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The significant synthetic potential and reactivity of tetracyanoethylene (TCNE) have captured the interest of numerous chemical communities. One of the most promising, readily achievable, yet least explored pathways for the reactivity of TCNE involves its interaction with arylamines. Typically, the reaction proceeds via tricyanovinylation (TCV); however, deviations from the standard chemical process have been observed in some instances. These include the formation of heterocyclic structures through tricyanovinyl intermediates, aliphatic dicarbonitriles through the cleavage of the C-C bond of a tetracyanoethyl substituent, complexation, and various pericyclic reactions. Therefore, the objective of this study is to review the diverse modes of interaction of TCNE with aromatic nitrogen-containing compounds and to focus the attention of the chemical community on the synthetic capabilities of this reagent, as well as the various biological and optical activities of the structures synthesized based on TCNE.

Animal models have been essential for advancing research of fetal alcohol spectrum disorder (FASD) in humans, but few animal species effectively replicate the behavioural and clinical signs of FASD. The honey bee ( Apis mellifera ) is a previously unexplored research model for FASD that offers the distinct benefit of highly social behaviour. In this study, we chronically exposed honey bee larvae to incremental concentrations of 0, 3, 6, and 10% ethanol in the larval diet using an in vitro rearing protocol and measured developmental time and survival to adult eclosion, as well as body weight and motor activity of newly emerged adult bees. Larvae reared on 6 and 10% dietary ethanol demonstrated significant, dose-responsive delays to pupation and decreased survival and adult body weight. All ethanol-reared adults showed significantly decreased motor activity. These results suggest that honey bees may be a suitable social animal model for future FASD research.

Background: Recurrent pregnancy loss (RPL) is a complicated reproductive disorder with underlying genetic and immunological causes. RPL may be influenced by hereditary thrombophilia, a class of blood clotting-related genetic abnormalities, via the vascular and immune systems. This study examines the immunological characteristics that hereditary thrombophilia patients have in common with RPL., Methods: A prospective cohort study included 300 patients split into two groups: a control group without hereditary thrombophilia and a group with the condition. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) levels were measured, along with demographic specifics, antiphospholipid antibodies, natural killer (NK) cell counts, and other cytokines. Group differences were found using statistical analysis., Results: Antiphospholipid antibodies were significantly more common in the thrombophilia group (42% testing positive, p=0.001) compared to the control group (12% testing positive), despite demographic factors being similar between groups (p=0.372 and p=0.093). When body mass index (BMI) was taken into account, the study found a statistically significant difference (p=0.046), with the thrombophilia group having a higher mean BMI (26.3 kg/m 2 , standard deviation (SD): 2.8) than the control group (24.7 kg/m 2 , SD: 3.1). IL-6 (14.8 pg/mL, SD: 3.2, p=0.029) were higher than the control group (12.4 pg/mL, SD: 2.1), and TNF-α levels were higher in the thrombophilia group (10.5 pg/mL, SD: 2.0, p=0.012) compared to the control group (8.9 pg/mL, SD: 1.5), but NK cell counts did not differ significantly (p=0.213)., Conclusion: This study emphasizes the role of elevated pro-inflammatory cytokines (IL-6 and TNF-α) and antiphospholipid antibodies in RPL among people with hereditary thrombophilia. In this population, early detection and immunomodulatory interventions may improve pregnancy outcomes. To fully comprehend these mechanisms and create customized treatments, collaborative research is required., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Kirovakov et al.)

"Heptil" (unsymmetrical dimethylhydrazine-UDMH) is extensively employed worldwide as a propellant for rocket engines. However, UDMH constantly loses its properties as a result of its continuous and uncontrolled absorption of moisture, which cannot be rectified. This situation threatens its long-term usability. UDMH is an exceedingly toxic compound (Hazard Class 1), which complicates its transportation and disposal. Incineration is currently the only method used for its disposal, but this process generates oxidation by-products that are even more toxic than the original UDMH. A more benign approach involves its immediate reaction with a formalin solution to form 1,1-dimethyl-2-methylene hydrazone (MDH), which is significantly less toxic by an order of magnitude. MDH can then be polymerized under acidic conditions, and the resulting product can be burned, yielding substantial amounts of nitrogen oxides. This review seeks to shift the focus of MDH from incineration towards its application in the synthesis of relatively non-toxic and readily available analogs of various pharmaceutical substances. We aim to bring the attention of the international chemical community to the distinctive properties of MDH, as well as other hydrazones (such as glyoxal, acrolein, crotonal, and meta-crolyl), wherein each structural fragment can initiate unique transformations that have potential applications in molecular design, pharmaceutical research, and medicinal chemistry.

