[3+2]-Cycloaddition of Nitrile Imines to Parabanic Acid Derivatives-An Approach to Novel Spiroimidazolidinediones.

Approximately 1,3-Dipolar cycloaddition of imidazolidine derivatives containing exocyclic double bonds is a convenient method of creating spiro-conjugated molecules with promising anticancer activity. In this work, the derivatives of parabanic acid (2-thioxoimidazolidine-4,5-diones and 5-aryliminoimidazolidine-2,4-diones) were first investigated as dipolarophiles in the reactions with nitrile imines. The generation of nitrile imines was carried out either by the addition of tertiary amine to hydrazonoyl chlorides «drop by drop¼ or using the recently proposed diffusion mixing technique, which led to ~5-15% increases in target compound yields. It was found that the addition of nitrile imines to C=S or C=N exocyclic double bonds led to 1,2,4-thiazolines or triazolines and occurred regioselectively in accordance with the ratio of FMO coefficients of reactants. The yield of the resulting spiro-compound depended on the presence of alkyl substituents in the nitrile imine structure and was significantly decreased in reactions with imines with strong electron donor or electron-withdrawing groups. Some of the obtained compounds showed reasonable in vitro cytotoxicity. IC50 values were calculated for HCT116 (colon cancer) cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test.

RECIPROCAL CLICKING LOCATION ANALYSIS IN THE INTRAARTICULAR TEMPOROMANDIBULAR DISORDERS AFTER AXIOGRAPHY INVESTIGATION.

OBJECTIVE: The aim: To investigate the peculiarities of hinge axis trajectories in patients with condyle-disc complex intraarticular Temporomandibular Disorders (TMD) and determine the average coordinates of the reciprocal clicking location by axiography. PATIENTS AND METHODS: Materials and methods: The results of axiographic examination of 151 patients (108 females and 43 males) with TMD confirmed by MRI were analyzed. This population included 44 persons with disc displacement with reduction (DDR), 45 persons with disc displacement with reduction and intermittent locking (DDRI), 62 persons with disc displacement without reduction (DDWR). Axiographic examination was carried out using CADIAX diagnostic device. Analysis of hinge axis movements was performed and the coordinates of articular disc reduction were determined. RESULTS: Results: The quality of hinge axis trajectories in persons with DDR, DDRI was defined mainly as average and in patients with DDWR as poor. Quantitative indicators of trajectories during protrusion-retrusion movements were not beyond the average level. The length of the mouth opening-closing trajectory in patients with DDRI and DDWR has shown a tendency to decrease. We found that on average the reciprocal closing clicking (disc reduction) occurs at a distance of 0-1.4 mm on the X-axis, 0.1-2.9 mm on the Z-axis, and 0-0.85 mm on the Y-axis. CONCLUSION: Conclusions: The obtained wide range of reciprocal clicking location parameters indicates the priority of a personalized approach when planning preliminary treatment in order to restore the disc-condylar complex of TMJ.

Epidural abscess imitating recurrent pilonidal sinus: a case report.

Pilonidal sinus or pilonidal cyst is a common benign disease, affecting mostly young working men. We present the first case of an epidural abscess imitating pilonidal sinus. A 33-year old male, suffering from previously undiagnosed and untreated diabetes mellitus (DM), presented to our emergency department (ER), one month after open surgical treatment of pilonidal sinus, due to weakness and fever. After re-operation of the pilonidal cyst and due to post-operative pus production and continuation of fever a computer tomogr aphy (CT )scan was performed revealing an epidural abscess extending from the thoracic vertebrae 12 (T-12) to the sacrococcygeal area. At that point he underwent new surgery for drainage of the epidural abscess. The patient received intravenous antimicrobial treatment and was discharged on the 23rd postoperative day without signs or symptoms of infection. At follow up for a whole year no signs of recurrence have been observed.

