS protocol for determining benzodiazepines and hypnotics at low concentration in hair specimens was developed and validated in order to target the allegedly administered drugs on a chronological basis. In the case hereby reported, a 26-year-old woman claimed to have been sexually assaulted after being administered an allegedly drugged coffee, but toxicological analysis of urine and blood provided no evidence of any drug intake. Subsequently, a second woman accused the same man of sexual abuse. Hence, the suspect was prosecuted. Specimens were collected from four subjects (two alleged victims, the suspect and his wife) and segmental hair analysis was performed. The results revealed that zolpidem was present at low picogram per milligram concentration in three out of eleven segments of hair specimen obtained from the first of the alleged victims, offering plain evidence of single or sporadic exposure, whereas the agent was detected in the high picogram per milligram range in the hair collected from suspect's wife, coherently with therapeutic administration. The presence of interfering signals typical of the keratin-containing matrix was found and possible hair degradation by cosmetic treatments was investigated by electron microscopy, so as to obtain a judicious interpretation of the analytical findings.

A case of intoxication by haloperidol is reported. Haloperidol is a butyrophenone derivative commonly used in many hospital units as an antipsychotic agent. Adverse reactions due to haloperidol intoxication include drowsiness, blurred vision, extrapyramidal effects, tardive dyskinesia, tachycardia, hypotension and muscular rigidity. In August 2008, a 49 year-old female nurse started feeling various symptoms such as muscular rigidity, drowsiness and buccal dyskinesia. After 3 months, she was hospitalized for the worsening of these symptoms. Four months later, she showed once more the same symptoms. Two open water bottles from which the nurse used to drink in the hospital were confiscated and analyzed. Moreover, the nurse was asked to give a sample of her hair for executing the inherent toxicological analyses. Haloperidol was found in both bottles 1 and 2 at a concentration of 31.5 μg/mL and 43.6 μg/mL, respectively. Based on segmental hair analysis, it was deduced that the nurse consumed haloperidol in the approximate period from August 2008 to March 2009. The higher levels of haloperidol in hair were found in accordance with the periods of most severe appearance of symptoms, requiring the hospitalization of the nurse. The analysis of preservatives and excipients led us to conclude that the pharmaceutical drug was probably added to the water bottles as "Haldol 2mg/mL oral solution"., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Organotypic slices cultured for weeks in vitro represent an extremely valuable strategy for the investigation of the long-term properties of neuronal circuits under physiological and pathological conditions. Here, we describe how to prepare rat organotypic hippocampal slice cultures and how to expose them for appropriate periods of time to excitotoxic agents or to oxygen and glucose deprivation conditions, in order to mimic the pattern of pyramidal cell damage which is observed in vivo and in other in vitro models. This preparation is very useful not only to study synaptic plasticity or the pathways and mechanisms of neurodegeneration but also to evaluate the effects of neuroprotective agents.

