Defects in AMPAR trafficking and microglia activation underlie socio-cognitive deficits associated to decreased expression of phosphodiesterase 2 a.

Phosphodiesterase 2 A (PDE2A) is an enzyme involved in the homeostasis of cAMP and cGMP and is the most highly expressed PDE in human brain regions critical for socio-cognitive behavior. In cerebral cortex and hippocampus, PDE2A expression level is upregulated in Fmr1-KO mice, a model of the Fragile X Syndrome (FXS), the most common form of inherited intellectual disability (ID) and autism spectrum disorder (ASD). Indeed, PDE2A translation is negatively modulated by FMRP, whose functional absence causes FXS. While the pharmacological inhibition of PDE2A has been associated to its pro-cognitive role in normal animals and in models of ID and ASD, homozygous PDE2A mutations have been identified in patients affected by ID, ASD and epilepsy. To clarify this apparent paradox about the role of PDE2A in brain development, we characterized here Pde2a+/- mice (homozygote animals being not viable) at the behavioral, cellular, molecular and electrophysiological levels. Pde2a+/- females display a milder form of the disorder with reduced cognitive performance in adulthood, conversely males show severe socio-cognitive deficits throughout their life. In males, these phenotypes are associated with microglia activation, elevated glutathione levels and increased externalization of Glutamate receptor (GluR1) in CA1, producing reduced mGluR-dependent Long-term Depression. Overall, our results reveal molecular targets of the PDE2A-dependent pathway underlying socio-cognitive performance. These results clarify the mechanism of action of pro-cognitive drugs based on PDE2A inactivation, which have been shown to be promising therapeutic approaches for Alzheimer's disease, schizophrenia, FXS as well as other forms of ASD.

Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Comprehensive Monitoring of Psychoactive Substances in Psychiatric Patients Using Liquid Chromatography-High-Resolution Mass Spectrometry: A Key Tool for Treatment Planning and Understanding Consumption Patterns in Rome, Italy.

BACKGROUND: The comprehensive monitoring of licit and illicit drug consumption plays a crucial role in understanding the complexities of patient conditions and designing effective treatment strategies. In this study, the prevalence of psychoactive prescription drugs, classical illicit drugs, and new psychoactive substances (NPS) were objectively assessed in individuals diagnosed with drug-related psychiatric disorders or episodes. METHODS: Blood, urine, and hair samples were collected from psychiatric patients admitted to the Mental Health Department and Drug Addiction Service of the North Rome Local Health Authority with declared or suspected psychoactive drug use. Comprehensive drug screening was conducted for all samples using ultra-high-performance liquid chromatography-high-resolution mass spectrometry. RESULTS: A total of 71 blood and urine and 50 hair samples were analyzed to confirm the suitability of the ultra-high-performance liquid chromatography-high-resolution mass spectrometry method for the study purposes. The main substances found in blood and urine were antipsychotics (71.8% and 66.2%) and benzodiazepines (62.0% and 59.2%), respectively, whereas cocaine (84.0%) and antipsychotics (74.0%) was more evident in hair. Z-drugs were detected in blood (7.0%), urine (5.6%), and hair (24%) samples; amphetamines were mainly detected in hair samples (14.0%). Synthetic cathinones were the most frequently detected NPS in hair specimens (8.0%), whereas synthetic cannabinoids were mainly found in blood samples (11.3%). These analyses showed that patients were polydrug users (77.5% detected in blood and urine, and 94.0% in hair). CONCLUSIONS: Comprehensive screening enabled the assessment of past, recent, and actual consumption of psychoactive substances, including licit and illicit drugs and NPS, by psychiatric patients. A thorough understanding of substance consumption patterns can enhance therapeutic interventions and management of psychiatric disorders associated with psychoactive substance use.

Role of Phosphodiesterases in Biology and Pathology 2.0.

Phosphodiesterases (PDEs) are ubiquitous enzymes that hydrolyse cAMP and cGMP second messengers temporally, spatially, and integratedly according to their expression and compartmentalization inside the cell [...].

New Synthetic Opioids Use among Patients in Treatment for an Opioid Use Disorder in Barcelona.