Dilated cardiomyopathy (DCM) is a disease of the myocardium defined by left ventricular enlargement and systolic dysfunction leading to heart failure. Danicamtiv, a new targeted myosin activator designed for the treatment of DCM, was characterised in in vitro and in vivo preclinical studies. Danicamtiv human hepatic clearance was predicted to be 0.5 mL/min/kg from in vitro metabolic stability studies in human hepatocytes. For human, plasma protein binding was moderate with a fraction unbound of 0.16, whole blood-to-plasma partitioning ratio was 0.8, and danicamtiv showed high permeability and no efflux in a Caco-2 cell line. Danicamtiv metabolism pathways in vitro included CYP-mediated amide-cleavage, N -demethylation, as well as isoxazole- and piperidine-ring-opening. Danicamtiv clearance in vivo was low across species with 15.5, 15.3, 1.6, and 5.7 mL/min/kg in mouse, rat, dog, and monkey, respectively. Volume of distribution ranged from 0.24 L/kg in mouse to 1.7 L/kg in rat. Oral bioavailability ranged from 26% in mouse to 108% in dog. Simple allometric scaling prediction of human plasma clearance, volume of distribution, and half-life was 0.64 mL/min/kg, 0.98 L/kg, and 17.7 h, respectively. Danicamtiv preclinical attributes and predicted human pharmacokinetics supported advancement toward clinical development.

Background & Aims: Although off-label use of sofosbuvir-containing regimens occurs regularly in patients with hepatitis C virus (HCV) infection undergoing dialysis for severe renal impairment or end-stage renal disease (ESRD), these regimens are not licensed for this indication, and there is an absence of dosing recommendations in this population. This study evaluated the safety and efficacy of sofosbuvir/velpatasvir in patients with HCV infection with ESRD undergoing dialysis., Methods: In this phase II, single-arm study, 59 patients with genotype 1-6 HCV infection with ESRD undergoing hemodialysis or peritoneal dialysis received open-label sofosbuvir/velpatasvir (400 mg/100 mg) once daily for 12 weeks. Patients were HCV treatment naive or treatment experienced without cirrhosis or with compensated cirrhosis. Patients previously treated with any HCV NS5A inhibitor were not eligible. The primary efficacy endpoint was the proportion of patients achieving sustained virologic response (SVR) 12 weeks after discontinuation of treatment (SVR12). The primary safety endpoint was the proportion of patients who discontinued study drug due to adverse events., Results: Overall, 56 of 59 patients achieved SVR12 (95%; 95% CI 86-99%). Of the 3 patients who did not achieve SVR12, 2 patients had virologic relapse determined at post-treatment Week 4 (including 1 who prematurely discontinued study treatment), and 1 patient died from suicide after achieving SVR through post-treatment Week 4. The most common adverse events were headache (17%), fatigue (14%), nausea (14%), and vomiting (14%). Serious adverse events were reported for 11 patients (19%), and all were deemed to be unrelated to sofosbuvir/velpatasvir., Conclusions: Treatment with sofosbuvir/velpatasvir for 12 weeks was safe and effective in patients with ESRD undergoing dialysis., Lay Summary: Sofosbuvir/velpatasvir is a combination direct-acting antiviral that is approved for treatment of patients with hepatitis C virus (HCV) infection. Despite the lack of dosing recommendations, sofosbuvir-containing regimens (including sofosbuvir/velpatasvir) are frequently used for HCV-infected patients undergoing dialysis. This study evaluated the safety and efficacy of sofosbuvir/velpatasvir for 12 weeks in patients with HCV infection who were undergoing dialysis. Treatment with sofosbuvir/velpatasvir was safe and well tolerated, resulting in a cure rate of 95% in patients with HCV infection and end-stage renal disease. Clinical Trial Number: NCT03036852., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug-drug interaction potential. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug-drug interaction risk. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.

Purpose: The purpose of this study was to evaluate the quality of automatically propagated contours of organs at risk (OARs) based on respiratory-correlated navigator-triggered four-dimensional magnetic resonance imaging (RC-4DMRI) for calculation of internal organ-at-risk volume (IRV) to account for intra-fractional OAR motion., Methods and Materials: T2-weighted RC-4DMRI images were of 10 volunteers acquired and reconstructed using an internal navigator-echo surrogate and concurrent external bellows under an IRB-approved protocol. Four major OARs (lungs, heart, liver, and stomach) were delineated in the 10-phase 4DMRI. Two manual-contour sets were delineated by two clinical personnel and two automatic-contour sets were propagated using free-form deformable image registration. The OAR volume variation within the 10-phase cycle was assessed and the IRV was calculated as the union of all OAR contours. The OAR contour similarity between the navigator-triggered and bellows-rebinned 4DMRI was compared. A total of 2400 contours were compared to the most probable ground truth with a 95% confidence level (S95) in similarity, sensitivity, and specificity using the simultaneous truth and performance level estimation (STAPLE) algorithm., Results: Visual inspection of automatically propagated contours finds that approximately 5-10% require manual correction. The similarity, sensitivity, and specificity between manual and automatic contours are indistinguishable (P > 0.05). The Jaccard similarity indexes are 0.92 ± 0.02 (lungs), 0.89 ± 0.03 (heart), 0.92 ± 0.02 (liver), and 0.83 ± 0.04 (stomach). Volume variations within the breathing cycle are small for the heart (2.6 ± 1.5%), liver (1.2 ± 0.6%), and stomach (2.6 ± 0.8%), whereas the IRV is much larger than the OAR volume by: 20.3 ± 8.6% (heart), 24.0 ± 8.6% (liver), and 47.6 ± 20.2% (stomach). The Jaccard index is higher in navigator-triggered than bellows-rebinned 4DMRI by 4% (P < 0.05), due to the higher image quality of navigator-based 4DMRI., Conclusion: Automatic and manual OAR contours from Navigator-triggered 4DMRI are not statistically distinguishable. The navigator-triggered 4DMRI image provides higher contour quality than bellows-rebinned 4DMRI. The IRVs are 20-50% larger than OAR volumes and should be considered in dose estimation., (© 2018 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.)

