Cranial anatomy of Acynodon adriaticus and extreme durophagous adaptations in Eusuchia (Reptilia: Crocodylomorpha).

Acynodon adriaticus, a small eusuchian from the Late Cretaceous of Italy, is known for its well-preserved cranial and postcranial material. Despite its excellent preservation, many details remain hidden due to the physical overlap between the elements and matrix obliteration. We used Micro-CT scans to reveal previously overlooked anatomical features and describe in detail the cranial and dental anatomy of this taxon, shedding new light on its palaeoecology. The holotypic specimen, SC 57248, represents a mature individual exhibiting signs of hyperossification, developed ornamentation, and various pathologies, including jaw arthritis and a possible dental anomaly. Acynodon adriaticus exhibits significant durophagous adaptations, including a robust, brevirostrine skull optimized for powerful biting and stress-load capacity. Its specialized dentition, lacking caniniform teeth, features anterior chisel-like teeth and hypertrophic posterior molariforms with thick enamel, indicative of a diet specializing in hard-shelled prey. The dentition pattern, accelerated molariform replacement rate, and reduced orbit size suggest adaptations for durophagous foraging in turbid, densely vegetated aquatic environments. The paleoecological context during the Late Cretaceous, characterized by increased freshwater habitats and high invertebrate diversity, likely facilitated the evolution of such specialized traits in A. adriaticus. This small crocodylomorph likely foraged slowly in shallow, benthic environments, using its powerful bite to process mollusks and large arthropods. The study of A. adriaticus, along with comparisons with other crocodylomorphs and ecomorphologically similar taxa like Iharkutosuchus makadii and Gnatusuchus pebasensis, provides a valuable morphofunctional model for understanding the evolutionary pathways of extinct crocodylians to durophagy.

Fingerprinting alkaloids for traceability: Semi-untargeted UHPLC-MS/MS approach in raw lupins as a case study.

Lupin seeds are recognized for their nutritional value and potential health benefits, but they contain also a significant amount of alkaloids, an anti-nutritive class of compounds, which vary significantly in composition within and between species due to environmental factors. In this study, we developed a predictive multi-experiment approach using ultra-high performance liquid chromatography coupled with triple quadrupole with linear ionic trap tandem mass spectrometry (UHPLC-QqQ-LIT-MS/MS) for comprehensive alkaloid profiling and geographical classification of Lupinus albus L. samples originating from four different Italian regions. Six targeted quinolizidine alkaloids were detected and 21 other alkaloids were putatively identified. Hierarchical Cluster Analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were applied to explore the data structure and successfully classify samples according to their geographical origin. The data demonstrate the efficacy of the developed approach in providing valuable insights in alkaloid profiles of lupin seeds and their potential as markers for geographical traceability.

Investigating the modulation of the endocannabinoid system by probiotic Lactiplantibacillus plantarum IMC513 in a zebrafish model of di-n-hexyl phthalate exposure.

Environmental pollutants used as plasticizers in food packaging and in thousands of everyday products have become harmful for their impact on human health. Among them, phthalates, recognized as emerging endocrine disruptors (EDs) can induce toxic effects leading to different health disorders. Only few studies evaluated the effects of di-n-hexyl phthalate (DnHP) in in vivo models and no studies have been conducted to investigate the effect of DnHP on the endocannabinoid system (ECS), one of the majors signaling pathways involved in the microbiota-gut-brain axis. Due to the current relevance of probiotic bacteria as beneficial dietary interventions, the present study was aimed at evaluating the potential neuroprotective impact of daily administration of Lactiplantibacillus (Lpb.) plantarum IMC513 on zebrafish adults exposed to DnHP, with a focus on ECS modulation. Gene expression analysis revealed a promising protective role of probiotic through the restoration of ECS genes expression to the control level, in the brain of zebrafish daily exposed to DnHP. In addition, the levels of main endocannabinoids were also modulated. These findings confirm the potential ability of probiotics to interact at central level by modulating the ECS, suggesting the use of probiotics as innovative dietary strategy to counteract alterations by EDs exposure.

Molecularly imprinted polymer coupled to UHPLC-MS/MS for the analysis of phomopsins in lupin samples.

