ALBERT

Beschreibung:    An inexpensive, simple and environmentally friendly method based on dispersive liquid liquid microextraction (DLLME) for rapid determination of benzene derivatives in water samples was proposed. A significant improvement of DLLME procedure was achieved. Trace volume ethyl acetate (60 μL) was exploited as dispersion solvent instead of common ones such as methanol and acetone, the volume of which was more than 0.5 mL, and the organic solvent required in DLLME was reduced to a great extent. Only 83-μL organic solvent was consumed in the whole analytic process and the preconcentration procedure was less than 10 min. The advantageous approach coupled with gas chromatograph-flame ionization detector was proposed for the rapid determination of benzene, toluene, ethylbenzene and xylene isomers in water samples. Results showed that the proposed approach was an efficient method for rapid determination of benzene derivatives in aqueous samples. Content Type Journal Article Category Short Communication Pages 1-5 DOI 10.1007/s10337-012-2215-7 Authors Chun Peng Diao, College of Environmental Science and Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou, 510006 China Chao Hai Wei, College of Environmental Science and Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou, 510006 China Chun Hua Feng, College of Environmental Science and Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou, 510006 China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Development of a reversed phase high performance liquid chromatographic method for determination of six related impurities in prilocaine substance is reported. The test of related impurities in European Pharmacopoeia (Ph. Eur.) cannot meet the demands with the chromatographic parameters given, therefore different types of chromatographic systems and eight columns have been evaluated in the present study. A new method with a Hypercarb column was developed and validated. This method fulfils the demands in the Ph. Eur., and the validation shows that the method is selective, reproducible, linear, accurate and robust with sufficient limits of detection (0.001–0.004% of 2.5 mg prilocaine mL −1 ) and quantification (0.002–0.009% of 2.5 mg prilocaine mL −1 ). Content Type Journal Article Category Original Pages 1-8 DOI 10.1007/s10337-012-2212-x Authors Janina Sroka-Markovic, Analytical Development, AstraZeneca, 15185 Södertälje, Sweden Linda Johansson, Analytical Development, AstraZeneca, 15185 Södertälje, Sweden Magnus B. O. Andersson, Analytical Development, AstraZeneca, 15185 Södertälje, Sweden Ingrid Granelli, Department of Analytical Chemistry, Stockholm University, 10691 Stockholm, Sweden Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    In the present study, baseline separation of the enantiomers of 16 β-carboline derivatives was successfully achieved using both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) techniques in short run times (〈15 min) and thus permit the determination of enantiomeric excess. In HPLC methodology, cellulose chiral stationary phase (Chiralcel OD-H) was used with a binary mobile phase constituted of n -hexane/ethanol 85/15 leading to a resolution factor of 12.6 in 15 min. Preparative HPLC allowed to obtain pure enantiomers of two compounds. In CE, chiral selectivity was developed with an in-capillary stacking strategy using anionic (highly sulfated-γ) cyclodextrins 5% (w/v) as chiral selectors and a 60 mM phosphate buffer (pH 2.5) resulting in a resolution of 10.26 in 14 min of analysis. The analytical characteristics of the two developed methods were studied in terms of repeatability, limits of detection and limits of quantification showing their suitability to be extended to all the other molecules. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2194-8 Authors Emmanuelle Lipka, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Saïd Yous, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Christophe Furman, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Pascal Carato, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Carole Deghaye, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Jean-Paul Bonte, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Claude Vaccher, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille Nord de France, EA 4481, 3 rue du Pr. Laguesse, 59000 Lille, France Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A capillary chromatography system was developed using an open capillary tube and a ternary solvents carrier solution of water-hydrophilic/hydrophobic organic solvent mixture. The chromatography is called a tube radial distribution chromatography (TRDC) system. The TRDC system works without applying high voltages or using specific columns, such as monolithic and packed columns. In this study, the effects of tube materials on separation performance were examined in the TRDC system, by using poly(tetrafluoroethylene) (PTFE; 100–400 μm inner diameter), polyethylene (PE; 200 μm inner diameter), and copolymer of (tetrafluoroethylene–perfluoroalcoxyethylene) (PTFE–PFAE; 100 μm inner diameter) capillary tubes. An analyte solution of 2,6-naphthalenedisulfonic acid and 1-naphthol as a model was subjected to the system with a water–acetonitrile–ethyl acetate carrier solution; 15:3:2 volume ratio (water-rich carrier) and 3:8:4 volume ratio (organic solvent-rich carrier). The flow rates were adjusted to be 0.5 μL min −1 for PTFE and PTFE–PFAE tubes as well as 2.0 μL min −1 for PE tube under laminar flow conditions. These analytes in the solution were separated in this order with the water-rich carrier solution with baseline separation in the three capillary tubes, while they were eluted in the reverse order or not separated with the organic solvent-rich carrier solution. The effects of tube temperature on separation were also examined with the water-rich carrier solution; the best resolutions were observed at 0 °C of the tube temperature. The obtained results were compared with those of fused-silica capillary tube and discussed. Content Type Journal Article Category Short Communication Pages 1-5 DOI 10.1007/s10337-012-2205-9 Authors Yudai Kudo, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Naoya Jinno, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Masahiko Hashimoto, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Kazuhiko Tsukagoshi, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: R.E. Hester and R.M. Harrison (Eds.): Nuclear Power and the Environment Content Type Journal Article Category Book Review Pages 1-2 DOI 10.1007/s10337-012-2208-6 Authors Ken Jones, Knutsford, Cheshire, UK Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    In this study, improved homogeneous liquid–liquid extraction (HLLE), equipped with GC–ECD has been developed for the extraction and determination of organochlorinated pesticides (OCPs) in water. The phase separation phenomenon occurred by temperature in a ternary solvent (water/methanol/chloroform) system. Several factors influencing the extraction efficiency were investigated and optimized with orthogonal array design. Furthermore, in this study, for the first time, before immiscible organic phase formation, different volumes of deionized water were subjected to homogeneous solution to investigate the effect of this factor on the extraction performance of HLLE. Optimal results were as follows: volume of the extracting solvent (chloroform), 50 μL; volume of the consolute solvent (methanol), 1.2 mL; volume of the sample, 2.5 mL; volume of the deionized water, 0.5 mL; time of centrifuge, 7 min. Under the optimum conditions, repeatability was obtained by spiking OCPs at concentration level of 20 μg L −1 , the RSDs varied between 4.8 and 10.7% ( n  = 4). The limits of detection of 0.02–0.12 μg L −1 were obtained for the OCPs. Enrichment factors and the extraction percent of the studied compounds were in the range of 240–300 and 69.2–84.0%, respectively. Finally, the results of the proposed HLLE method were compared with the same HLLE method without addition of deionized water. The results indicated that the proposed method has higher enrichment factors and lower detection limits. Content Type Journal Article Category Original Pages 1-7 DOI 10.1007/s10337-012-2206-8 Authors Farahnaz Rezaei, Department of Analytical Chemistry, Faculty of Chemistry, Iran University of Science and Technology, Narmak, 16846 Tehran, Iran Mohammad-Reza Milani Hosseini, Department of Analytical Chemistry, Faculty of Chemistry, Iran University of Science and Technology, Narmak, 16846 Tehran, Iran Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A sensitive and rapid liquid chromatography–mass spectrometry (LC–MS) assay was established for the quantitation of CYC-116, an antitumor drug, in rat plasma. The chromatographic separation was accomplished on a Kromasil C 18 column (150 mm × 4.6 mm i.d., 5 μm particle size) at an isocratic flow rate of 0.8 mL min −1 using acetonitrile–water–formic acid (23.5:76.5:0.1, v/v/v) as the mobile phase. The plasma extraction was performed by liquid–liquid extraction using ethyl acetate as the solvent, and the extracts were subjected to MS analysis using a quadrupole mass spectrometer. The calibration curve of CYC-116 was linear over the concentration range of 5–2,500 ng mL −1 ( r  = 0.9955). The mean recovery was 85.0 ± 8.0%, and the matrix effect ranged from 90.0 to 110.0%. The intra- and interday precisions were less than 11.8 and 6.6%, respectively, and the accuracy was within ±5.8%. The method was successfully applied to study the pharmacokinetics of CYC-116 in rats after oral administration. Content Type Journal Article Category Original Pages 1-6 DOI 10.1007/s10337-011-2175-3 Authors Jing Su, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Xiaohui Chen, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Qing Li, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Zhiguo Yu, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Xiaoduo Guan, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Lulu Geng, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Kaishun Bi, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016 People’s Republic of China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Tube radical distribution chromatography (TRDC) uses an untreated open tubular capillary tube and a ternary mixture of solvents (water and hydrophilic/hydrophobic organic solvents) as a carrier solution. A model analyte mixture comprising 1-naphthol, 1-naphthoic acid, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was examined by the TRDC and capillary zone electrophoresis (CZE) systems that comprised mainly a capillary tube and a detector. In the TRDC system the elution order of analytes could be changed by altering the component ratios of the solvents, whereas in the CZE system the elution order was changed by altering the electroosmotic flow direction. The experimental data obtained provide clues about the features and utility of TRDC as a new separation method. Content Type Journal Article Category Short Communication Pages 1-6 DOI 10.1007/s10337-012-2203-y Authors Kisuke Tabata, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Naoya Jinno, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Keiichi Noda, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Masahiko Hashimoto, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Kazuhiko Tsukagoshi, Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: K. Kamienska-Trela (Ed.): Nuclear Magnetic Resonance. Volume 40. Specialist Periodical Reports of the Royal Society of Chemistry Content Type Journal Article Category Book Review Pages 1-2 DOI 10.1007/s10337-012-2209-5 Authors John C. Lindon, Imperial College London, London, UK Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A simple, selective and highly sensitive method was developed and optimized to determine the most commonly used UV filters with endocrine-disrupting potential in water, namely benzophenone-3 (BP-3), octocrylene (OC), ethylhexyl dimethyl p -aminobenzoate (OD-PABA), ethylhexyl methoxycinnamate, ethylhexyl salicylate (EHS) and homosalate (HMS). Samples were extracted by stir bar sorptive extraction followed by liquid desorption (SBSE-LD). The important factors influencing SBSE-LD were optimized. Under optimal conditions, assays were performed on 50 mL of water sample using stir bars (0.5 mm in film thickness, 10 mm in length) at room temperature. The analytes were determined by liquid chromatography–tandem mass spectrometry with triple quadrupole analyzer using atmospheric pressure chemical ionization. The main parameters in HPLC–APCI–MS/MS were also optimized to provide the best performances for all analytes. Moreover, matrix effect was investigated using two methods the post-column infusion system and the method of spiked matrices after extraction. As a result, no significant matrix effect on the analysis was observed. The method showed good linearity ( R 2 coefficients greater than 0.996 in different water samples after SBSE-LD). Recoveries of the analytes were close to 90%, except for BP-3 (64%) and OC (76%) with relative standard deviation lower than 11%. Detection limits were between 0.6 and 3.3 ng L −1 for all the analytes except for HMS (94 ng L −1 ) and EHS (114 ng L −1 ). This methodology was applied to measure UV filters in seawater, river water and wastewater in different sites of Liguria; BP-3 and OC were found in most of the considered samples at rather low concentration level. Content Type Journal Article Category Original Pages 1-10 DOI 10.1007/s10337-012-2202-z Authors Emanuele Magi, Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso 31, 16146 Genoa, Italy Marina Di Carro, Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso 31, 16146 Genoa, Italy Carlo Scapolla, Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso 31, 16146 Genoa, Italy Kieu T. N. Nguyen, Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso 31, 16146 Genoa, Italy Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Water-soluble sodium poly(aspartate- co -lactide) (PALNa) copolymers with a molar ratio of aspartate-to-lactide units equal to 1:0.6, 1:1.0 and 1:1.5 were studied using NMR spectroscopy to determine the composition as well as SEC-MALS and static light-scattering measurements to determine the molar-mass characteristics of the copolymers. In the copolymer aqueous solutions, high-molar-mass species were detected, most probably due to the incomplete dissolution of the samples. The molar-mass averages determined in water with added simple electrolyte, i.e., NaCl, were much lower than the values determined in pure water. The concentration of the salt, which allows dissolution on a molecular level, and the separation predominantly according to a size-exclusion mechanism depend on the chemical composition of the PALNa copolymers. The optimal mobile phase for the PALNa-1/0.6 and the PALNa-1/1.0 copolymers was 0.1 M NaCl at pH 9, and for the PALNa-1/1.5 copolymer with a higher content of lactide units it was 0.05 M NaCl at pH 9. The molar-mass averages of the PALNa-1/1.0 copolymer, determined by SEC-MALS and static light-scattering measurements, were comparable. Content Type Journal Article Category Original Pages 1-8 DOI 10.1007/s10337-012-2180-1 Authors Maja Gričar, Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia Majda Žigon, Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia Tina Šmigovec Ljubič, Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia Ema Žagar, Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Luigi Mondello (Ed.): Comprehensive Chromatography in Combination with Mass Spectrometry Content Type Journal Article Category Book Review Pages 1-2 DOI 10.1007/s10337-012-2189-5 Authors Edward R. Adlard, Burton, South Wirral, UK Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Formaldehyde has been highlighted as potential genotoxic impurity (GTI). Trace-level quantification of GTIs in drug substances requires sensitive, precise and accurate analytical methodologies for their estimation in drug substances and control. Analysis and estimation of formaldehyde is very challenging due to its properties namely volatility, high polarity, low molecular weight and over and above the absence of chromophore. This article presents a validated HPLC–UV method which is sensitive to quantification of formaldehyde in active pharmaceutical ingredient. As formaldehyde does not possess chromophore, the developed HPLC method involves derivatization with 2,4-dinitrophenylhydrazine. Using this method, the detection and quantitation limits achieved are 0.5 and 1.5 ppm, respectively. The calibration curve of formaldehyde was linear over the concentration range of 1.5–20 ppm. The method was found to be sensitive, precise and accurate and the proposed method has been successfully applied to estimate formaldehyde content in scale-up batches of bulk drug. Content Type Journal Article Category Original Pages 1-6 DOI 10.1007/s10337-012-2186-8 Authors A. Nageswari, Center of Pharmaceutical Sciences, IST, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, 500072 India K. V. S. R. Krishna Reddy, Aptuit Laurus Private Limited, ICICI Knowledge Park, Turkapally, Hyderabad, 500078 India K. Mukkanti, Center of Pharmaceutical Sciences, IST, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, 500072 India Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A method combining solid-phase extraction and high-performance liquid chromatography (SPE–HPLC) has been developed for analysis of ten rare ginsenosides including 20( R )-Rh 1 , 20( S )-Rh 1 , 20( R )-Rg 3 , 20( S )-Rg 3 , Rg 5 , Rk 1 , Rg 6 , F 4 , Rk 3 , and Rh 4 in three kinds of injection (Shenfu, Shenmai, and Shengmai). Waters Oasis HLB SPE columns were used to clean and enrich the sample. An Eclipse XDB-C 18 column was used to separate the analytes, with water and acetonitrile as mobile phase components under gradient elution conditions at 30 °C. There were good linear correlations between the signals measured and the concentrations of the ten analytes ( r 2  ≥ 0.9996). Intraday and interday precision were both better than 2.19% and average recovery ranged from 95.25 to 108.83%. The proposed method was successfully applied to determination of the ten analytes in different batches of the injections and the results indicated the method is simple, rapid, and accurate. The proposed method could be used for quality control during manufacture of the injections. Content Type Journal Article Category Original Pages 1-7 DOI 10.1007/s10337-012-2183-y Authors Rui-Jie Yang, College of Chemistry, Jilin University, ChangChun, 130012 Jilin Province, China Xu-Wen Li, College of Chemistry, Jilin University, ChangChun, 130012 Jilin Province, China Hua Yao, College of Chemistry, Jilin University, ChangChun, 130012 Jilin Province, China Mu-Chun Zhang, Department of Urology, China-Japan Union Hospital, Jilin University, ChangChun, 130033 Jilin Province, China Yong-Ri Jin, College of Chemistry, Jilin University, ChangChun, 130012 Jilin Province, China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Frontonasal dysplasia (FND) refers to a class of midline facial malformations caused by abnormal development of the facial primordia. The term encompasses a spectrum of severities but characteristic features include combinations of ocular hypertelorism, malformations of the nose and forehead and clefting of the facial midline. Several recent studies have drawn attention to the importance of Alx homeobox transcription factors during craniofacial development. Most notably, loss of Alx1 has devastating consequences resulting in severe orofacial clefting and extreme microphthalmia. In contrast, mutations of Alx3 or Alx4 cause milder forms of FND. Whilst Alx1 , Alx3 and Alx4 are all known to be expressed in the facial mesenchyme of vertebrate embryos, little is known about the function of these proteins during development. Here, we report the establishment of a zebrafish model of Alx -related FND. Morpholino knock-down of zebrafish alx1 expression causes a profound craniofacial phenotype including loss of the facial cartilages and defective ocular development. We demonstrate for the first time that Alx1 plays a crucial role in regulating the migration of cranial neural crest (CNC) cells into the frontonasal primordia. Abnormal neural crest migration is coincident with aberrant expression of foxd3 and sox10 , two genes previously suggested to play key roles during neural crest development, including migration, differentiation and the maintenance of progenitor cells. This novel function is specific to Alx1, and likely explains the marked clinical severity of Alx1 mutation within the spectrum of Alx -related FND.