Purpose: To develop and evaluate a super-resolution approach to reconstruct time-resolved 4-dimensional magnetic resonance imaging (TR-4DMRI) with a high spatiotemporal resolution for multi-breathing cycle motion assessment., Methods and Materials: A super-resolution approach was developed to combine fast 3-dimensional (3D) cine MRI with low resolution during free breathing (FB) and high-resolution 3D static MRI during breath hold (BH) using deformable image registration. A T1-weighted, turbo field echo sequence, coronal 3D cine acquisition, partial Fourier approximation, and SENSitivity Encoding parallel acceleration were used. The same MRI pulse sequence, field of view, and acceleration techniques were applied in both FB and BH acquisitions; the intensity-based Demons deformable image registration method was used. Under an institutional review board-approved protocol, 7 volunteers were studied with 3D cine FB scan (voxel size: 5 × 5 × 5 mm 3 ) at 2 Hz for 40 seconds and a 3D static BH scan (2 × 2 × 2 mm 3 ). To examine the image fidelity of 3D cine and super-resolution TR-4DMRI, a mobile gel phantom with multi-internal targets was scanned at 3 speeds and compared with the 3D static image. Image similarity among 3D cine, 4DMRI, and 3D static was evaluated visually using difference image and quantitatively using voxel intensity correlation and Dice index (phantom only). Multi-breathing-cycle waveforms were extracted and compared in both phantom and volunteer images using the 3D cine as the references., Results: Mild imaging artifacts were found in the 3D cine and TR-4DMRI of the mobile gel phantom with a Dice index of >0.95. Among 7 volunteers, the super-resolution TR-4DMRI yielded high voxel-intensity correlation (0.92 ± 0.05) and low voxel-intensity difference (<0.05). The detected motion differences between TR-4DMRI and 3D cine were -0.2 ± 0.5 mm (phantom) and -0.2 ± 1.9 mm (diaphragms)., Conclusion: Super-resolution TR-4DMRI has been reconstructed with adequate temporal (2 Hz) and spatial (2 × 2 × 2 mm 3 ) resolutions. Further TR-4DMRI characterization and improvement are necessary before clinical applications. Multi-breathing cycles can be examined, providing patient-specific breathing irregularities and motion statistics for future 4D radiation therapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart muscle characterized by otherwise unexplained thickening of the left ventricle. Left ventricular outflow tract (LVOT) obstruction is present in approximately two-thirds of patients and substantially increases the risk of disease complications. Invasive treatment with septal myectomy or alcohol septal ablation can improve symptoms and functional status, but currently available drugs for reducing obstruction have pleiotropic effects and variable therapeutic responses. New medical treatments with more targeted pharmacology are needed, but the lack of preclinical animal models for HCM with LVOT obstruction has limited their development. HCM is a common cause of heart failure in cats, and a subset exhibit systolic anterior motion of the mitral valve leading to LVOT obstruction. MYK-461 is a recently-described, mechanistically novel small molecule that acts at the sarcomere to specifically inhibit contractility that has been proposed as a treatment for HCM. Here, we use MYK-461 to test whether direct reduction in contractility is sufficient to relieve LVOT obstruction in feline HCM. We evaluated mixed-breed cats in a research colony derived from a Maine Coon/mixed-breed founder with naturally-occurring HCM. By echocardiography, we identified five cats that developed systolic anterior motion of the mitral valve and LVOT obstruction both at rest and under anesthesia when provoked with an adrenergic agonist. An IV MYK-461 infusion and echocardiography protocol was developed to serially assess contractility and LVOT gradient at multiple MYK-461 concentrations. Treatment with MYK-461 reduced contractility, eliminated systolic anterior motion of the mitral valve and relieved LVOT pressure gradients in an exposure-dependent manner. Our findings provide proof of principle that acute reduction in contractility with MYK-461 is sufficient to relieve LVOT obstruction. Further, these studies suggest that feline HCM will be a valuable translational model for the study of disease pathology, particularly LVOT obstruction., Competing Interests: This work was supported in part by an unrestricted research grant from MyoKardia, Inc. to SPH, SM, MJE and EMG are employees and own shares of, and JBK is a former employee and owns shares of, MyoKardia, Inc., a biotechnology company developing small molecules that target the sarcomere for treatment of inherited cardiomyopathy. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Although arsenic trioxide (arsenite, As(III)) has shown a remarkable efficacy in the treatment of acute promyelocytic leukemia patients, multidrug resistance is still a major concern for its clinical use. Multidrug resistance-associated protein 4 (MRP4), which belongs to the ATP-binding cassette (ABC) superfamily of transporters, is localized to the basolateral membrane of hepatocytes and the apical membrane of renal proximal tubule cells. Due to its characteristic localization, MRP4 is proposed as a candidate in the elimination of arsenic and may contribute to resistance to As(III). To test this hypothesis, stable HEK293 cells overexpressing MRP4 or MRP2 were used to establish the role of these two transporters in As(III) resistance. The IC50 values of As(III) in MRP4 cells were approximately 6-fold higher than those in MRP2 cells, supporting an important role for MRP4 in resistance to As(III). The capacity of MRP4 to confer resistance to As(III) was further confirmed by a dramatic decrease in the IC50 values with the addition of MK571, an MRP4 inhibitor, and cyclosporine A, a well-known broad-spectrum inhibitor of ABC transporters. Surprisingly, the sensitivity of the MRP2 cells to As(III) was similar to that of the parent cells, although insufficient formation of glutathione and/or Se conjugated arsenic compounds in the MRP2 cells might limit transport. Given that MRP4 is a major contributor to arsenic resistance in vitro, further investigation into the correlation between MRP4 expression and treatment outcome of leukemia patients treated with arsenic-based regimens is warranted.