The demand for plant-based protein sources in the food industry has significantly increased in recent years, leading to the introduction of legume-based products as meat substitutes. However, concerns regarding food quality have emerged, particularly related to the presence of mycotoxins. This study addresses the need for the sensitive detection of phomopsins (PHOs), a class of peptide-based toxins. A selective extraction method using molecularly imprinted polymer (MIP) coupled with ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) was focused on the most toxic Phomopsin A (PHO-A). A rapid ultrasonochemical synthesis of MIP (5 min) was proposed and its performance was optimized in response to various factors, including the choice of dummy template and the selection of the monomer. The methacrylic acid-vinyl pyridine (MAA-VP) MIP exhibited high selectivity and affinity for PHO-A. The method was tested in lupin samples and the validation, according to SANTE/11312/2021 international guidelines, gave excellent recovery (80-90 %), low matrix effects, and high accuracy and precision. Real samples analysis confirmed the presence of PHO-A in artificially fungal inoculated lupins, with levels ranging from 0.377 to 0.576 mg kg-1. In order to identify further PHOs, a semi-untargeted approach using multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) was developed. PHO-B, PHO-D, PHO-E and PHO-P, rarely previously reported in lupin matrix, were tentatively identified. This study accounts for the effectiveness of MIP-based extraction coupled with UHPLC-triple quadrupole with linear ionic trap-MS/MS (UHPLC-QqQ-LIT-MS/MS) for quantification of PHO-A and putative detection of other PHOs, offering a promising method for investigating this class of toxins in food.

Protective effects of fatty acid amide hydrolase inhibition in UVB-activated microglia.

Neuroinflammation is a hallmark of several neurodegenerative disorders that has been extensively studied in recent years. Microglia, the primary immune cells of the central nervous system (CNS), are key players in this physiological process, demonstrating a remarkable adaptability in responding to various stimuli in the eye and the brain. Within the complex network of neuroinflammatory signals, the fatty acid N-ethanolamines, in particular N-arachidonylethanolamine (anandamide, AEA), emerged as crucial regulators of microglial activity under both physiological and pathological states. In this study, we interrogated for the first time the impact of the signaling of these bioactive lipids on microglial cell responses to a sub-lethal acute UVB radiation, a physical stressor responsible of microglia reactivity in either the retina or the brain. To this end, we developed an in vitro model using mouse microglial BV-2 cells. Upon 24 h of UVB exposure, BV-2 cells showed elevated oxidative stress markers and, cyclooxygenase (COX-2) expression, enhanced phagocytic and chemotactic activities, along with an altered immune profiling. Notably, UVB exposure led to a selective increase in expression and activity of fatty acid amide hydrolase (FAAH), the main enzyme responsible for degradation of fatty acid ethanolamides. Pharmacological FAAH inhibition via URB597 counteracted the effects of UVB exposure, decreasing tumor necrosis factor α (TNF-α) and nitric oxide (NO) release and reverting reactive oxidative species (ROS), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10) levels to the control levels. Our findings support the potential of enhanced fatty acid amide signaling in mitigating UVB-induced cellular damage, paving the way to further exploration of these lipids in light-induced immune responses.

Selective alterations of endocannabinoid system genes expression in obsessive compulsive disorder.

Obsessive Compulsive Disorder (OCD) is listed as one of the top 10 most disabling neuropsychiatric conditions in the world. The neurobiology of OCD has not been completely understood and efforts are needed in order to develop new treatments. Beside the classical neurotransmitter systems and signalling pathways implicated in OCD, the possible involvement of the endocannabinoid system (ECS) has emerged in pathophysiology of OCD. We report here selective downregulation of the genes coding for enzymes allowing the synthesis of the endocannabinoids. We found reduced DAGLα and NAPE-PLD in blood samples of individuals with OCD (when compared to healthy controls) as well as in the amygdala complex and prefrontal cortex of dopamine transporter (DAT) heterozygous rats, manifesting compulsive behaviours. Also mRNA levels of the genes coding for cannabinoid receptors type 1 and type 2 resulted downregulated, respectively in the rat amygdala and in human blood. Moreover, NAPE-PLD changes in gene expression resulted to be associated with an increase in DNA methylation at gene promoter, and the modulation of this gene in OCD appears to be correlated to the progression of the disease. Finally, the alterations observed in ECS genes expression appears to be correlated with the modulation in oxytocin receptor gene expression, consistently with what recently reported. Overall, we confirm here a role for ECS in OCD at both preclinical and clinical level. Many potential biomarkers are suggested among its components, in particular NAPE-PLD, that might be of help for a prompt and clear diagnosis.