Prevalence of drug resistance among HIV-1 treatment-naive patients in Greece during 2003-2015: Transmitted drug resistance is due to onward transmissions.

BACKGROUND: The prevalence of HIV-1 drug resistance among treatment-naïve patients ranges between 8.3% and 15% in Europe and North America. Previous studies showed that subtypes A and B were the most prevalent in the Greek HIV-1 epidemic. Our aim was to estimate the prevalence of resistance among drug naïve patients in Greece and to investigate the levels of transmission networking among those carrying resistant strains. METHODS: HIV-1 sequences were determined from 3428 drug naïve HIV-1 patients, in Greece sampled during 01/01/2003-30/6/2015. Transmission clusters were estimated by means of phylogenetic analysis including as references sequences from patients failing antiretroviral treatment in Greece and sequences sampled globally. RESULTS: The proportion of sequences with SDRMs was 5.98% (n=205). The most prevalent SDRMs were found for NNRTIs (3.76%), followed by N(t)RTIs (2.28%) and PIs (1.02%). The resistance prevalence was 22.2% based on all mutations associated with resistance estimated using the HIVdb resistance interpretation algorithm. Resistance to NNRTIs was the most common (16.9%) followed by PIs (4.9%) and N(t)RTIs (2.8%). The most frequently observed NNRTI resistant mutations were E138A (7.7%), E138Q (4.0%), K103N (2.3%) and V179D (1.3%). The majority of subtype A sequences (89.7%; 245 out of 273) with the dominant NNRTI resistance mutations (E138A, K103N, E138Q, V179D) were found to belong to monophyletic clusters suggesting regional dispersal. For subtype B, 68.1% (139 out of 204) of resistant strains (E138A, K103N, E138Q V179D) belonged to clusters. For N(t)RTI-resistance, evidence for regional dispersal was found for 27.3% and 21.6% of subtype A and B sequences, respectively. CONCLUSIONS: The TDR rate based on the prevalence of SDRM is lower than the average rate in Europe. However, the prevalence of NNRTI resistance estimated using the HIVdb approach, is high in Greece and it is mostly due to onward transmissions among drug-naïve patients.

Validation of the new Sepsis-3 definitions: proposal for improvement in early risk identification.

OBJECTIVES: Sepsis-3 definitions generated controversies regarding their general applicability. The Sepsis-3 Task Force outlined the need for validation with emphasis on the quick Sequential Organ Failure Assessment (qSOFA) score. This was done in a prospective cohort from a different healthcare setting. METHODS: Patients with infections and at least two signs of systemic inflammatory response syndrome (SIRS) were analysed. Sepsis was defined as total SOFA ≥2 outside the intensive care unit (ICU) or as an increase of ICU admission SOFA ≥2. The primary endpoints were the sensitivity of qSOFA outside the ICU and sepsis definition both outside and within the ICU to predict mortality. RESULTS: In all, 3346 infections outside the ICU and 1058 infections in the ICU were analysed. Outside the ICU, respective mortality with ≥2 SIRS and qSOFA ≥2 was 25.3% and 41.2% (p <0.0001); the sensitivities of qSOFA and of sepsis definition to predict death were 60.8% and 87.2%, respectively. This was 95.9% for sepsis definition in the ICU. The sensitivity of qSOFA and of ≥3 SIRS criteria for organ dysfunction outside the ICU was 48.7% and 72.5%, respectively (p <0.0001). Misclassification outside the ICU with the 1991 and Sepsis-3 definitions into stages of lower severity was 21.4% and 3.7%, respectively (p <0.0001) and 14.9% and 3.7%, respectively, in the ICU (p <0.0001). Adding arterial pH ≤7.30 to qSOFA increased sensitivity for prediction of death to 67.5% (p 0.004). CONCLUSIONS: Our analysis positively validated the use of SOFA score to predict unfavourable outcome and to limit misclassification into lower severity. However, qSOFA score had inadequate sensitivity for early risk assessment.