Objective: Our goal was to compare direct quantitation of circulating free 25-hydroxyvitamin D (25(OH)D)levels to calculated free 25(OH)D levels and their relationships to intact PTH (iPTH), a biomarker of 25(OH)D effect, in humans with a range of clinical conditions., Patients and Methods: Serum samples and clinical data were collected from 155 people: 111 without cirrhosis or pregnancy (comparison group), 24 cirrhotic patients with albumin <2.9 g/dL, and 20 pregnant women (second and third trimester). Total 25(OH)D (LC/MS/MS), free 25(OH)D (immunoassay), vitamin D binding protein (DBP) (immunoassay), albumin, and iPTH (immunoassay) were measured., Results: Total 25(OH)D, DBP, and albumin were lowest in patients with cirrhosis, but measured free 25(OH)D was highest in this group (P < .001). DBP was highest in pregnant women (P < .001), but measured free 25(OH)D did not differ from the comparison group. Calculated free 25(OH)D was positively correlated with measured free 25(OH)D (P < .0001) but explained only 13% of the variability with calculated values higher than measured. African Americans had lower DBP than other ethnic populations within all clinical groups (P < .03), and differences between measured and calculated free 25(OH)D were greatest in African Americans (P < .001). Measured free 25(OH)D was correlated with total 25(OH)D (P < .0001; r(2) = 0.51), but calculated free 25(OH)D was not. Similarly, both measured free 25(OH)D (P < .02) and total 25(OH)D (P < .05) were correlated with iPTH, but calculated free 25(OH)D was not., Conclusions: Calculated free 25(OH)D levels varied considerably from direct measurements of free 25(OH)D with discrepancies greatest in the data for African Americans. Differences in DBP binding affinity likely contributed to estimation errors between the races. Directly measured free 25(OH)D concentrations were related to iPTH, but calculated estimates were not. Current algorithms to calculate free 25(OH)D may not be accurate. Further evaluation of directly measured free 25(OH)D levels to determine its role in research and clinical management of patients is needed.

Multidrug resistance protein 2 (MRP2, ABCC2) is an efflux membrane transporter highly expressed in liver, kidney and intestine with important physiological and pharmacological roles. The goal of this study was to investigate the functional significance of promoter region polymorphisms in ABCC2 and potential allele-specific expression. Twelve polymorphisms in the 1.6 kb region upstream of the translation start site were identified by resequencing 247 DNA samples from ethnically diverse individuals. Luciferase reporter gene assays showed that ABCC2 -24C>T both alone and as part of a common haplotype (-24C>T/-1019A>G/-1549G>A) increased promoter function 35% compared with the reference sequence (P<0.0001). No other common variants or haplotypes affected ABCC2 promoter activity. Allele-specific expression was also investigated as a mechanism to explain reported associations of the synonymous ABCC2 3972C>T variant with pharmacokinetic phenotypes. In Caucasian liver samples (n=41) heterozygous for the 3972C>T polymorphism, the 3972C allele was preferentially transcribed relative to the 3972T allele (P<0.0001). This allelic imbalance was particularly apparent in samples with haplotypes containing two or three promoter/untranslated region variants (-1549G>A, -1019A>G and -24C>T). The observed allelic imbalance was not associated with hepatic or renal ABCC2 mRNA expression. Additional mechanisms will need to be explored to account for the interindividual variation in ABCC2 expression and MRP2 function.