Simulated Microgravity Affects Pro-Resolving Properties of Primary Human Monocytes.

Space-related stressors such as microgravity are associated with cellular and molecular alterations of the immune and inflammatory homeostasis that have been linked to the disorders that astronauts suffer from during their missions. Most of the research of the past 30 years has consistently established that innate adaptive immune cells represent a target of microgravity, which leads to their defective or dysfunctional activation, as well as to an altered ability to produce soluble mediators-e.g., cytokines/chemokines and bioactive lipids-that altogether control tissue homeostasis. Bioactive lipids include a vast array of endogenous molecules of immune origin that control the induction, intensity and outcome of the inflammatory events. However, none of the papers published so far focus on a newly characterized class of lipid mediators called specialized pro-resolving mediators (SPMs), which orchestrate the "resolution of inflammation"-i.e., the active control and confinement of the inflammatory torrent mostly driven by eicosanoids. SPMs are emerging as crucial players in those processes that avoid acute inflammation to degenerate into a chronic event. Given that SPMs, along with their metabolism and signaling, are being increasingly linked to many inflammatory disorders, their study seems of the outmost importance in the research of pathological processes involved in space-related diseases, also with the perspective of developing therapeutic countermeasures. Here, we show that microgravity, simulated in the rotary cell culture system (RCCS) developed by NASA, rearranges SPM receptors both at the gene and protein level, in human monocytes but not in lymphocytes. Moreover, RCCS treatment reduces the biosynthesis of a prominent SPM like resolvin (Rv) D1. These findings strongly suggest that not only microgravity can impair the functioning of immune cells at the level of bioactive lipids directly involved in proper inflammation, but it does so in a cell-specific manner, possibly perturbing immune homeostasis with monocytes being primary targets.

Salt elicitation to enhance phytochemicals in durum wheat seedlings.

BACKGROUND: Salt has been identified as an elicitor that can increase the accumulation of phytochemicals in seedlings during the germination process. However, the salinity level required to maximize the yield of phytochemicals, particularly phenolic compounds, needs further investigation for several plant species. To address this issue, we imposed increasing levels of salinity (NaCl solutions) on the sprouting substrate of Triticum durum (var. Platone) grains, at concentrations of 0, 50, 100, 150, 200, 250, and 300 mM (0_S, 50_S, 100_S, 150_S, 200_S, 250_S, and 300_S, respectively). RESULTS: The highest NaCl doses (250_S and 300_S) significantly impacted germination performance and were excluded from further analysis. The seedlings harvested at 8 days after sowing exhibited different growth stages depending on the salinity level: wheatgrass for 0_S, early wheatgrass for 50_S, intermediate between sprout and wheatgrass for 100_S, sprout for 150_S, and very early sprout for 200_S. Furthermore, salinity induced the concentration of phenolic compounds (PhCs) in the seedlings' tissues (i.e., both roots and shoots) in a salinity-dependent manner. The highest values were observed at 200_S, with an increase of 187% of the total investigated PhCs in comparison with 0_S, averaged over shoots and roots. In particular, in 200_S, the accumulation of phenolic acids was up to fourfold higher in roots, and that of flavonoids was up to twofold higher in shoots. CONCLUSION: Our findings suggest that the use of 200 mM NaCl applied to the sprouting substrate is excessive for producing edible sprouts but may be suitable for phytochemical extraction purposes. © 2023 Society of Chemical Industry.

Aß Chronic Exposure Promotes an Activation State of Microglia through Endocannabinoid Signalling Imbalance.