A fast liquid chromatographic/tandem mass spectrometric method was developed for the simultaneous determination in human urine of seventeen benzodiazepines, four relevant metabolites together plus zolpidem and zopiclone. The sample preparation, optimized to take into account the matrix effect, was based on enzymatic hydrolysis and liquid-liquid extraction. The separation of the twenty-three analytes was achieved in less than eight minutes. The whole methodology was fully validated according to UNI EN ISO/IEC 17025:2005 rules and 2006 SOFT/AAFS guidelines. Selectivity, linearity range, identification (LOD) and quantitation (LOQ) limits, precision, accuracy and recovery were evaluated. For all the species the signal/concentration linearity was satisfactory in the 50-1000 ng/mL concentration range. The limits of detection ranged from 0.5 to 30 ng/mL and LOQs from 1.7 to 100.0 ng/mL. Precisions were in the ranges 5.0-11.8%, 1.5-11.0% and 1.1-4.4% for low (100 ng/mL), medium (300 ng/mL) and high (1000 ng/mL) concentration, respectively. The accuracy, expressed as bias% was within ± 25 % for all the analytes. The recovery values, evaluated at 300 ng/mL concentration, ranged from 56.2% to 98.8%. The present method for the determination of several benzodiazepines, zolpidem and zopiclone in human urine proved to be simple, fast, specific and sensitive. The quantification by LC-MS/MS was successfully applied to 329 forensic cases among driving re-licensing, car accidents and alleged sexual violence cases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although type I interferons (IFN-α/β) have been traditionally associated with antiviral responses, their importance in host defense against bacterial pathogens is being increasingly appreciated. Little is known, however, about the occurrence and functional role of IFN-α/β production in response to pathogenic yeasts. Here, we found that conventional DCs, but not macrophages nor plasmacytoid DCs, mounted IFN-β responses after in vitro stimulation with Candida spp. or Saccharomyces cerevisiae. These responses absolutely required MyD88, a Toll-like receptor (TLR) adaptor molecule, and were partially dependent on TLR9 and TLR7. Moreover, Candida DNA, as well as RNA, could recapitulate the IFN-β response. After intravenous challenge with Candida albicans, most mice lacking the IFN-α/β receptor died from their inability to control fungal growth, whereas all WT controls survived. These data suggest that recognition of yeast nucleic acids by TLR7 and TLR9 triggers a host-protective IFN-α/β response., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen-glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1-30 μM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 μM. AM 251 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1-1 μM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids "on demand" may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Selective androgen receptor modulators (SARMs) have great therapeutic potential in various diseases including cancer cachexia, sarcopenia, and osteoporosis, and the number of drug candidates has been growing over the last decade. The SARM drug candidates S-22 and S-23 belong to one of the most advanced groups of androgen receptor modulators and are based on an arylpropionamide-derived core structure. Due to their anabolic effects, SARMs have been prohibited in elite sports and have been a subject of sports drug testing programmes since January 2008. Consequently, the structure of analytically useful urinary metabolites should be elucidated to provide targets for sensitive and retrospective analysis. In the present study, the phase-I and -II metabolism of S-22 and S-23 was simulated using hepatic human enzymes, and resulting metabolites were characterized by means of state-of-the-art mass spectrometric approaches employing high resolution/high accuracy Orbitrap mass spectrometry. Subsequently, the newly defined target compounds including the glucuronic acid conjugates of S-22 and S-23, their corresponding monohydroxylated and bishydroxylated analogs, as well as their B-ring depleted counterparts were implemented into an existing routine doping control procedure, which was examined for its specificity for the added substances. In order to obtain proof-of-concept data for authentic urine specimens, canine urine samples collected up to 72 h after oral administration of S-22 to dogs were analyzed using the established approach outlining the capability of the presented assay to detect the glucuronide of S-22 as well as the B-ring-depleted metabolite (M3) in all samples following therapeutic (31.4 µg/kg) dosing. Finally, M3 was chemically synthesized, characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry, and chosen as primary target for future doping control analyses., (Copyright © 2010 John Wiley & Sons, Ltd.)

Experimental evidence indicates that metabotropic glutamate (mGlu) receptors of the mGlu1 and mGlu5 subtypes play a differential role in models of cerebral ischemia and that only mGlu1 receptors are implicated in the pathways leading to postischemic neuronal injury. The localization of mGlu1 receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted experimental studies that have demonstrated mGlu1 receptor antagonist agents attenuate postischemic injury by enhancing GABA-mediated neurotransmission, thus providing a new viewpoint on the neuroprotective mechanism of these pharmacological agents. In view of the recent discovery of a functional interaction between group I mGlu receptors and the cannabinoid system in the modulation of synaptic transmission, we propose a novel mechanism that predicts that the neuroprotective effects of mGlu1 receptor antagonists on CA1 pyramidal cells are mediated by a mechanism that overcomes the "synaptic circuit break" operated by endocannabinoids on GABAergic transmission.

Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia. In this study, postconditioning was investigated by first exposing rat organotypic hippocampal slices to 30min oxygen-glucose deprivation (OGD), which promotes selective CA1 pyramidal cell death, and 5min later to either a brief period (3min) of OGD or to a low dose (10microM) of 3,5-dihydroxyphenylglycine (DHPG) for 30min. Both protocols attenuated CA1 neuronal injury, as revealed 24h later by measuring the intensity of propidium iodide fluorescence in this region. The beneficial effects were observed when DHPG postconditioning was applied up to 15min after OGD, but not at later time points, and was not additive with the neuroprotective effects of a preconditioning DHPG treatment. The attenuation of the OGD-induced CA1 injury evoked by postconditioning was prevented when mGlu1 and mGlu5 receptor antagonists and inhibitors of phosphatidylinositol 3-kinase and Akt activity were present in the incubation medium during the 5min recovery period after OGD and the 30min exposure to DHPG. The PI3K inhibitor was also able to prevent the reduction of NMDA toxicity induced by the DHPG treatment. Finally, DHPG increased the phosphorylation of Akt in a transient and mGlu1/mGlu5-dependent manner. Our results show that activation of the mGlu1/mGlu5-PI3K-Akt signaling pathway plays a crucial role in the mechanisms of postconditioning evoked by DHPG and point to this strategy as a possible novel therapeutic tool for stroke and cerebral ischemia.