Docetaxel-related neutropenia was associated with polymorphisms in the drug transporters ABCC2 and SLCO1B3 in Japanese cancer patients. We hypothesized that this association is because of reduced docetaxel clearance, associated with polymorphisms in those genes. We studied 64 US cancer patients who received a single cycle of 75 mg/m of docetaxel monotherapy. We found that the ABCC2 polymorphism at rs-12762549 trended to show a relationship with reduced docetaxel clearance (P=0.048), but not with neutropenia. There was no significant association of the SLCO1B3 polymorphisms with docetaxel clearance or neutropenia. We conclude that the relationship between docetaxel-associated neutropenia and polymorphisms in drug transporters identified in Japanese patients was not confirmed in this cohort of US cancer patients., (© 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.)

ATP-binding cassette (ABC) membrane transporters determine the disposition of many drugs, metabolites and endogenous compounds. Coding region variation in ABC transporters is the cause of many genetic disorders, but much less is known about the genetic basis and functional outcome of ABC transporter expression level variation. We used genotype and mRNA transcript level data from human lymphoblastoid cell lines to assess population and gender differences in ABC transporter expression, and to guide the discovery of genomic regions involved in transcriptional regulation. Nineteen of 49 ABC genes were differentially expressed between individuals of African, Asian and European descent, suggesting an important influence of race on expression level of ABC transporters. Twenty-four significant associations were found between transporter transcript levels and proximally located genetic variants. Several of the associations were experimentally validated in reporter assays. Through influencing ABC expression levels, these single-nucleotide polymorphisms may affect disease susceptibility and response to drugs.

Acute kidney injury is associated with a significant inflammatory response that has been the target of renoprotection strategies. Epoxyeicosatrienoic acids (EETs) are anti-inflammatory cytochrome P450-derived eicosanoids that are abundantly produced in the kidney and metabolized by soluble epoxide hydrolase (sEH; Ephx2) to less active dihydroxyeicosatrienoic acids. Genetic disruption of Ephx2 and chemical inhibition of sEH were used to test whether the anti-inflammatory effects of EETs, and other lipid epoxide substrates of sEH, afford protection against cisplatin-induced nephrotoxicity. EET hydrolysis was significantly reduced in Ephx2(-/-) mice and was associated with an attenuation of cisplatin-induced increases in serum urea nitrogen and creatinine levels. Histological evidence of renal tubular damage and neutrophil infiltration was also reduced in the Ephx2(-/-) mice. Likewise, cisplatin had no effect on renal function, neutrophil infiltration, or tubular structure and integrity in mice treated with the potent sEH inhibitor 1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea) (AR9273). Consistent with the ability of EETs to interfere with nuclear factor-κB (NF-κB) signaling, the observed renoprotection was associated with attenuation of renal NF-κB activity and corresponding decreases in the expression of tumor necrosis factor (TNF) α, TNF receptor (TNFR) 1, TNFR2, and intercellular adhesive molecule-1 before the detection of tubular injury. These data suggest that EETs or other fatty acid epoxides can attenuate cisplatin-induced kidney injury and sEH inhibition is a novel renoprotective strategy.

Regulatory elements play an important role in the variability of individual responses to drug treatment. This has been established through studies on three classes of elements that regulate RNA and protein abundance: promoters, enhancers and microRNAs. Each of these elements, and genetic variants within them, are being characterized at an exponential pace by next-generation sequencing (NGS) technologies. In this review, we outline examples of how each class of element affects drug response via regulation of drug targets, transporters and enzymes. We also discuss the impact of NGS technologies such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq), and the ramifications of new techniques such as high-throughput chromosome capture (Hi-C), chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and massively parallel reporter assays (MPRA). NGS approaches are generating data faster than they can be analyzed, and new methods will be required to prioritize laboratory results before they are ready for the clinic. However, there is no doubt that these approaches will bring about a systems-level understanding of the interplay between genetic variants and drug response. An understanding of the importance of regulatory variants in pharmacogenomics will facilitate the identification of responders versus non-responders, the prevention of adverse effects and the optimization of therapies for individual patients.

The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex., (© 2011 Blackwell Publishing Ltd.)