Dysfunctional phenotype of microglia, the primary brain immune cells, may aggravate Alzheimer's disease (AD) pathogenesis by releasing proinflammatory factors, such as nitric oxide (NO). The endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are bioactive lipids increasingly recognised for their essential roles in regulating microglial activity both under normal and AD-driven pathological conditions. To investigate the possible impact of chronic exposure to ß-amyloid peptides (Aß) on the microglial endocannabinoid signalling, we characterised the functional expression of the endocannabinoid system on neonatal microglia isolated from wild-type and Tg2576 mice, an AD-like model, which overexpresses Aß peptides in the developing brain. We found that Aß-exposed microglia produced 2-fold more 2-AG than normal microglia. Accordingly, the expression levels of diacylglycerol lipase-α (DAGLα) and monoacylglycerol lipase (MAGL), the main enzymes responsible for synthesising and hydrolysing 2-AG, respectively, were consistently modified in Tg2576 microglia. Furthermore, compared to wild-type cells, transgenic microglia basally showed increased expression of the cannabinoid 2 receptor, typically upregulated in an activated proinflammatory phenotype. Indeed, following inflammatory stimulus, Aß-exposed microglia displayed an enhanced production of NO, which was abolished by pharmacological inhibition of DAGLα. These findings suggested that exposure to Aß polarises microglial cells towards a pro-AD phenotype, possibly by enhancing 2-AG signalling.

Determination of endocannabinoids and their conjugated congeners in the brain by means of µSPE combined with UHPLC-MS/MS.

The present study encompasses the development of a fast and reliable analytical method to quantify the main endocannabinoids and some of their conjugated congeners, particularly N-arachidonoyl amino acids, in brain tissue. Samples were homogenized and a micro solid phase extraction (µSPE) procedure was developed for brain homogenate clean-up. Miniaturized SPE was selected as it allowed to work with reduced sample amounts, while maintaining high sensitivity; this last feature was mandatory due to the low concentration of endocannabinoids in biological matrices that makes their determination a challenging analytical task. UHPLC-MS/MS was used for the analysis as it provided a great sensitivity, especially for conjugated forms that were detected by negative ionization. Polarity switching was applied during the run; low limits of quantification were between 0.003 ng g-1 and 0.5 ng g-1. This method provided also low matrix effect (lower than 30%) and good extraction recoveries in the brain. To the best of our knowledge, this is the first time that µSPE is applied on this matrix for this class of compounds. The method was validated according to international guidelines, and then tested on real cerebellum samples from mice, which were sub-chronically treated with URB597, a well-known inhibitor of the fatty acid amide hydrolase.

Development of an HPLC-MS/MS Method for the Determination of Alkaloids in Lupins.

Lupin alkaloids (LAs) represent a class of toxic secondary metabolites in plants, in particular in Lupinus spp.; they are produced as a defense mechanism due to their strong bitter taste and are very dangerous for human and animals. In this work, a sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analytical method for the identification and quantification of thirteen lupin alkaloids was developed and validated according to FDA guidelines. Efficient extraction and clean-up steps, carried out by solid-phase extraction, were finely tuned on the basis of the characteristics of the analytes and lupin samples, providing good selectivity with minimized matrix interference. The effectiveness of the method was proven by the satisfactory recovery values obtained for most of the analytes and a matrix effect ≤23% for all tested levels. In addition, a sensitive and reliable determination of the target compounds was obtained; LOQs were between 1 and 25 µg Kg-1, i.e., below the requested maximum levels (<200 mg Kg-1). The method was applied to evaluate the LAs profile in different batches of raw L. albus L. samples, varying in size and across farming treatments.

Study on Molecularly Imprinted Polymers Obtained Sonochemically for the Determination of Aflatoxins in Food.

Aflatoxins (AFs) are fungi secondary metabolites produced by the Aspergillus family. These compounds can enter the food chain through food contamination, representing a risk to human health. Commercial immunoaffinity columns are widely used for the extraction and cleanup of AFs from food samples; however, their high cost and large solvent consumption create a need for alternative strategies. In this work, an alternative strategy for producing molecularly imprinted polymers (MIPs) was proposed to extract aflatoxins AFB1, AFB2, AFG1, and AFG2 from complex food samples, using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The MIPs were synthesized via a low-cost and rapid (5 min) sonochemical free-radical polymerization, using 1-hydroxy-2-naphthoic acid as a dummy template. MIPs-based solid phase extraction performance was tested on 17 dietary supplements (vegetables, fruits, and cereals), obtaining appreciable recovery rates (65-90%) and good reproducibility (RSD ≤ 6%, n = 3); the selectivity towards other mycotoxins was proved and the data obtained compared with commercial immunoaffinity columns. The proposed strategy can be considered an alternative affordable approach to the classical immunoaffinity columns, since it is more selective and better performing.

Description of the first definitive Corythosaurus (Dinosauria, Hadrosauridae) specimens from the Judith River Formation in Montana, USA and their paleobiogeographical significance.