Beschreibung: Activating somatic and germline mutations of closely related RAS genes (H, K, N) have been found in various types of cancer and in patients with developmental disorders, respectively. The involvement of the RAS signalling pathways in developmental disorders has recently emerged as one of the most important drivers in RAS research. In the present study, we investigated the biochemical and cell biological properties of two novel missense KRAS mutations (Y71H and K147E). Both mutations affect residues that are highly conserved within the RAS family. KRAS Y71H showed no clear differences to KRAS wt , except for an increased binding affinity for its major effector, the RAF1 kinase. Consistent with this finding, even though we detected similar levels of active KRAS Y71H when compared with wild-type protein, we observed an increased activation of MEK1/2, irrespective of the stimulation conditions. In contrast, KRAS K147E exhibited a tremendous increase in nucleotide dissociation generating a self-activating RAS protein that can act independently of upstream signals. As a consequence, levels of active KRAS K147E were strongly increased regardless of serum stimulation and similar to the oncogenic KRAS G12V . In spite of this, KRAS K147E downstream signalling did not reach the level triggered by oncogenic KRAS G12V , especially because KRAS K147E was downregulated by RASGAP and moreover exhibited a 2-fold lower affinity for RAF kinase. Here, our findings clearly emphasize that individual RAS mutations, despite being associated with comparable phenotypes of developmental disorders in patients, can cause remarkably diverse biochemical effects with a common outcome, namely a rather moderate gain-of-function.

