Focusing on the 5F-MDMB-PICA, 4F-MDMB-BICA synthetic cannabinoids and their primary metabolites in analytical and pharmacological aspects.

Nowadays, more and more new synthetic cannabinoids (SCs) appearing on the illicit market present challenges to analytical, forensic, and toxicology experts. For a better understanding of the physiological effect of SCs, the key issue is studying their metabolomic and psychoactive properties. In this study, our validated targeted reversed phase UHPLC-MS/MS method was used for determination of urinary concentration of 5F-MDMB-PICA, 4F-MDMB-BICA, and their primary metabolites. The liquid-liquid extraction procedure was applied for the enrichment of SCs. The pharmacological characterization of investigated SCs were studied by radioligand competition binding and ligand stimulated [35S]GTPγS binding assays. For 5F-MDMB-PICA and 4F-MDMB-BICA, the median urinary concentrations were 0.076 and 0.312 ng/mL. For primary metabolites, the concentration range was 0.029-881.02* ng/mL for 5F-MDMB-PICA-COOH, and 0.396-4579* ng/mL for 4F-MDMB-BICA-COOH. In the polydrug aspect, the 22 urine samples were verified to be abused with 6 illicit drugs. The affinity of the metabolites to CB1R significantly decreased compared to the parent ligands. In the GTPγS functional assay, both 5F-MDMB-PICA and 4F-MDMB-BICA were acting as full agonists, while the metabolites were found as weak inverse agonists. Additionally, the G-protein stimulatory effects of the full agonist 5F-MDMB-PICA and 4F-MDMB-BICA were reduced by metabolites. These results strongly indicate the dose-dependent CB1R-mediated weak inverse agonist effects of the two butanoic acid metabolites. The obtained high concentration of main urinary metabolites of 5F-MDMB-PICA and 4F-MDMB-BICA confirmed the relevance of their routine analysis in forensic and toxicological practices. Based on in vitro binding assays, the metabolites presumably might cause a lower psychoactive effect than parent compounds.

[Tempora mutantur... et nos? The future of the Hungarian anatomy teaching in reflection of the German trends]. / Tempora mutantur et nos? A magyar anatómiaoktatás jövoje a német trendek tükrében.

The traditional four-semester anatomy is a subject to change: next to the external pressure, there is an intrinsic need to shift the emphasis. The mapping of the strengths, weaknesses and threats of the Hungarian anatomy teaching helps to formulate the directions of possible development. Current trends in the German medical education should be carefully followed. Nowadays, nearly 25% of the medical students in Germany are studying according to the new, integrated "Modellstudiengang", i.e. all the conventional subjects are reorganised into organ system thematic blocks. The unified German written final exam system provides an objective assessment parameter: to rank the 36 German medical schools according to the results of the anatomy exams. The homepage-published data, the number of semesters or teaching hours, or the thematic concept of the subject alone cannot explain the rankings of the medical schools according to the anatomy exam results. The greatest challenges of the Hungarian anatomy teaching today are: the development of an outcome-oriented, unified, practical system of requirements, the redefinition of the subject, the more effective interaction with the clinical colleagues, solving the problems of faculty recruitment and establishing the vertical integration of anatomy.

Single dose irradiation of defined region of rat brain with stereotactic brainlab system.

UNLABELLED: Background and purpose of our study was to develop a precise dose delivery technique for partial brain irradiation of two rats simultaneously. METHODS: Using a self-developed frame stereotactic radiotherapy with single doses of 30-90 Gy was delivered to the frontal lobe of 22 animals. Tolerability and reproducibility of the method were evaluated and dosimetric measurements were conducted to verify the treatment plans. 2, 4 and 6 months after the irradiation magnetic resonance imaging (MRI) scans and histopathological examinations were performed to detect late radiation induced biological changes. RESULTS: Immobilization device provided excellent reproducibility and tolerability. Dosimetry revealed good correspondence with planned dose distribution, but the measured absorbed dose was 30% lower than the planned dose. During the 6 months follow-up period the procedure related death of subject animals after 30 Gy, 70 Gy and 90 Gy were 0%, 20% and 100% respectively. T2 signal and structural changes on MRI scans found to be dose and time dependent. While 30 Gy caused no detectable structural changes, 70 Gy lead to cystic necrosis in 2 cases after 4 month. Histopathology revealed signs of necrosis on macroscopic examination after 70 Gy in the high dose region involving both frontal lobes, and no obvious microscopic changes in the surrounding area were detectable. CONCLUSION: Our technique of rat cranial irradiation using human stereotactic system provided high accuracy of single dose delivery for a pair of small animals, resulting in brain injury in the defined area. This method proved to be a reproducible model for preclinical studies on radiation effects.