Impact of common ABCB1 polymorphism on pharmacokinetics and pharmacodynamics of clopidogrel and its metabolites.

WHAT IS KNOWN AND OBJECTIVE: The reasons of clopidogrel (CLP) resistance are still unclear. The response to CLP may be influenced by both genetic and non-genetic factors. Among genetic factors, common polymorphisms in the gene coding glycoprotein-P (P-gp, MDR1 and ABCB1) are considered as potential determinants of the efficacy of CLP treatment. The aim of this study was to evaluate the influence of ABCB1 3435C>T genetic polymorphism on the pharmacokinetics and pharmacodynamics of CLP and its metabolites: diastereoisomers of thiol metabolite (the inactive H3 and the active H4) and inactive carboxylic derivative. METHODS: The study group included 42 patients undergoing elective coronary angiography and percutaneous coronary intervention. The plasma concentrations of CLP and its metabolites were measured by a validated HPLC-MS/MS method. Whole-blood aggregation was determined with Multiplate analyzer. For evaluation of ABCB1 3435C>T polymorphism, PCR-RFLP method was applied. RESULTS AND DISCUSSION: It was found that Exposition to the unchanged CLP, measured by AUC0-t of the drug, was significantly lower (P = 0·012) in TT homozygotes comparing to that observed in CC and CT genotypes, although no correlation was found between platelet aggregation and ABCB1 genetic polymorphism. WHAT IS NEW AND CONCLUSION: Our findings show that the presence of 3435C>T allele has an impact on CLP pharmacokinetics but not on the drug pharmacodynamics. Therefore, the 3435C>T genotype may not be the primary determinant influencing the pharmacokinetics of the active H4 metabolite and antiplatelet effect of the drug.

The problem of medical dispatchers' responsibility functioning in the emergency medical services system.

AIM OF THE STUDY: Deaths due to inappropriate functioning of the emergency medical services system, as recently described by Polish mass media, has drawn the attention of society to the activities of medical dispatchers. Legal regulations impose obligations on those persons associated with receiving phone calls and dispensing appropriate emergency medical teams. In this paper an analysis of chosen medicolegal opinions from the practice of the Department of Forensic Medicine and Forensic Toxicology, Medical University of Silesia in Katowice, towards malpractices committed by dispatchers of EMS, was performed. MATERIAL AND METHODS: The authors analysed 12 of medicolegal opinions, issued from 2007 to 2012 by a team of experts. RESULTS: The errors noted in the work of dispatchers consisted of delays in giving appropriate assistance due to the inability to properly converse, a propensity to downplay patients' symptoms, and dispatchers crossing their own competences. CONCLUSIONS: The problem may be resolved by the subsidy of EMS, fine-tuning the algorithms for conduct, and proper education of both staff and public.

Pharmacokinetic studies of enantiomers of ibuprofen and its chiral metabolites in humans with different variants of genes coding CYP2C8 and CYP2C9 isoenzymes.

The pharmacokinetics of ibuprofen enantiomers and its chiral metabolites, namely (R,S)-29-hydroxyibuprofen and (RR,RS,SR,SS)-29-carboxyibuprofen, was studied in healthy volunteers carrying different alleles coding cytochrome P450 (CYP) 4502C isoenzymes. Following administration of 400 mg of racemic ibuprofen, enantiomers of the parent compound and their metabolites were isolated from plasma and urine samples using solid-phase extraction and were quantified by the validated capillary zone electrophoresis method. The levels of the analytes in biological fluids were used to calculate their pharmacokinetic parameters in subjects with different variants of CYP2C8 and CYP2C9 isoenzymes. The analysis of each subject's genotype was carried out using polymerase chain reaction-restriction fragment length polymorphism. Impaired metabolism of ibuprofen enantiomers was associated with the presence of CYP2C8*3, CYP2C9*2 and CYP2C9*3 alleles. The greatest effect of mutated alleles on pharmacokinetics was observed in a subject with a CYP2C8*1/*3, CYP2C9*1/*2 genotype. This subject appeared to have lower value of clearance, greater area under the curve (AUC) and longer time t(0.5) in comparison with the wild-type.