Beschreibung: Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2 Q127 -complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs.

Beschreibung: Balancing selection has maintained human leukocyte antigen (HLA) allele diversity, but it is unclear whether this selection is symmetric (all heterozygotes are comparable and all homozygotes are comparable in terms of fitness) or asymmetric (distinct heterozygote genotypes display greater fitness than others). We tested the hypothesis that HLA is under asymmetric balancing selection in populations by estimating allelic branch lengths from genetic sequence data encoding peptide-binding domains. Significant deviations indicated changes in the ratio of terminal to internal branch lengths. Such deviations could arise even if no individual alleles present a strikingly altered branch length (e.g. if there is an overall distortion, with all or many terminal branches being longer than expected). DQ and DP loci were also analyzed as haplotypes. Using allele frequencies for 419 distinct populations in 10 geographical regions, we examined population differentiation in alleles within and between regions, and the relationship between allelic branch length and frequency. The strongest evidence for asymmetrical balancing selection was observed for HLA-DRB1 , HLA-B and HLA-DPA1 , with significant deviation ( P ≤ 1.1 x 10 –4 ) in about half of the populations. There were significant results at all loci except HLA-DQB1 / DQA1 . We observed moderate genetic variation within and between geographic regions, similar to the rest of the genome. Branch length was not correlated with allele frequency. In conclusion, sequence data suggest that balancing selection in HLA is asymmetric (some heterozygotes enjoy greater fitness than others). Because HLA polymorphism is crucial for pathogen resistance, this may manifest as a frequency-dependent selection with fluctuation in the fitness of specific heterozygotes over time.

Beschreibung: Birt–Hogg–Dubé syndrome (BHD) is a human cancer disorder caused by mutations in the tumor suppressor gene Folliculin ( FLCN ) with unknown biological functions. Here, we show that the Drosophila homolog of FLCN, dFLCN (a.k.a. dBHD ) localizes to the nucleolus and physically interacts with the 19S proteasomal ATPase, Rpt4, a nucleolar resident and known regulator of rRNA transcription. Downregulation of dFLCN resulted in an increase in nucleolar volume and upregulation of rRNA synthesis, whereas dFLCN overexpression reduced rRNA transcription and counteracted the effects of Rpt4 on rRNA production by preventing the association of Rpt4 with the rDNA locus. We further show that human FLCN exhibited evolutionarily conserved function and that Rpt4 knockdown inhibits the growth of FLCN-deficient human renal cancer cells in mouse xenografts. Our study suggests that FLCN functions as a tumor suppressor by negatively regulating rRNA synthesis.

Beschreibung: KitL, via its receptor cKit, supports primordial germ cell (PGC) growth, survival, migration and reprogramming to pluripotent embryonic germ cells (EGCs). However, the signaling downstream of KitL and its regulation in PGCs remain unclear. A constitutively activating mutation, cKit V558 , causes gain-of-function phenotypes in mast cells and intestines, and gastrointestinal stromal tumors (GISTs) when heterozygous. Unexpectedly, we find that PGC growth is not significantly affected in cKit V558 heterozygotes, whereas in homozygotes, increased apoptosis and inefficient migration lead to the depletion of PGCs. Through genetic studies, we reveal that this oncogenic cKit allele exhibits loss-of-function behavior in PGCs distinct from that in GIST development. Examination of downstream signaling in GISTs from cKit V558/+ mice confirmed hyperphosphorylation of AKT and ERK, but both remain unperturbed in cKit V558/+ PGCs and EGCs. In contrast, we find reduced activation of ERK1/2 and JNK1 in cKit V558 homozygous PGCs and EGCs. Inhibiting JNK, though not ERK1/2, increased apoptosis of wild-type PGCs, but did not further affect the already elevated apoptosis of cKit V558 / V558 PGCs. These results demonstrate a cell-context-dependent response to the cKit V558 mutation. We propose that AKT overload protection and JNK-mediated survival comprise PGC-specific mechanisms for regulating cKit signaling.