INTRODUCTION: New synthetic opioids (NSO), a class of new psychoactive substances (NPS), have recently emerged and pose an upcoming global public health challenge. The effects produced by NSO are similar to those from morphine, but they present greater pharmacological potency and abuse potential. Due to the increasing number of fatal overdoses and seizures in which NSO have been detected as heroin substitutes or adulterants, individuals with Opioid Use Disorder (OUD) represent a vulnerable population. The aim of our study was to describe and characterize from a gender perspective a Spanish cohort of potential conscious or unconscious NSO users. METHODS: A cross-sectional study was conducted in a cohort of OUD participants under treatment in addiction care services in Barcelona and Badalona, Spain. Clinical evaluation was performed through an ad hoc survey, a scale to evaluate reasons to use an opioid without prescription (range 0-4) and the Wellbeing Index (WHO-5) (range 0-100). Objective consumption of NSO was assessed by urinalysis carried out by two validated methods: high-sensitivity gas chromatography-mass spectrometry (MS) and ultra-high-performance liquid chromatography-high-resolution MS. RESULTS: A total of 154 participants with OUD were enrolled. They were mainly men (72.7%), mean age 47.8 years. Methadone was the predominant medication for opioid agonist treatment (mean dose 61.25 mg/day). A total of 32 (20.8%) participants reported having consumed some opioid to become "high" in the previous 3 months. The principal reasons for consuming illicit opioids were Replacing other drugs (mean 2.03) and Availability (mean 1.62), although Low price, was more highly valued by men (p = 0.045) and Shorter effect duration, most highly rated by women (p = <0.001). In the WHO-5, the mean score was 55 (SD = 30.1) without differences by gender. Fentanyl and derivatives or/and metabolites were detected in 7 (6.1%) participants, but illicit/non-prescribed NSOs were found in 5 out of 114 patients (4.4%), and other non-fentanyl opioids in 36 participants (26 men and 10 women). CONCLUSION: A non-negligible consumption of NSO-fentanyl's (positive detection in 6.1% of biological samples) was detected. The reasons for using these substances and also the well-being differed between the genders. There is therefore both voluntary and involuntary NSO consumption in our country which highlights the importance of approaching this potential public health problem.

Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology.

Phosphodiesterase 5A (PDE5A) is involved in cGMP hydrolysis, regulating many physiological processes. Increased activity of PDE5A has been found in several pathological conditions, and the pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications. We have identified the presence of three different Pde5a isoforms in cardiomyocytes, and we have found that the expression of specific Pde5a isoforms may have a causal role in the onset of pathological responses in these cells. In our previous study, we demonstrated that PDE5A inhibition could ameliorate muscular dystrophy by acting at different levels, as assessed by the altered genomic response of muscular cells following treatment with the PDE5A inhibitor tadalafil. Thus, considering the importance of PDE5A in various pathophysiological conditions, we further investigated the regulation of this enzyme. Here, we analysed the expression of Pde5a isoforms in the pathophysiology of skeletal muscle. We found that skeletal muscle tissues and myogenic cells express Pde5a1 and Pde5a2 isoforms, and we observed an increased expression of Pde5a1 in damaged skeletal muscles, while Pde5a2 levels remained unchanged. We also cloned and characterized the promoters that control the transcription of Pde5a isoforms, investigating which of the transcription factors predicted by bioinformatics analysis could be involved in their modulation. In conclusion, we found an overexpression of Pde5a1 in compromised muscle and identified an involvement of MyoD and Runx1 in Pde5a1 transcriptional activity.

Atrophy, oxidative switching and ultrastructural defects in skeletal muscle of the ataxia telangiectasia mouse model.

Ataxia telangiectasia is a rare, multi system disease caused by ATM kinase deficiency. Atm-knockout mice recapitulate premature aging, immunodeficiency, cancer predisposition, growth retardation and motor defects, but not cerebellar neurodegeneration and ataxia. We explored whether Atm loss is responsible for skeletal muscle defects by investigating myofiber morphology, oxidative/glycolytic activity, myocyte ultrastructural architecture and neuromuscular junctions. Atm-knockout mice showed reduced muscle and fiber size. Atrophy, protein synthesis impairment and a switch from glycolytic to oxidative fibers were detected, along with an increase of in expression of slow and fast myosin types (Myh7, and Myh2 and Myh4, respectively) in tibialis anterior and solei muscles isolated from Atm-knockout mice. Transmission electron microscopy of tibialis anterior revealed misalignments of Z-lines and sarcomeres and mitochondria abnormalities that were associated with an increase in reactive oxygen species. Moreover, neuromuscular junctions appeared larger and more complex than those in Atm wild-type mice, but with preserved presynaptic terminals. In conclusion, we report for the first time that Atm-knockout mice have clear morphological skeletal muscle defects that will be relevant for the investigation of the oxidative stress response, motor alteration and the interplay with peripheral nervous system in ataxia telangiectasia.

Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers.