Pharmacokinetics of high-dose i.v. treosulfan in children undergoing treosulfan-based preparative regimen for allogeneic haematopoietic SCT.

Pharmacokinetic studies of high-dose treosulfan were carried out in seven paediatric patients (age range: 2-15 years) undergoing treosulfan-based conditioning regimen prior to allogeneic haematopoietic SCT. Treosulfan was administered intravenously in a daily dose of 10, 12 or 14 g/m(2) within 2 h. Five out of seven patients received 12 g/m(2). The plasma concentrations of treosulfan and its quantity eliminated with urine were determined using a validated HPLC method with refractometric detection. Pharmacokinetic parameters were evaluated following first dose using a two-compartment disposition model. These studies demonstrated a dose-dependent increase of area under the concentration (AUC) and maximum concentrationplasma (C(max)), but there was variability of these parameters. Rapid clearance of tresoulfan was observed, especially in 10 and 12 g/m(2) doses. Terminal half-life (t(0.5)) of treosulfan was in the range of 1.71-2.15 h, but the mean percent of parent drug eliminated with urine was 30%, range 16.3-45.4% of the total dose eliminated during the first 12 h after administration. The results of this study confirmed the linear pharmacokinetics of treosulfan, as used in children. However, variability of pharmacokinetic results observed in children studied demonstrates the need for pharmacokinetic evaluation in each paediatric patient undergoing the treosulfan-based preparative regimen, including those using different doses. This approach could enable further reduction of the risk of early and late organ toxicity related to high-dose treosulfan in paediatric patients.

Evidence for neuronal origin and metabotropic receptor-mediated regulation of extracellular glutamate and aspartate in rat striatum in vivo following electrical stimulation of the prefrontal cortex.

Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5-s intervals in the striatum of chloral hydrate-anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser-induced fluorescence. Application of a single 10-s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200-300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 microM tetrodotoxin or depletion of extracellular Ca2+, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (200 microM) increased resting Glu and Asp levels by 300-500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylate (200 microM). This effect was blocked by the metabotropic Glu receptor antagonist (RS)-alpha-methylcarboxyphenylglycine (MCPG; 200 microM). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of L-trans-pyrrolidine-2,4-dicarboxylic acid, MCPG produced a five- to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.

In vivo monitoring of glutathione and cysteine in rat caudate nucleus using microdialysis on-line with capillary zone electrophoresis-laser induced fluorescence detection.

A fully-automated method for monitoring thiols in vivo using microdialysis coupled on-line with capillary zone electrophoresis with laser-induced fluorescence detection was developed. Dialysates were derivatized on-line with monobromobimane and automatically transferred to the separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nl/min resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits for these analytes were in the 20-40 nM range and the response was linear up to 20 microM. The system was applied to the measurement of glutathione and cysteine in the extracellular space of the caudate nucleus of anesthetized rats. The measured basal concentrations of glutathione and cysteine were 2.0 +/- 0.1 microM and 2.3 +/- 0.3 microM, respectively which agree well with literature values. Increases in glutathione and cysteine were monitored with 180 s temporal resolution during stimulation by infusion of potassium. The average concentration of glutathione and cysteine during stimulation was 3.0 +/- 0.9 and 3.3 +/- 0.5 microM (n = 3), respectively. This system is the first to obtain high relative recoveries and high temporal resolution simultaneously for multiple thiols with microdialysis sampling in the brain.

High temporal resolution monitoring of glutamate and aspartate in vivo using microdialysis on-line with capillary electrophoresis with laser-induced fluorescence detection.