Background: Previously, MDR1 T-129C polymorphism, encoding multidrug resistant transporter MDR1/P-glycoprotein, was reported to be predictive of poorly-differentiated colorectal adenocarcinomas. Here, VEGF T-1498C, C-634G and C-7T polymorphisms, encoding vascular endothelial growth factor (VEGF), were investigated in terms of their association with differentiation grade., Methods: VEGF genotypes were determined by TaqMan(R) MGB probe based polymerase chain reaction and evaluated were confirmed by direct sequencing in 36 Japanese patients., Results: VEGF T-1498C, but not C-634G or C-7T, was predictive of poorly-differentiated ones, and thereby a poor prognosis (p = 0.064 for genotype, p = 0.037 for allele), and this effect can be explained by that on VEGF expression. Treatment of a colorectal adenocarcinoma cell line, HCT-15, with sodium butyrate, a typical differentiating agent, resulted in an increase of alkaline phosphatase activity and MDR1 mRNA expression, but in a decrease of VEGF mRNA expression. The transfection of VEGF small interfering RNA (siRNA) induced the expression of MDR1 mRNA to 288-332% of the control level, whereas MDR1 siRNA had no effect on VEGF mRNA expression., Conclusions: VEGF T-1498C polymorphism is also a candidate marker predictive of poorly-differentiated colorectal adenocarcinomas, but further investigations with a large number of patients should be addressed to draw a conclusion.

Preserving of tubal pregnancy after first trimester is rare and very often ends fatal. Nevertheless new methods of diagnosis and therapy of ectopic pregnancy, in industrial countries it remains on first place as reason for mother mortality in first three months of pregnancy and represents 10% of all mother mortality.

Biological membranes consist of lipid bilayers with liquid-ordered and liquid-disordered phases. It is believed that cholesterol controls the size of the microdomains in the liquid-ordered phase and thereby affects the mobility as well as the permeability of the membrane. We study this process in a model system consisting of the nonionic surfactant C(12)E(5) and water in the lamellar phase. We measure the diffusion of fluorescent probe molecules (rhodamine B) by fluorescence correlation spectroscopy. For different surfactant to water ratios, we measure how the molecular mobility varies with the amount of cholesterol added. We find that a reduction of the diffusion coefficient is already detectable at a molar ratio of 8 mol % cholesterol.

Glucocorticoids such as prednisolone are used for their anti-inflammatory properties. But there is evidence to suggest that under certain conditions, glucocorticoids have pro-inflammatory effects, for example, enhancement of IL-1beta production. To date, it has been reported that IL-1beta production intensity was associated with single nucleotide polymorphisms at positions -1470, -511, and -31 in the promoter region and at position 3954 in exon 5 of the IL-1beta gene. In the present study, it was examined whether these IL-1beta genotypes were associated with the suppressive effect of prednisolone on IL-1beta production in human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS). A midrange concentration (10(-6) M) of prednisolone suppressed the LPS-induced increase in IL-1beta mRNA expression and protein release, while higher concentrations (10(-5) M, 10(-4) M) exhibited less suppression or had a synergistic stimulative effect on IL-1beta production in certain subjects. Under treatment with 10(-4) M prednisolone, the levels of IL-1beta protein production stimulated by LPS in PBMC extracted from the subjects with the IL-1beta TT(-31), TC(-31), and CC(-31) genotypes were suppressed to 6.0+/-3.4%, 31.4+/-57.0%, and 87.7+/-84.8%, respectively, of the level in prednisolone-untreated control cells (TT(-31) vs. CC(-31), p<0.05). Glucocorticoid-based anti-inflammatory therapy might be less effective in patients with the IL-1beta TC(-31) and CC(-31) genotypes than those with the TT(-31) genotype.

In our previous study, the MDR1/Pglycoprotein-overexpressing multidrug resistant subline, Hvr100-6, was established from the human cervical carcinoma cell line HeLa-Ohio (HeLa) by stepwise exposure to an anti-microtubule agent, vinblastine sulfate, a typical substrate of MDR1. Their gene and protein expression profiles were analyzed herein, and 148 genes were identified to be differentially expressed by cDNA microarray analysis. The up-regulation of sorcin, a soluble resistance-related calcium-binding protein of 22 kDa, was confirmed in Hvr100-6 cells by the proteome analysis. To clarify the relationship between MDR1 and sorcin, HeLa cells were treated with small interfering RNAs (siRNAs) targeted for theirs mRNAs. The siRNA for MDR1 mRNA resulted in its decrease by 86% and 61% on the days 1 and 2 after the treatment, whereas the expression level of sorcin mRNA was not changed. On the other hand, the siRNA for sorcin mRNA suppressed its expression by 80-90% on days 1-3 after the treatment. Interestingly; suppression of sorcin induced a more than 3-fold increase in the expression level for MDR1 mRNA. An efflux function of MDR1 evaluated with using rhodamine 123 as a probe showed a tendency to be increased in HeLa cells treated with siRNA for sorcin, compared with that in the cells treated with scramble siRNA. The activity and the expression of caspase-3 in the sorcin knock-down HeLa cells were relatively higher than those in the cells treated with scramble siRNA. Thus, we demonstrated that sorcin might be a partial suppressor of MDR1 expression. Furthermore, the present study suggested that sorcin repressed apoptosis via dysfunction of caspase-3.