The use of the new psychoactive substances is continuously growing and the implementation of accurate and sensible analysis in biological matrices of users is relevant and fundamental for clinical and forensic purposes. Two different analytical technologies, high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) were used for a screening analysis of classic drugs and new psychoactive substances and their metabolites in urine of formed heroin addicts under methadone maintenance therapy. Sample preparation involved a liquid-liquid extraction. The UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2 mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2 mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100-1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m, 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40-550 m/z). Urine samples from 296 patients with a history of opioid use disorder were examined. Around 80 different psychoactive substances and/or metabolites were identified, being methadone and metabolites the most prevalent ones. The possibility to screen for a huge number of psychotropic substances can be useful in suspected drug related fatalities or acute intoxication/exposure occurring in emergency departments and drug addiction services.

Regulation of Kit Expression in Early Mouse Embryos and ES Cells.

Kit is a growth factor receptor that regulates proliferation and/or survival of many embryonic and postnatal stem cell types. When mutated, it can induce malignant transformation of the host cells. To dissect the Kit role in the control of ESC pluripotency, we studied its expression during early mouse embryogenesis and during the process of ESC derivation from inner cell mass (ICM) cells. We followed the in vitro development of early mouse embryos obtained from transgenic mice carrying Kit promoter regions fused to EGFP (Kit-EGFP) and found that they initiate EGFP expression at morula stage. EGFP expression is then maintained in the blastocyst, within the ICM, and its levels increase when cultured in the presence of MAPK and GSK3ß inhibitors (2i) plus LIF compared with the LIF-only condition. Kit-EGFP ESCs showed nonhomogeneous EGFP expression pattern when cultured in LIF condition, but they upregulated EGFP expression, as well as that of Sox2, Nanog, Prdm14, when shifted to 2i-LIF culture. Similarly, primordial germ cells (PGCs) in the process of embryonic germ cell (EGC) conversion showed enhanced EGFP expression in 2i-LIF. Kit expression was affected by manipulating Sox2 levels in ESCs. Chromatin immunoprecipitation experiments confirmed that Sox2 binds Kit regulatory regions containing Sox2 consensus sequences. Finally, Kit constitutive activation induced by the D814Y mutation increased ESC proliferation and cloning efficiency in vitro and in teratoma assays in vivo. Our results identify Kit as a pluripotency-responsive gene and suggest a role for Kit in the regulation of ESC proliferation. Stem Cells 2019;37:332-344.

THC and CBD concentrations in blood, oral fluid and urine following a single and repeated administration of "light cannabis".

Background "Light cannabis" is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration lower than 0.2% and variable cannabidiol (CBD) content. We studied THC and CBD excretion profiles in blood, oral fluid (OF) and urine after smoking one or four light cannabis cigarettes. Methods Blood, OF and urine samples were obtained from six healthy light cannabis consumers after smoking one 1 g cigarette containing 0.16% THC and 5.8% CBD and from six others after smoking four 1 g cigarettes within 4 h. Sample collection began 0.5 and 4.5 h after smoking one or four cigarettes, respectively. Cannabinoid concentrations were quantified by gas chromatography-mass spectrometry (GC-MS). Results At the first collection, the highest THC and CBD concentrations occurred in blood (THC 7.0-10.8 ng/mL; CBD 30.2-56.1 ng/mL) and OF (THC 5.1-15.5 ng/mL; CBD 14.2-28.1 ng/mL); similar results occurred 0.5 h after the last of four cigarettes in blood (THC 14.1-18.2 ng/mL, and CBD 25.6-45.4 ng/mL) and OF (THC 11.2-24.3 ng/mL; CBD 14.4-37.0 ng/mL). The mean OF to blood ratio ranged from 0.6 to 1.2 after one and 0.6 to 1.9 after four light cannabis cigarettes. THC/CBD ratios in blood and OF were never greater than 2. Urinary 11-nor-9-carboxy-THC concentrations peaked 8 h after one and four cigarettes. Conclusions OF was a valuable alternative to blood in monitoring consumption of light cannabis. Blood and OF THC/CBD concentration ratios, never exceeded 2, possibly providing a useful biomarker to identify light cannabis vs illegal higher THC cannabis use, where THC/CBD ratios are generally greater than 10.

Determination of the Synthetic Cannabinoids JWH-122, JWH-210, UR-144 in Oral Fluid of Consumers by GC-MS and Quantification of Parent Compounds and Metabolites by UHPLC-MS/MS.