An automated method for high temporal resolution monitoring of the neurotransmitters glutamate and aspartate in vivo using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was developed. Microdialysis probes placed in the striatum of anesthetized rats were coupled on-line with the CE system by an automated flow-gated interface. Analytes were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and detected by LIF using the 354 nm line (7 mW) of a He-Cd laser for excitation. With dialysis flow rates of 1.2 microL/ min, the detection limit at the dialysis probe was 200 nM for glutamate and aspartate. Glutamate and aspartate could be resolved in less than 5 s with over 200,000 theoretical plates. The sampling time was limited by the separation time while the temporal resolution was limited to approximately 12 s because of band broadening that occurs within the probe and its associated tubing. The high temporal resolution allowed the first simultaneous monitoring of glutamate and aspartate overflow during acute electrical stimulation in the rat brain.

Monitoring of met-enkephalin in vivo with 5-min temporal resolution using microdialysis sampling and capillary liquid chromatography with electrochemical detection.

A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 microl/min. Separations were performed using 7 cm x 25 microm I.D. fused-silica capillary columns packed with 5 microm Alltima C18 particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 microl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K+-induced depolarization conditions.

Quantitative in vivo monitoring of primary amines in rat caudate nucleus using microdialysis coupled by a flow-gated interface to capillary electrophoresis with laser-induced fluorescence detection.

A method for monitoring primary amines in vivo using microdialysis coupled on-line with capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection was explored. Dialysates were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and automatically transferred to a separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nL/min, resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits were in the 20-50 nM range, and the response was linear up to 50 microM. Temporal resolution was between 45 s and 3 min and was limited by separation time or broadening of sample zones during transfer to the separation capillary, depending on the operational parameters. The system was applied to measurement of primary amines in the caudate nucleus of anesthetized rats. Using CZE for separation, it was possible to resolve and monitor several compounds, including aspartate and glutamate. The measured basal concentrations of aspartate and glutamate were 1.2 +/- 0.1 and 5.0 +/- 0.4 microM, respectively, which agrees well with literature values. Increases in in vivo aspartate and glutamate were monitored with 90 s temporal resolution during K+ depolarization using dialysis flow rates of 79 nL/min; however, temporal resolution of 45 s was possible at the expense of lower relative recovery if the dialysis flow rate was increased to 155 nL/min. The use of MEKC as the separation mode significantly increased the number of compounds that could be resolved and detected. Using MEKC to separate the dialysate samples allowed aspartate, glutamate, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, tyrosine, and valine to be resolved and detected. The basal concentrations for these compounds using MEKC were 1.9 +/- 0.2, 4.1 +/- 0.2, 4.6 +/- 0.7, 2.6 +/- 0.3, 5.4 +/- 0.4, 1.8 +/- 0.2, 2.0 +/- 0.2, 11.3 +/- 1.3, 3.3 +/- 0.9, and 5.3 +/- 0.3 microM, respectively. The concentrations of these primary amines in the striatum were monitored after K+ depolarization with 3 min temporal resolution. This is the first microdialysis system to generate high relative recoveries and good temporal resolution simultaneously for multiple neurotransmitters.

Quantitative in vivo measurements using microdialysis on-line with capillary zone electrophoresis.

A system which couples microdialysis with capillary zone electrophoresis (CZE) on-line is used to monitor ascorbate and lactate in the caudate nucleus of rat brain. On-line interface of microdialysis probe and electrophoresis capillary, along with the high mass sensitivity of CZE, allows the probe to be operated at flow rates as low as 40 nl/min. Under these conditions, the relative recovery is nearly 100% and quantitative monitoring is possible. The microscale system also facilitates calibration by the low flow rate method. In spite of the low flow rate, temporal resolution in the 45-125 s range is possible for these compounds. The system is demonstrated by observing changes in ascorbate due to infusions of elevated K+ through the dialysis probe and systemic injections of amphetamine and an anesthetic (ketamine/xylazine/acepromazine mixture). Lactate is monitored in response to elevated K+ infusions.