Despite the long history of research in the late Campanian Judith River Formation in northern Montana, most of the vertebrate fossils are represented by fragmentary remains, making precise taxonomic identifications difficult. Contrary to this, the partially contemporaneous Dinosaur Park Formation, Alberta, Canada is known for its tremendous fossil preservation, permitting rigorous studies of dinosaur diversity, evolution, and biostratigraphy. Hadrosaurids comprise one of the most abundant dinosaur clades in the Dinosaur Park Formation, but taxonomic affinities of hadrosaurid specimens remain poorly understood in the Judith River Formation. Corythosaurus is the most common hadrosaurid in the Dinosaur Park Formation and, to date, has been restricted to this formation. This study reports the first definitive Corythosaurus specimens from the Judith River Formation, which were discovered on two private ranches in northern Montana. The attribution of the most complete skeleton to Corythosaurus is indicated by: wide crest-snout angle, presence of premaxilla-nasal fontanelle, dorsoventrally expanded nasal, laterally exposed ophthalmic canal of the laterosphenoid, and tall neural spines. A second specimen preserves a large ilium that can be positively identified as Corythosaurus based on its associated skull, which is now in private hands. The specimens were recovered from the Coal Ridge Member of the Judith River Formation, which is approximately time equivalent to the Dinosaur Park Formation. Thus, the discovery of Corythosaurus in the Judith River Formation extends the biogeographic range of this genus and establishes a framework for future interformational biostratigraphic studies of Late Cretaceous dinosaur faunas in North America.

LC-MS/MS Analysis of AEA and 2-AG.

LC-MS/MS is a powerful analytical technique that provides unequivocal identification and reliable quantification of the analytes, using Selected Reaction Monitoring or Multi Reaction Monitoring acquisition mode.Anandamide (N-arachidonoylethanolamine, AEA) and 2-Arachidonoylglycerol (2-AG) are the most abundant endocannabinoids (eCBs), which play a major role in a wide variety of physiological and pathological processes. Analysis of those compounds by means of LC-MS/MS allows the detection of very low concentrations in biological samples. Here, we describe how to determine AEA and 2-AG levels in tiny samples of tissues and plasma through LC-MS/MS, by using very quick and easy-to-perform extraction procedures, with reduced solvent consumption.

Endocannabinoid Metabolism and Transport as Drug Targets.

The wide distribution of the endocannabinoid system (ECS) throughout the body and its pivotal pathophysiological role offer promising opportunities for the development of novel therapeutic drugs for treating several diseases. However, the need for strategies to circumvent the unwanted psychotropic and immunosuppressive effects associated with cannabinoid receptor agonism/antagonism has led to considerable research in the field of molecular alternatives, other than type-1 and type-2 (CB1/2) receptors, as therapeutic targets to indirectly manipulate this pro-homeostatic system. In this context, the use of selective inhibitors of proteins involved in endocannabinoid (eCB) transport and metabolism allows for an increase or decrease of the levels of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) in the sites where these major eCBs are indeed needed. This chapter will briefly review some preclinical and clinical evidence for the therapeutic potential of ECS pharmacological manipulation.

Chemical characterization and evaluation of antioxidant activity from different cultivars of Cannabis sativa L. of Abruzzo's region.

In this work, the chemical composition and the antioxidant evaluation of the inflorescences from 12 Cannabis sativa L. monoecious cultivars (Carmagnola Lemon CL, Ferimon F, Gran Sasso Kush GSK, Antal A, Carmagnola C, Kompolti K, Futura 75 F75, Villanova V, Tiborzallasi T, Finola FL, Kc Virtus KV and Pineapple P) cultivated at the same condition, were investigated. GC-MS analysis was carried out to evaluate the volatile fraction, while HPLC-MS/MS was used for cannabinoids and polyphenolic compounds. The evaluation of antioxidant activity was carried out using ABTS*+, Trolox equivalence antioxidant capacity (TEAC), ferric reducing antioxidant property (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH*) assays in vitro. The obtained data, demonstrated that each cultivar has a characteristic chemical profile, with highest antioxidant capacity for CL, F75, GSK and F. Based on the in vitro antioxidant activity the plant extracts can be considered as promising candidates for different applications in food field.

Endocannabinoid System Regulation in Female Rats with Recurrent Episodes of Binge Eating.