The consumption of synthetic cannabinoids (SCs) has significantly increased in the last decade and the analysis of SCs and their metabolites in human specimens is gaining interest in clinical and forensic toxicology. A pilot study has been carried out using a combination of an initial last generation gas chromatography-mass spectrometry (GC-MS) screening method for the determination of JWH-122, JWH-210, UR-144) in oral fluid (OF) of consumers and an ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) confirmatory method for the quantification of the parent compounds and their metabolites in the same biological matrix. OF samples were simply liquid-liquid extracted before injecting in both chromatographic systems. The developed methods have been successfully validated and were linear from limit of quantification (LOQ) to 50 ng/mL OF. Recovery of analytes was always higher than 70% and matrix effect always lower than 15% whereas intra-assay and inter-assay precision and accuracy were always better than 16%. After smoking 1 mg JWH-122 or UR-144 and 3 mg JWH-210, maximum concentration of 4.00-3.14 ng/mL JWH-122, 8.10-7.30 ng/mL JWH-210 ng/mL and 7.40 and 6.81 ng/mL UR-144 were measured by GC-MS and UHPLC-HRMS respectively at 20 min after inhalation. Metabolites of JWH 122 and 210 were quantified in OF by UHPLC-HRMS, while that of UR144 was only detectable in traces. Our results provide for the first time information about disposition of these SCs and their metabolites in consumers OF. Last generation GC-MS has proven useful tool to identify and quantify parent SCs whereas UHPLC-HRMS also confirmed the presence of SCs metabolites in the OF of SCs consumers.

PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis.

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A-/-) is embryonic lethal. Notably, livers of PDE2A-/- embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A-/- liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A-/- livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A-/- embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A-/- embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis.

UHPLC-HRMS and GC-MS Screening of a Selection of Synthetic Cannabinoids and Metabolites in Urine of Consumers.

Background and Objectives: The use of synthetic cannabinoids has increased around the world. As a result, the implementation of accurate analysis in human biological matrices is relevant and fundamental. Two different analytical technologies, ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) and high-sensitivity gas chromatography-mass spectrometry (GC-MS) were used for the determination of three synthetic cannabinoids JWH-122, JWH 210, UR-144 and their metabolites in urine of consumers. Materials and Methods: Sample preparation included an initial hydrolysis with ß-glucuronidase and liquid-liquid extraction. The UHPLC-HRMS method included a Kinetex 2.6 u Biphenyl 100A (100 × 2.1 mm, 2.6 µm) (Phenomenex, Italy) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode was used (mass range 100-1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m × 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40-550 m/z). Results: Both methods have been successfully used for screening of parent synthetic cannabinoids and their metabolites in urine samples of consumers. Conclusions: The screening method applied JWH-122, JWH-210, UR-144 and their metabolites in urine of consumers can be applied to other compounds of the JWH family.

Cannabinoids determination in bronchoalveolar lavages of cannabis smokers with lung disease.

Background Cannabis smoke affects the lungs similarly to tobacco smoke, causing symptoms such as increased cough, sputum, hyperinflation and chronic bronchitis. Chronic use can also cause serious lung diseases and airway obstruction. We developed and validated a method for the identification and quantification of cannabinol (CBN), cannabidiol (CBD), Δ-9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) in bronchoalveolar lavages (BALs) from hospitalized former or current tobacco smoking patients with lung disease and a long history of cannabis consumption and limited current tobacco use. Methods For the extraction of cannabinoids from BALs, a 1 mL sample was added with 300 µL of 0.1 N NaOH and 3 mL of hexane/ethyl acetate (9:1). The solvent was then evaporated to dryness. Trimethylsilyl derivatives were prepared and then analyzed by gas chromatography/mass spectrometry. Results The method was linear for the analytes under investigation with coefficients of determination of at least 0.99. Absolute analytical recovery was always better than 80%, imprecision and inaccuracy was always under 15%. Six cases out of 15 were positive for THC, CBN and CBD. In two BALs samples, the presence of 11-OH-THC was also measured while THC-COOH was not detected. In the six positive cases, the last cannabis smoking occurred in the previous 2-14 days. Conclusions This is the first time that cannabinoids have been detected in BALs, demonstrating the presence of a drug with its metabolites in a target organ of consumers who present with a lung disease. This occurrence let us hypothesize a role of cannabinoids in the development of the disease and prompted an investigation on possible associations between cannabis smoking and clinical outcomes in patients with lung disease and eventually evaluate a cytotoxic effect of cannabinoids themselves.

Development and Validation of a Method for the Analysis of Bisoprolol and Atenolol in Human Bone.