The Ca(2+)-regulated photoprotein obelin has been examined as a label for bioluminescence immunoassay of infective agents. The hepatitis B virus (HbsAg) and the bacteria Escherichia coli and Shigella sonnei lipopolysaccharide (LPS) were chosen as model antigens. Chemically synthesized obelin-corresponding antibody conjugates were used in a solid-phase microplate immunoassay. The sensitivities achieved by the assay were 0.25 ng/mL for S. sonnei LPS and 0.375 ng/mL for HbsAg. A novel, filter-based immunoassay to determine bacterial admixtures in the environment was proposed. The NanoCeram filters were effectively applied to 'trap' and pre-concentrate pathogens from samples under study for the purposes of further detection and measurement of the absorbed material by bioluminescence immunoassay., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

The lack of space abilities predetermine the fate of tubal pregnancy. Very often it disturbs in first few weeks. Preserving the tubal pregnancy after first trimester is very rear and often ends fatal. During 1970 in France the extrauterine pregnancy is met once on every 100 births. Now it is 2% from all births. In our country the relative part of ectopic pregnancy for last 20 years has grown twice. The authors represent two cases of tubal pregnancy, developed until second trimester, one of them ended fatal. The difficulties during diagnosing, characteristics of clinic and therapeutic problems are shown. Analysis are made on tendency of growing frequency of ectopic pregnancy, nevertheless modern diagnostic methods and also its role for maternal mortality.

The authors report retrospective investigation of causes for maternal mortality in the Department of Obstetric and Gynecology in Hospital in Pleven for the period 1977-2001 and comparison between indexes for a different period of time. Objects of investigation were patient histories necropsy report and forensic expertise. Vital births were 73922 for a period of 25 years in Pleven. Dead pregnant and maternity were 45 and the rate of maternal mortality was 60,07/per 100 000 vital births. Causes of maternal mortality were divided in immediate 40 cases (88,88%) and indirect--5 cases (11,11%). The hemorrhage was the most common cause of maternal mortality--18 cases (45%). The authors mention that the absolute number of maternal losses is comparatively constant but the rate of maternal mortality in creases because of tendency of decrease of birthrate and concentration of pathology in Department of Obstetric and Gynecology in Pleven.

Recent advances in pharmacogenomics have suggested the association of clinical outcome of glucocorticoid-based anti-inflammatory therapy with a single nucleotide polymorphism at position 3435 in exon 26 (C3435T) of the MDR1 gene. In the present study, the effects of the MDR1 C3435T genotype on the time-dependent profiles of gene expression and function of MDR1/P-glycoprotein were evaluated in peripheral blood mononuclear cells (PBMCs) under lipopolysaccharide (LPS)-induced experimental acute inflammation. LPS treatment resulted in the rapid elevation of IL-1beta and TNF-alpha mRNA levels relative to beta-actin mRNA at 1 h, with a subsequent slight decrease at 3 h after the treatment, while the down-regulation of the relative concentration of MDR1 mRNA was found at 3 h, not at 1 h, after LPS treatment. Here, the C3435T genotype-dependent down-regulations of MDR1 mRNA level were found for CC(3435) and CT(3435), but not for TT(3435), and were 64.1+/-10.1%, 71.4+/-5.9% and 100.0+/-22.5% (+/-S.D.), respectively, of their respective baseline levels, which were independent of C3435T (0.010+/-0.005, 0.011+/-0.013 and 0.009+/-0.006 (+/-S.D.), respectively). The C3435T genotype-dependent down-regulation was supported by the increase of the intracellular accumulation of calcein in PBMCs treated with LPS for 72 h, and the increase was more predominant for CC(3435) than TT(3435). These data suggested that glucocorticoid-based anti-inflammatory therapy might be more effective for C(3435)-allele carriers than non-carriers.

Unlabelled: The most of European registries of congenital anomalies (CA) collected information of CA in livebirths, stillbirths and terminated pregnancies following prenatal/ultrasound diagnosis., Objectives: to assess terminated pregnancies after prenatal/ ultrasound diagnosis of CA as a part of register of CA performed in University Hospital-Pleven. Among 21 202 births monitored during the study period (1996-2005), 679 CA were detected. The total prevalence of CA was 32/ 1000 births. The outcome of pregnancy for all cases of selected CA by register was 620 livebirths (91.3%), 36 stillbirths (5.3%), 23 terminated pregnancies (TP) (3.4%). The percentage of pregnancy termination was higher in the case of isolated anomalies, mainly lethal and CA associated with a low survival rate (61%), than with multiple ones. The most common CA detected after prenatal/ ultrasound diagnosis were neural tube defects (NTD) - the main reason for TP (52% of cases). The low proportion of these CA in TP (1/3) compared to their proportion in livebirths (50%) demonstrated an insufficiency of prenatal diagnosis of NTD as a part of register of CA performed in University Hospital-Pleven. Prenatal diagnosis of CA allows an early genetic counseling of mother presenting information on neonatal prognosis and recurrence risk for subsequent pregnancies. It helps family to take an adequate decision for termination of pregnancy with bad prognosis about heavy fetal CA.