In-depth protein profiling of the postsynaptic density from mouse hippocampus using data-independent acquisition proteomics.

Located at neuronal terminals, the postsynaptic density (PSD) is a highly complex network of cytoskeletal scaffolding and signaling proteins responsible for the transduction and modulation of glutamatergic signaling between neurons. Using ion-mobility enhanced data-independent label-free LC-MS/MS, we established a reference proteome of crude synaptosomes, synaptic junctions, and PSD derived from mouse hippocampus including TOP3-based absolute quantification values for identified proteins. The final dataset across all fractions comprised 49 491 peptides corresponding to 4558 protein groups. Of these, 2102 protein groups were identified in highly purified PSD in at least two biological replicates. Identified proteins play pivotal roles in neurological and synaptic processes providing a rich resource for studies on hippocampal PSD function as well as on the pathogenesis of neuropsychiatric disorders. All MS data have been deposited in the ProteomeXchange with identifier PXD000590 (http://proteomecentral.proteomexchange.org/dataset/PXD000590).

Breast cancer under 40 years of age: increasing number and worse prognosis.

Breast cancer at a relatively young age with a poor prognosis is currently exhibiting an increasing incidence. In a retrospective cohort analysis of early breast cancer cases after surgery from our institutional patient registry, 141 patients aged ≤ 40 years constituted the younger group, with 300 randomly selected patients aged >40 years as controls. A significant and steady increase was found in the relative number of younger cases during the years 2004-2009. The histological type and grade and the lymph node status of the cancers differed significantly between the two groups, with more aggressive biological behaviour, a more advanced stage and a worse prognosis in the younger group. Half of the cancers in the younger cohort were ER-negative, while two-thirds in the control group were ER-positive. Comparatively more tumours were PR-positive and HER2-negative in the control group than in the younger group. The rates of triple-negative cases were 25% and 13% in the younger age and the control group, respectively (p = 0.026). Significantly higher mastectomy and axillary block dissection rates were observed in the younger age group, and more chemotherapy was administered than in the control group. Our findings demonstrate the significance of breast cancer in cases aged <40 years, and draw attention to the need for appropriate care in these cases.

Blockade of AMPA-receptors attenuates 4-aminopyridine seizures, decreases the activation of inhibitory neurons but is ineffective against seizure-related astrocytic swelling.

The neurotransmitter glutamate plays a pivotal role in the development of the neuropathological sequelae following acute seizures. Our previous data proved the efficacy of the NMDA-receptor antagonists on the symptoms, survival and neuronal activation in the 4-aminopyridine- (4-AP) induced seizures. In this study, we examined the effects of two different doses of a non-competitive, selective, allosteric AMPA-receptor antagonist, GYKI 52466. GYKI 52466 was effective in prolonging the latency to generalised seizures and reduction of seizure mortality. However, the effects on neuronal c-fos expression and astrocyte swelling were complex. The 25mg/kg dose of GYKI 52466 was effective in reducing the c-fos immunoreactivity (IR) in the hippocampus only. In the neocortex the overall c-fos-IR cell counts were increased significantly. Investigation of the neocortical parvalbumin-containing interneuron population proved that GYKI 52466 decreased c-fos expression. The 50mg/kg dose of GYKI 52466 significantly reduced the c-fos-IR in the neo- and allocortex, not only in principal neurons, but also in the parvalbumin-positive interneurons. The GYKI 52466-pretreatment did not prevent the astrocyte swelling in the investigated cortical areas; thus we conclude that the AMPA-receptors have little if any involvement in the in the mediation of neuropathological alterations in acute convulsions.

Neocortical c-fos mRNA transcription in repeated, brief, acute seizures: is c-fos a coincidence detector?