Recurrent Binge Eating (BE) episodes characterize several eating disorders. Here, we attempted to reassemble a condition closer to BE disorder, and we analyzed whether recurrent episodes might evoke molecular alterations in the hypothalamus of rats. The hypothalamus is a brain region which is sensitive to stress and relevant in motivated behaviors, such as food intake. A well-characterized animal model of BE, in which a history of intermittent food restriction and stress induce binge-like palatable food consumption, was used to analyze the transcriptional regulation of the endocannabinoid system (ECS). We detected, in rats showing the BE behavior, an up-regulated gene expression of cannabinoid type-1 receptor (CB1), sn-1-specific diacylglycerol lipase, as well as fatty acid amide hydrolase (Faah) and monoacylglycerol lipase. A selective reduction in DNA methylation was also observed at the promoter of Faah, which is consistent with the changes in the gene expression. Moreover, BE behavior in rats was associated with an increase in anandamide (AEA) levels. Our findings support the relevant role of the ECS in the regulation of food intake in rats subjected to repeated BE episodes, and, in particular, on AEA signaling, acting via CB1 and FAAH modulation. Notably, the epigenetic regulation of the Faah gene might suggest this enzyme as a possible target for developing new therapeutical approaches.

Targeting mGlu1 Receptors in the Treatment of Motor and Cognitive Dysfunctions in Mice Modeling Type 1 Spinocerebellar Ataxia.

Type 1 spinocerebellar ataxia (SCA1) is a progressive neurodegenerative disorder with no effective treatment to date. Using mice modeling SCA1, it has been demonstrated that a drug that amplifies mGlu1 receptor activation (mGlu1 receptor PAM, Ro0711401) improves motor coordination without the development of tolerance when cerebellar dysfunction manifests (i.e., in 30-week-old heterozygous ataxin-1 [154Q/2Q] transgenic mice). SCA1 is also associated with cognitive dysfunction, which may precede cerebellar motor signs. Here, we report that otherwise healthy, 8-week-old SCA1 mice showed a defect in spatial learning and memory associated with reduced protein levels of mGlu1α receptors, the GluN2B subunit of NMDA receptors, and cannabinoid CB1 receptors in the hippocampus. Systemic treatment with Ro0711401 (10 mg/kg, s.c.) partially corrected the learning deficit in the Morris water maze and restored memory retention in the SCA1 mice model. This treatment also enhanced hippocampal levels of the endocannabinoid, anandamide, without changing the levels of 2-arachidonylglycerol. These findings suggest that mGlu1 receptor PAMs may be beneficial in the treatment of motor and nonmotor signs associated with SCA1 and encourage further studies in animal models of SCA1 and other types of SCAs.

The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

Oral fluid as a new investigative matrix for the determination of organic gunshot residue exposure.

In recent years, increased use of ammunition without lead and heavy metals was observed, leading to a growing interest in the detection of organic gunshot residues (OGSR) as evidence of firearms related crimes. The wide range of compounds belonging to the OGSR class hinders their mass spectrometric detection as different ionization techniques may be needed to obtain good results for all compounds. The purpose of this work was the development of a reliable analytical method by means of UHPLC-HRMS for the determination in oral fluid (OF) of the most common explosives and the most used stabilizers, arising from fire discharge and post-deflagration residues. For this purpose, SPE was used for OF clean-up before UHPLC-HRMS analysis. All target analytes were chromatographically separated by means of a Polar-C18 column. A chlorinated compound was added to the mobile phases in order to promote the formation of chloride adduct ions in the electrospray ion source operating in polarity switching to allow the best conditions for each analyte. The detection was conducted by means of a high-resolution mass spectrometer equipped with Orbitrap technology working in data dependent acquisition mode, in order to detect both the precursor ions and/or the most intense fragments for stabilizers. To verify its potential, the method was tested on real samples: a shooting session was performed in an open shooting range; the shooters fired from 2 to 20 rounds with a 9x21 caliber, thereafter OF was sampled. Samples were analyzed confirming that explosives may be detected in OF; the use of this matrix may be of great interest for investigative purposes as it is less affected by secondary transfer when compared to other commonly sampled matrices. The developed method could be a useful tool for law enforcement authorities for the detection of explosives in forensic potential scenarios, including biological matrices.