We report our clinical opinion for recombinant activated factor VII (NovoSeven, Novo Nordisk, Copenhagen, Denmark) administration in puerperae with massive haemorrhage caused by uterine hypotonia. Four women with severe bleeding in post-placental period received NovoSeven in bolus IV. The blood loss and laboratory changes in hematology and haemostasis parameters are monitored. The right time of application and the mean effective dose of the medication are discussed. The bleeding was ceased in all cases. Decrease in values of Hb, Er and PTT was noted. The mean administered dose of 72 micrograms/kg BW was effective. The laboratory values showed tendency for improvement on the 24 hour after administration and normal levels on discharge. The use of recombinant factor VIIA in puerperae with severe bleeding in the postplacental period is effective and safe enough and could be an alternative to the extreme surgical procedures.

The authors report retrospective investigation of causes for maternal mortality in the Department of Obstetric and Gynecology in Hospital in Pleven for the period 1977-2001 and comparison between indexes for a different period of time. Objects of investigation were patient histories necropsy report and forensic expertise. Vital births were 73922 for a period of 25 years in Pleven. Dead pregnant and materinity were 45 and the rate of maternal mortality was 60,07/per 100 000 vital births. Causes of maternal mortality were divided in immediate 40 cases (88,88%) and indirect - 5 cases (11,11%). The hemorrhage was the most common cause of maternal mortality - 18 cases (45%). The authors mention that the absolute number of maternal losses is comparatively constant but the rate of maternal mortality in creases because of tendency of decrease of birthrate and concentration of pathology in Department of Obstertic and Gynecology in Pleven.

Magnesium sulphate (MgSO4) is with a proved effectiveness in cases of praeclampsia/eclampsia, but its use as a tocolytic is discussed in the last few years. That is why we had for an object to study its effect as a tocolytic in cases of abortions and premature labours. The study is prospective and if is made in I-st obstetric clinic of High Medical School--Pleven. Treatment with Cormagnesin was carried out to pregnant women for suppressing the uterine activity. Cormagnesin 200 (1 amp-10 ml) contains 1000 mg MgSO4, and Cormagnesin 400 (1 amp-10 ml) contains 2000 mg MgSO4. The medicine was administered in dosage of 4 or 5 g for 30 min, and after that if there were any uterine contractions the infusion was carried on with additional 5 g MgSO4 for 6 to 12 h. The total dosage was from 4 to 60 g MgSO4. The authors reported on very good effect in cases with pains and increased uterine tone--18 (36.73%), as well as in cases with pains and irregular uterine contractions 5 (10.21%), while the treatment was without any effect in cases with uterine contractions on 15-20 min, increased uterine tone, bleeding and Pelvic score 1-3 points in spite of high dosages of MgSO4 and longer duration of treatment. The authors made the conclusion, that the subjective complaints should not be accepted as an indication for administration of MgSO4, and MgSO4 should be administered in cases with increased uterine tone and irregular uterine contractions. Every genital bleeding and suspicion for placental abruption should be defined more precisely, because lately diagnosed placental abruption and unjustified expectation for suppression of uterine activity by MgSO4, may lead to increase of perinatal morbidity and mortality. In spite of the controversial data about MgSO4 as a tocolytic, its administration is justified and necessary.

The aim of the study was to present out experience with the registration of congenital anomalies (CA) and to assess the effect of the preventive genetic-consultative activities in affected families. In the period 1990-1996, 19174 infants born or hospitalized at the Clinic of Obstetrics, Pleven were screened for CA, showing frequency of 26.1%. Structural analysis of the CA is presented. 226 out of 500 (45%) families with and affected child were consulted by a geneticist. Data an prenatal diagnosis (PD) offered to 142 families at high risk and their reproductive decision are submitted. The low rate of families made use of invasive PD is pointed out; the real benefits of ultrasonography as a screening test for detection of fetal anomalies has been recommended.

Captopril is the first oral inhibitor of angiotensin-converting enzyme, which has a strong effect on patients with hypertonic states. The influence of the preparation was studied on liver drug-metabolizing enzymic systems of rats in two ways of oral administration-single and a 10-day treatment with a dose of 75 mg/kg. The effect on the levels of microsomal cytochrome B5 and P450 as well as on that of hem was investigated. The activities of some enzymes from the first and second phases of biotransformation were determined. There no changes in the examined parameters 1 h after single oral treatment with 75 mg/kg of captopril. Induction of some of the examined enzymes was observed after a 10-day treatment with 75 mg/kg r. o. of captopril once daily: there was statistically significant elevation of the level of cytochrome P450 as well as of the activity of ethylmorphindelethylase and HA-DPH-cytochrome-C-reductase. The results show that there is probably induction at the first phase of phenobarbital type of drug metabolism.