A method based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of bisoprolol and atenolol in human bone. After the addition of lobivolol as internal standard, pulverized samples were incubated in acetonitrile for 1 h under ultrasounds. After adjusting the pH of the samples to 6, they were centrifuged, and the supernatants were subjected to solid phase extraction. Elution was achieved by using 3 mL of 2% ammonium hydroxide in 80:20 dichloromethane:isopropanol solution. Eluted samples were evaporated and derivatized. Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 0.1-0.3 ng/mg (depending on the drug) to 150 ng/mg, the mean absolute recoveries were 60% for bisoprolol and 106% for atenolol, the matrix effect was 69% for bisoprolol and 70% for atenolol and process efficiency was 41% for bisoprolol and 80% for atenolol. The intra- and inter-assay accuracy values were always better than 12%. The validated method was then applied to bone samples from two real forensic cases in which toxicological analysis in blood were positive for atenolol in the first case (0.65 µg/mL) and bisoprolol in the second case (0.06 µg/mL). Atenolol was found in bone samples from the corresponding case at the approximate concentration of 148 ng/mg and bisoprolol was found at 8 ng/mg.

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line.

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.

Determination of cannabinoids in oral fluid and urine of "light cannabis" consumers: a pilot study.

Background In those countries where cannabis use is still illegal, some manufacturers started producing and selling "light cannabis": dried flowering tops containing the psychoactive principle Δ-9-tetrahydrocannabinol (THC) at concentrations lower than 0.2% together with variable concentration of cannabidiol (CBD). We here report a pilot study on the determination of cannabinoids in the oral fluid and urine of six individuals after smoking 1 g of "light cannabis". Methods On site screening for oral fluid samples was performed, as a laboratory immunoassay test for urine samples. A validated gas chromatography-mass spectrometry (GC-MS) method was then applied to quantify THC and CBD, independently from results of screening tests. Results On site screening for oral fluid samples, with a THC cut-off of 25 ng/mL gave negative results for all the individuals at different times after smoking. Similarly, negative results for urine samples screening from all the individuals were obtained. Confirmation analyses showed that oral fluid THC was in the concentration range from 2.5 to 21.5 ng/mL in the first 30 min after smoking and then values slowly decreased. CBD values were usually one order of magnitude higher than those of THC. THC-COOH, the principal urinary THC metabolite, presented the maximum urinary value of 1.8 ng/mL, while urinary CBD had a value of 15.1 ng/mL. Conclusions Consumers of a single 1 g dose of "light cannabis" did not result as positive in urine screening, assessing recent consumption, so that confirmation would not be required. Conversely, they might result as positive to oral fluid testing with some on-site kits, with THC cut-off lower than 25 ng/mL, at least in the first hour after smoking and hence confirmation analysis can be then required. No conclusions can be drawn of eventual chronic users.

A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11.

Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11 (+/-) spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11 (-/-) background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination.

ß-Adrenergic response is counteracted by extremely-low-frequency pulsed electromagnetic fields in beating cardiomyocytes.

Proper ß-adrenergic signaling is indispensable for modulating heart frequency. Studies on extremely-low-frequency pulsed electromagnetic field (ELF-PEMF) effects in the heart beat function are contradictory and no definitive conclusions were obtained so far. To investigate the interplay between ELF-PEMF exposure and ß-adrenergic signaling, cultures of primary murine neonatal cardiomyocytes and of sinoatrial node were exposed to ELF-PEMF and short and long-term effects were evaluated. The ELF-PEMF generated a variable magnetic induction field of 0-6mT at a frequency of 75Hz. Exposure to 3mT ELF-PEMF induced a decrease of contraction rate, Ca(2+) transients, contraction force, and energy consumption both under basal conditions and after ß-adrenergic stimulation in neonatal cardiomyocytes. ELF-PEMF exposure inhibited ß-adrenergic response in sinoatrial node (SAN) region. ELF-PEMF specifically modulated ß2 adrenergic receptor response and the exposure did not modify the increase of contraction rate after adenylate cyclase stimulation by forskolin. In HEK293T cells transfected with ß1 or ß2 adrenergic receptors, ELF-PEMF exposure induced a rapid and selective internalization of ß2 adrenergic receptor. The ß-adrenergic signaling, was reduced trough Gi protein by ELF-PEMF exposure since the phosphorylation level of phospholamban and the PI3K pathway were impaired after isoproterenol stimulation in neonatal cardiomyocytes. Long term effects of ELF-PEMF exposure were assessed in cultures of isolated cardiomyocytes. ELF-PEMF counteracts cell size increase, the generation of binucleated of cardiomyocytes and prevents the up-regulation of hypertrophic markers after ß-adrenergic stimulation, indicating an inhibition of cell growth and maturation. These data show that short and long term exposure to ELF-PEMF induces a reduction of cardiac ß-adrenergic response at molecular, functional and adaptative levels.