Beschreibung: TDP-43 is an evolutionarily conserved RNA-binding protein currently under intense investigation for its involvement in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is normally localized in the nucleus, but translocated to the cytoplasm in diseased neurons. The endogenous functions of TDP-43 in the nervous system remain poorly understood. Here, we show that the loss of Drosophila TDP-43 (dTDP-43) results in an increased production of sensory bristles and sensory organ precursor (SOP) cells on the notum of some but not all flies. The location of ectopic SOPs varies among mutant flies. The penetrance of this novel phenotype is dependent on the gender and sensitive to environmental influences. A similar SOP phenotype was also observed on the wing and in the embryos. Overexpression of dTDP-43 causes both loss and ectopic production of SOPs. Ectopic expression of ALS-associated mutant human TDP-43 (hTDP-43 M337V and hTDP-43 Q331K ) produces a less severe SOP phenotype than hTDP-43 WT , indicating a partial loss of function of mutant hTDP-43. In dTDP-43 mutants, miR-9a expression is significantly reduced. Genetic interaction studies further support the notion that dTDP-43 acts through miR-9a to control the precision of SOP specification. These findings reveal a novel role for endogenous TDP-43 in neuronal specification and suggest that the FTD/ALS-associated RNA-binding protein TDP-43 functions to ensure the robustness of genetic control programs.

Beschreibung: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in Caenorhabditis elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2 and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.

Beschreibung: Mitochondrial DNA (mtDNA) mutations leading to the disruption of respiratory complex I (CI) have been shown to exhibit anti-tumorigenic effects, at variance with those impairing only the function but not the assembly of the complex, which appear to contribute positively to cancer development. Owing to the challenges in the analysis of the multi-copy mitochondrial genome, it is yet to be determined whether tumour-associated mtDNA lesions occur as somatic modifying factors or as germ-line predisposing elements. Here we investigated the whole mitochondrial genome sequence of 20 pituitary adenomas with oncocytic phenotype and identified pathogenic and/or novel mtDNA mutations in 60% of the cases. Using highly sensitive techniques, namely fluorescent PCR and allele-specific locked nucleic acid quantitative PCR, we identified the most likely somatic nature of these mutations in our sample set, since none of the mutations was detected in the corresponding blood tissue of the patients analysed. Furthermore, we have subjected a series of 48 pituitary adenomas to a high-resolution array comparative genomic hybridization analysis, which revealed that CI disruptive mutations, and the oncocytic phenotype, significantly correlate with low number of chromosomal aberrations in the nuclear genome. We conclude that CI disruptive mutations in pituitary adenomas are somatic modifiers of tumorigenesis most likely contributing not only to the development of oncocytic change, but also to a less aggressive tumour phenotype, as indicated by a stable karyotype.

Beschreibung: Functional loss of SMN1 causes proximal spinal muscular atrophy (SMA), the most common genetic condition accounting for infant lethality. Hence, the hypomorphic copy gene SMN2 is the only resource of functional SMN protein in SMA patients and influences SMA severity in a dose-dependent manner. Consequently, current therapeutic approaches focus on SMN2 . Histone deacetylase inhibitors (HDACi), such as the short chain fatty acid VPA (valproic acid), ameliorate the SMA phenotype by activating the SMN2 expression. By analyzing blood SMN2 expression in 16 VPA-treated SMA patients, about one-third of individuals were identified as positive responders presenting increased SMN2 transcript levels. In 66% of enrolled patients, a concordant response was detected in the respective fibroblasts. Most importantly, by taking the detour of reprograming SMA patients' fibroblasts, we showed that the VPA response was maintained even in GABAergic neurons derived from induced pluripotent stem cells (iPS) cells. Differential expression microarray analysis revealed a complete lack of response to VPA in non-responders, which was associated with an increased expression of the fatty acid translocase CD36. The pivotal role of CD36 as the cause of non-responsiveness was proven in various in vitro approaches. Most importantly, knockdown of CD36 in SMA fibroblasts converted non- into pos-responders. In summary, the concordant response from blood to the central nervous system (CNS) to VPA may allow selection of pos-responders prior to therapy. Increased CD36 expression accounts for VPA non-responsiveness. These findings may be essential not only for SMA but also for other diseases such as epilepsy or migraine frequently treated with VPA.

Beschreibung: Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by mutations of the X-linked MECP2 gene. Although the loss of MeCP2 function affects many neural systems, impairments of catecholaminergic function have been hypothesized to underlie several of the cardinal behavioral deficits of RTT patients and Mecp2-deficient mice. Although recent Mecp2 reactivation studies indicate that RTT may be a reversible condition, it remains unclear whether specifically preserving Mecp2 function within a specific system will be sufficient to convey beneficial effects. Here, we test whether the selective preservation of Mecp2 within catecholaminergic cells will improve the phenotype of Mecp2-deficient mice. Our results show that this targeted preservation of Mecp2 significantly improves the lifespan, phenotypic severity and cortical epileptiform discharge activity of both male and female Mecp2-deficient mice. Further, we found that the catecholaminergic preservation of Mecp2 also improves the ambulatory rate, rearing activity, motor coordination, anxiety and nest-building performances of Mecp2-deficient mice of each gender. Interestingly, our results also revealed a gender-specific improvement, as specific cortical and hippocampal electroencephalographic abnormalities were significantly improved in male, but not female, rescue mice. Collectively, these results support the role of the catecholaminergic system in the pathogenesis of RTT and provide proof-of-principle that restoring MeCP2 function within this specific system could represent a treatment strategy for RTT.

Beschreibung: Mutations in COL4A1 have been identified in families with hereditary small vessel disease of the brain presumably due to a dominant-negative mechanism. Here, we report on two novel mutations in COL4A1 in two families with porencephaly, intracerebral hemorrhage and severe white matter disease caused by haploinsufficiency. Two families with various clinical presentations of cerebral microangiopathy and autosomal dominant inheritance were examined. Clinical, neuroradiological and genetic investigations were performed. Electron microscopy of the skin was also performed. In one of the families, sequence analysis revealed a one base deletion, c.2085del, leading to a frameshift and a premature stopcodon, p.(Gly696fs). In the other family, a splice site mutation was identified, c.2194-1G〉A, which most likely leads to skipping of an exon with a frameshift and premature termination as a result. In fibroblasts of affected individuals from both the families, nonsense-mediated decay (NMD) of the mutant COL4A1 messenger RNAs (mRNAs) and a clear reduction of COL4A1 protein expression were demonstrated, indicating haploinsufficiency of COL4A1. Moreover, thickening of the capillary basement membrane in the skin was documented, similar to reports in patients with COL4A1 missense mutations. These findings suggest haploinsufficiency, a different mechanism from the commonly assumed dominant-negative effect, for COL4A1 mutations as a cause of (antenatal) intracerebral hemorrhage and white matter disease.