The antiviral activities of type I IFNs have long been established. However, comparatively little is known of their role in defenses against nonviral pathogens. We examined here the effects of type I IFNs on host resistance against the model pathogenic yeast Cryptococcus neoformans. After intratracheal or i.v. challenge with this fungus, most mice lacking either the IFN-alpha/beta receptor (IFN-alpha/betaR) or IFN-beta died from unrestrained pneumonia and encephalitis, while all wild-type controls survived. The pulmonary immune response of IFN-alpha/betaR-/- mice was characterized by increased expression of IL-4, IL-13, and IL-10, decreased expression of TNF-alpha, IFN-gamma, inducible NO synthetase, and CXCL10, and similar levels of IL-12 mRNA, compared with wild-type controls. Histopathological analysis showed eosinophilic infiltrates in the lungs of IFN-alpha/betaR-/- mice, although this change was less extensive than that observed in similarly infected IFN-gammaR-deficient animals. Type I IFN responses could not be detected in the lung after intratracheal challenge. However, small, but statistically significant, elevations in IFN-beta levels were measured in the supernatants of bone marrow-derived macrophages or dendritic cells infected with C. neoformans. Our data demonstrate that type I IFN signaling is required for polarization of cytokine responses toward a protective type I pattern during cryptococcal infection.

No vaccine is available for preventing infections by serogroup B Neisseria meningitidis (MenB), which accounts for a major portion of meningococcal cases in developed countries, because of the poor immunogenicity of the capsular polysaccharide (CP) even after protein conjugation. We have previously induced anticapsular antibodies by immunization with a single chain variable fragment (scFv), which mimics a protective CP epitope. This surrogate antigen, however, was ineffective at inducing serum bactericidal activity, an accepted marker of protection in humans. Serum bactericidal activity was consistently achieved by immunizing mice with the scFv-encoding gene. Immunization with vectors without a secretory signal sequence before the scFv resulted in markedly higher bactericidal activity relative to those with such a sequence. The induced antibodies were capsule specific, as shown by complete inhibition of bactericidal activity by purified MenB CP and by resistance to killing of MenA or MenC. Moreover, these antibodies were predominantly of the IgG2a isotype, reflecting a T helper type 1 response. Administration of sera from scFv gene-vaccinated animals protected infant rats against MenB bacteremia. These data illustrate the potential of vaccination with genes encoding capsular mimics in providing protection against MenB and other encapsulated bacteria.

Host defenses against the encapsulated yeast Cryptococcus neoformans involve both humoral and cell-mediated immunity. Mannoproteins (MPs) are a heterogeneous class of immunodominant glycoproteins which have been only incompletely characterized. In this study, we report on the molecular features of two novel MPs that are recognized by serum antibodies during cryptococcosis. After fractionation of extracellular cryptococcal products, MPs reacted more strongly than other components with sera from C. neoformans-infected AIDS patients. Further fractionation and Western blot analysis of MPs evidenced the presence of highly reactive bands with molecular masses of 250, 125, 115, and 84 kDa. The 115- and 84-kDa bands contained significant amounts of N-linked oligosaccharides, as shown by decreased molecular mass after peptide-N-glycosidase F treatment. N-terminal amino acid sequences of the two bands were used to search C. neoformans nucleotide databases. Homologous genomic sequences were used to synthesize DNA probes and isolate cDNA clones containing the full-length genes, which were designated MP84 and MP115. Both genes showed the presence of a serine/threonine-rich region, a potential site for heavy glycosylation. MP84 and MP115 showed homology with, respectively, polysaccharide deacetylases and carboxylesterases from other organisms. Recombinant, deglycosylated proteins expressed in Escherichia coli still reacted with sera from patients, albeit more weakly than natural MPs, indicating that at least some of the reactive epitopes were retained in the recombinant forms. In conclusion, we identified two novel MPs that are important targets of antibody responses during cryptococcosis. These data may be useful to devise alternative immunity-based strategies to control the disease.