The effect of acute brief seizures on neocortical c-fos expression was investigated in rats injected with 5 mg/kg 4-aminopyridine. Electroencephalography in freely moving animals with implanted neocortical electrodes detected an average of 2.67 tonic-clonic convulsions within 1 h following the 4-AP treatment. Tissue samples of the somatosensory neocortex were collected at 30 min, 1 h, 3 h, 5 h and 8 h following the treatment for PCR and immunohistochemistry. The c-fos mRNA displayed the first significant rise at 1 h, and remained significantly higher through 3 h. The number of c-fos protein immunoreactive cells was significantly elevated already at 30 min, peaked at 1 h, and declined by 5 h. We conclude that in repetitive, brief seizures, the first convulsion does not increase c-fos RNA transcription, whilst the second causes a long-lasting gene expression and a large increase of c-fos protein synthesis. The phenomenon may have implications in the pathogenesis of human and animal epilepsies.

Seizure, neurotransmitter release, and gene expression are closely related in the striatum of 4-aminopyridine-treated rats.

The present experiments aimed to compare the length of seizure activity with the time-related increase of transmitter release and the induction of c-fos gene expression in the striatum of the rat. Anesthetized Wistar rats were intraperitoneally treated with 7 mg/kg 4-aminopyridine, and the transmitter levels in the striatum were measured by means of in vivo microdialysis, 30, 60, 90, 120, and 150 min following the treatment. Striatal and neocortical electric activity was monitored with depth and surface electrodes, respectively. The expression level of the c-fos gene was estimated by counting the striatal c-fos-immunostained cell nuclei at the time intervals of the microdialysis. 4-aminopyridine elicited high-frequency seizure discharges in the EEG and significantly increased glutamate, aspartate, GABA, serotonin, noradrenaline, and dopamine levels in the extracellular dialysates. The number of c-fos-stained cell nuclei in the striatum displayed a prolonged increase, showing significantly elevated numbers throughout the experiment. The increase of c-fos expression in time correlated best with the increase of glutamate release, which was also significantly elevated at every sampling time. The GABA release, culminating at 60 min after the seizure onset, correlated best with the cessation of the electrographic seizure. Aspartate, norepinephrine, serotonin, and dopamine displayed transient but significant elevations. We conclude that glutamate plays the essential role (most probably through ionotropic and metabotropic receptors) in the extracellular signaling, which eventually leads to intracellular cascades and c-fos gene expression in the striatum during convulsions.

Non-competitive NMDA receptor antagonists moderate seizure-induced c-fos expression in the rat cerebral cortex.

We examined the effects of non-competitive NMDA glutamate receptor antagonists on seizures elicited by 4-aminopyridine (4-AP), and in particular, on the expression of the transcription factor c-fos induced by these seizures. Induction of c-fos mRNA due to 4-AP-elicited seizures was ascertained by reverse transcription polymerase chain reaction in samples of the neocortex. Adult rats were pretreated with the NMDA receptor antagonists amantadine (40 mg/kg), ketamine (3mg/kg), dizocilpine (MK-801; 1mg/kg) or dextrometorphan (40 mg/kg); 4-AP (5mg/kg) was then injected i.p. Controls were treated with either antagonist only or with 4-AP only. Pretreatment with the antagonists (with the exception of amantadine) increased the latency of behavioural seizures, but not all of the antagonists caused symptomatic seizure protection. In the brains which were processed for Fos immunohistochemistry, quantitative evaluation of immunostained cells was performed in the neocortex and hippocampus. Treatment with either antagonist did not induce by itself c-fos expression, with the exception of amantadine, which caused slight Fos induction in the neocortex. Pretreatment with all the antagonists resulted in decrease of seizure-induced Fos immunoreactivity with respect to non-pretreated animals. Decrease of immunostained cells was significant in the neocortex, in the granule cell layer and hilus of the dentate gyrus, in hippocampal areas CA1 and CA2. MK-801, ketamine and dextrometorphan decreased significantly Fos immunoreactivity also in area CA3. The decrease of Fos immunostaining was not directly correlated with a suppression of behavioural seizures. The results support an important role of NMDA receptors in c-fos gene induction in acute 4-AP seizures.