Beschreibung:    In this study, an end-point-based fluorescence assay for soluble epoxide hydrolase (sEH) was transformed into an on-line continuous-flow format. The on-line biochemical detection system (BCD) was coupled on-line to liquid chromatography (LC) to allow mixture analysis. The on-line BCD was based on a flow system wherein sEH activity was detected by competition of analytes with the substrate hydrolysis. The reaction product was measured by fluorescence detection. In parallel to the BCD data, UV and MS data were obtained through post-column splitting of the LC effluent. The buffer system and reagent concentrations were optimized resulting in a stable on-line BCD with a good assay window and good sensitivity (S/N 〉 60). The potency of known sEH inhibitors (sEHis) obtained by LC–BCD correlates well with published values. The LC–BCD system was applied to test how oxidative microsomal metabolism affects the potency of three sEHis. After incubation with pig liver microsomes, several metabolites of sEHis were characterized by MS, while their individual potencies were measured by BCD. For all compounds tested, active metabolites were observed. The developed method allows for the first time the detection of sEHis in mixtures providing new opportunities in the development of drug candidates. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2343-0 Authors David Falck, Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands Nils Helge Schebb, Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA, USA Setyo Prihatiningtyas, Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands Jiawen Zhang, Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands Ferry Heus, Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands Christophe Morisseau, Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA, USA Jeroen Kool, Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands Bruce D. Hammock, Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA, USA Wilfried M. A. Niessen, Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A quantitative structure–retention relationship study was performed for 286 flavor compounds with highly structural diversity on different stationary phases of different polarities. Firstly, the structure–retention relationship was statistically explored using constitutional, topological, molecular properties, charge descriptors and quantum chemical descriptors. Some recently developed chemometric methods, such as the ones for robust analysis and variable selection were firstly employed to predict accurately the gas chromatographic retention indices. The stability and validity of models have been tested by internal and external validation, and good robustness and predictive ability were obtained. The resulting QSRR models were well correlated, with the square of correlation coefficient for cross validation, Q 2 , values of 0.9847, 0.9838, 0.9745 and 0.9646 on stationary phase DB5, OV101, OV17 and C20M, respectively. The molecular properties known to be relevant for GC retention index, such as molecular size, branching, electron density distribution and hydrogen bond effect were well covered by generated descriptors. The descriptors used in models on four stationary phases were compared, and some reasonable explanations about gas chromatographic retention mechanism were obtained. The developed model may be useful for the prediction of flavor compounds while experimental data are unavailable. Content Type Journal Article Category Original Pages 1-13 DOI 10.1007/s10337-012-2349-7 Authors Xian Chen, Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Hong-Dong Li, Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Fang-Qiu Guo, Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Jun Yan, Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Dong-Sheng Cao, Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Yi-Zeng Liang, Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    In this paper, the review on application of factorial-based designs in liquid chromatography (LC) is given. The most useful and applicable full factorial design and reduced forms of full factorial design (fractional factorial design and Plackett–Burman design) applied in LC are presented. Literature survey shows that experimental design presents very often used tool in screening, optimization and robustness testing of LC methods. Content Type Journal Article Category Original Pages 1-14 DOI 10.1007/s10337-012-2350-1 Authors Biljana Jančić Stojanović, Department of Drug Analysis, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A heart-cut two-dimensional high-performance liquid chromatography method for enantiomeric determination of salbutamol, salmeterol and atenolol in urine is presented. It involves the use of two separations in a liquid chromatography–liquid chromatography achiral–chiral coupling. Target compounds were previously separated in a primary column (Kinetex™ HILIC, 2.6 μm, 150 × 2.1 mm I.D.) with a mixture of MeOH:ACN:ammonium acetate buffer (5 mM, pH 6) 90:5:5 (v/v/v) as mobile phase at a flow rate of 0.40 mL min −1 . Enantiomeric separation was carried out by transferring peak of each compound through a switching valve to a vancomycin chiral column (Chirobiotic™ V, 2.6 μm, 150 × 2.1 mm I.D.) using MeOH:ammonium acetate buffer (2 mM, pH 4) 97:3 (v/v) as mobile phase at a flow rate of 0.50 mL min −1 . Ultraviolet detection was done at 227 nm. The method was applied to determine target analytes in urine samples after enzymatic hydrolysis with β-glucuronidase from Helix pomatia , followed by a solid-phase extraction procedure using Isolute ® HCX mixed-mode cartridges. Extraction recoveries ranged from 82 to 90 % in urine samples. Detection limits were 0.091–0.095 μg for each enantiomer of atenolol and between 0.058 and 0.076 and 0.18–0.14 μg for enantiomers of salbutamol and salmeterol, respectively (3 mL of urine). Linearity ranges were between 0.5 and 10 μg mL −1 . Intraday and interday reproducibilities of enantiomeric ratio and enantiomeric fraction, expressed as relative standard deviation, were between 1.9 and 9.0 %. The optimized method was successfully applied to the analysis of urine samples obtained from excretion studies in volunteers and in freeze-dried urine samples, containing urinary components with MW 〈 10,000 and components with MW 〉 10,000, spiked with different amounts of studied drugs. Content Type Journal Article Category Original Pages 1-11 DOI 10.1007/s10337-012-2353-y Authors Yung Yang, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Noelia Rosales-Conrado, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Vanesa Guillén-Casla, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain María Eugenia León-González, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Luis Vicente Pérez-Arribas, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Luis María Polo-Díez, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Purpose   In this study, direct separation of ketoprofen enantiomers was performed on a Chirobiotic T column. Methods   The effects of the type and amount of the organic modifier, buffer concentration, pH value, temperature and flow rate on retention and selectivity were investigated. Experiments were carried out in the temperature range of 20–40 °C to study the effects of temperature. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/ T . Molecular dynamics simulation was done to investigate interactions between ketoprofen enantiomers and the chiral selector—teicoplanin. Results   It was observed that pH and flow rate had a large influence on resolution. Baseline separation of ketoprofen enantiomers could be achieved with low amounts of methanol, high temperature and high buffer concentrations. Conclusions   Results from a thermodynamic study and molecular dynamics simulation show that steric hindrance effect, π–π complexation, hydrogen bonding and electrostatic forces are the main driving forces which cause chiral recognition of ketoprofen enantiomers. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2352-z Authors Xiaomei He, China Pharmaceutical University, 24 Tongjia Lane, Nanjing, 210009 Jiangsu province, China Rui Lin, Yancheng Health Vocational and Technical College, Yancheng, 224005 China Hua He, China Pharmaceutical University, 24 Tongjia Lane, Nanjing, 210009 Jiangsu province, China Meiling Sun, China Pharmaceutical University, 24 Tongjia Lane, Nanjing, 210009 Jiangsu province, China Deli Xiao, China Pharmaceutical University, 24 Tongjia Lane, Nanjing, 210009 Jiangsu province, China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    In this study, we focused on the studying of taurine complexes with phenol and sodium hypochlorite, and of taurine with sodium hypobromite by spectrometry, reverse phase chromatography and ion-exchange chromatography. The formed complexes were studied under various conditions such as temperature (10, 20, 30, 40, 50 and 60 °C), and/or time of interaction (0, 5, 10, 15, 20, 25 and 30 min). In addition, we optimized high performance liquid chromatography coupled with UV detector for detection of taurine and its complexes with the acids. Taurine–phenol–hypochlorite complex was effectively separated under isocratic elution, mobile phase water:methanol 30:70 %, v : v , flow rate 1 mL min −1 and 55 °C. Taurine-bromamine complex was isolated under the following optimized conditions as isocratic elution, mobile phase water:methanol 85:15 % v : v , flow rate 1 mL min −1 and 55 °C. The limits of detection (3 S/N) were estimated as 1 μM for both types of complexes, i.e. for taurine. Further, we estimated recovery in one sample of urine (male 25 years), commercially achieved energy drink and tea leaves and varied from 79 to 86 %. Further, we aimed our attention at investigating the ability of the above characterized taurine and taurine complexes to scavenge reactive oxygen species. For this purpose, an ion-exchange liquid chromatography with post-column derivatization with ninhydrin and VIS detector was used. It clearly follows from the results obtained that taurine itself reacts with peroxide more intensely than in a bound form, which can be associated with the highest signal decrease. Complexes stabilized structure taurine against peroxide radicals, resulting in slower decreasing of peak heights. The most stable was taurine complexes with phenol and hypobromite. Content Type Journal Article Category Original Pages 1-11 DOI 10.1007/s10337-012-2354-x Authors Lukas Nejdl, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Jiri Sochor, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Ondrej Zitka, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Natalia Cernei, Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno, Czech Republic Branislav Ruttkay-Nedecky, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Pavel Kopel, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Petr Babula, Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno, Czech Republic Vojtech Adam, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Jaromir Hubalek, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Rene Kizek, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Although biallelic mutations in non-collagen genes account for 〈10% of individuals with osteogenesis imperfecta, the characterization of these genes has identified new pathways and potential interventions that could benefit even those with mutations in type I collagen genes. We identified mutations in FKBP10 , which encodes the 65 kDa prolyl cis–trans isomerase, FKBP65, in 38 members of 21 families with OI. These include 10 families from the Samoan Islands who share a founder mutation. Of the mutations, three are missense; the remainder either introduce premature termination codons or create frameshifts both of which result in mRNA instability. In four families missense mutations result in loss of most of the protein. The clinical effects of these mutations are short stature, a high incidence of joint contractures at birth and progressive scoliosis and fractures, but there is remarkable variability in phenotype even within families. The loss of the activity of FKBP65 has several effects: type I procollagen secretion is slightly delayed, the stabilization of the intact trimer is incomplete and there is diminished hydroxylation of the telopeptide lysyl residues involved in intermolecular cross-link formation in bone. The phenotype overlaps with that seen with mutations in PLOD2 (Bruck syndrome II), which encodes LH2, the enzyme that hydroxylates the telopeptide lysyl residues. These findings define a set of genes, FKBP10 , PLOD2 and SERPINH1 , that act during procollagen maturation to contribute to molecular stability and post-translational modification of type I procollagen, without which bone mass and quality are abnormal and fractures and contractures result.

Beschreibung: The GUCY2D gene encodes retinal membrane guanylyl cyclase (RetGC1), a key component of the phototransduction machinery in photoreceptors. Mutations in GUCY2D cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive human retinal blinding disease. The effects of RetGC1 deficiency on human rod and cone photoreceptor structure and function are currently unknown. To move LCA1 closer to clinical trials, we characterized a cohort of patients (ages 6 months—37 years) with GUCY2D mutations. In vivo analyses of retinal architecture indicated intact rod photoreceptors in all patients but abnormalities in foveal cones. By functional phenotype, there were patients with and those without detectable cone vision. Rod vision could be retained and did not correlate with the extent of cone vision or age. In patients without cone vision, rod vision functioned unsaturated under bright ambient illumination. In vitro analyses of the mutant alleles showed that in addition to the major truncation of the essential catalytic domain in RetGC1, some missense mutations in LCA1 patients result in a severe loss of function by inactivating its catalytic activity and/or ability to interact with the activator proteins, GCAPs. The differences in rod sensitivities among patients were not explained by the biochemical properties of the mutants. However, the RetGC1 mutant alleles with remaining biochemical activity in vitro were associated with retained cone vision in vivo . We postulate a relationship between the level of RetGC1 activity and the degree of cone vision abnormality, and argue for cone function being the efficacy outcome in clinical trials of gene augmentation therapy in LCA1.

Beschreibung:    A capillary gas chromatography (GC) procedure has been developed for the determination of four pharmaceutical preparations (famotidine, ranitidine, cimetidine, and metformin) after precolumn derivatization with methylglyoxal (MGo). GC was carried out using an HP-5 column (30 m × 0.32 mm id) at an initial column temperature of 90 °C for 2 min, followed by heating rate of 25 °C min −1 up to 265 °C. Nitrogen flow rate was 2.5 mL min −1 with split ratio 10:1. A linear calibration curve was obtained within 50–1,000 ng mL −1 and the limit of detection (LOD) was within 17–25 ng mL −1 . The derivatization, GC elution, and separation were repeatable in terms of retention time and peak height/peak area with relative standard deviation within ±4.6 %. The procedure was applied to the determination of the drugs in pharmaceutical preparations and the sera of volunteers who were given oral doses of the drugs. The results of the analysis agreed with the labeled values of the pharmaceutical preparations and were 147–4,903 ng mL −1 in serum with an RSD within 1.0–4.2 %, after ingestion of a single dose of 40–500 mg of active ingredient in a tablet. Content Type Journal Article Category Original Pages 1-7 DOI 10.1007/s10337-012-2321-6 Authors Subhan Ali Majidano, Institute of Advanced Research Studies in Chemical Sciences (IARSCS), University of Sindh, Jamshoro, Pakistan Muhammad Yar Khuhawar, Institute of Advanced Research Studies in Chemical Sciences (IARSCS), University of Sindh, Jamshoro, Pakistan Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Male patients with Peutz–Jeghers syndrome (PJS) have defective spermatogenesis and are at increased risk of developing Sertoli cell tumors. Mutations in the Liver Kinase B1 ( LKB1/STK11) gene are associated with the pathogenesis of PJS and have been identified in non-PJS patients with sporadic testicular cancers. The mechanisms controlled by LKB1 signaling in Sertoli cell functions and testicular biology have not been described. We have conditionally deleted the Lkb1 gene ( Lkb1 cko ) in somatic testicular cells to define the molecular mechanisms involved in the development of the testicular phenotype observed in PJS patients. Focal vacuolization in some of the seminiferous tubules was observed in 4-week-old mutant testes but germ cell development appeared to be normal. However, similar to PJS patients, we observed progressive germ cell loss and Sertoli cell only tubules in Lkb1 cko testes from mice older than 10 weeks, accompanied by defects in Sertoli cell polarity and testicular junctional complexes and decreased activation of the MAP/microtubule affinity regulating and focal adhesion kinases. Suppression of AMP kinase and activation of mammalian target of rapamycin (mTOR) signaling were also observed in Lkb1 cko testes. Loss of Tsc1 or Tsc2 copies the progressive Lkb1 cko phenotype, suggesting that dysregulated activation of mTOR contributes to the pathogenesis of the Lkb1 cko testicular phenotype. Pten cko mice had a normal testicular phenotype, which could be explained by the comparative lack of mTOR activation detected. These studies describe the importance of LKB1 signaling in testicular biology and the possible molecular mechanisms driving the pathogenesis of the testicular defects observed in PJS patients.

Beschreibung: The human X-linked macrosatellite DXZ4 is a large tandem repeat located at Xq23 that is packaged into heterochromatin on the male X chromosome and female active X chromosome and, in response to X chromosome, inactivation is organized into euchromatin bound by the insulator protein CCCTC-binding factor (CTCF) on the inactive X chromosome (Xi). The purpose served by this unusual epigenetic regulation is unclear, but suggests a Xi-specific gain of function for DXZ4. Other less extensive bands of euchromatin can be observed on the Xi, but the identity of the underlying DNA sequences is unknown. Here, we report the identification of two novel human X-linked tandem repeats, located 58 Mb proximal and 16 Mb distal to the macrosatellite DXZ4. Both tandem repeats are entirely contained within the transcriptional unit of novel spliced transcripts. Like DXZ4, the tandem repeats are packaged into Xi-specific CTCF-bound euchromatin. These sequences undergo frequent CTCF-dependent interactions with DXZ4 on the Xi, implicating DXZ4 as an epigenetically regulated Xi-specific structural element and providing the first putative functional attribute of a macrosatellite in the human genome.

Beschreibung: The apolipoprotein E ( APOE ) genotype is the major genetic risk factor for Alzheimer's disease (AD). We have access to cerebrospinal fluid (CSF) and plasma APOE protein levels from 641 individuals and genome-wide genotyped data from 570 of these samples. The aim of this study was to test whether CSF or plasma APOE levels could be a useful endophenotype for AD and to identify genetic variants associated with APOE levels. We found that CSF ( P = 8.15 x 10 –4 ) but not plasma ( P = 0.071) APOE protein levels are significantly associated with CSF Aβ 42 levels. We used Mendelian randomization and genetic variants as instrumental variables to confirm that the association of CSF APOE with CSF Aβ 42 levels and clinical dementia rating (CDR) is not because of a reverse causation or confounding effect. In addition the association of CSF APOE with Aβ 42 levels was independent of the APOE 4 genotype, suggesting that APOE levels in CSF may be a useful endophenotype for AD. We performed a genome-wide association study to identify genetic variants associated with CSF APOE levels: the APOE 4 genotype was the strongest single-genetic factor associated with CSF APOE protein levels ( P = 6.9 x 10 –13 ). In aggregate, the Illumina chip single nucleotide polymorphisms explain 72% of the variability in CSF APOE protein levels, whereas the APOE 4 genotype alone explains 8% of the variability. No other genetic variant reached the genome-wide significance threshold, but nine additional variants exhibited a P -value 〈10 –6 . Pathway mining analysis indicated that these nine additional loci are involved in lipid metabolism ( P = 4.49 x 10 –9 ).

Beschreibung:    A liquid chromatography-mass spectrometry (LC-MS) assay was developed and validated for the quantification of lurasidone, an atypical antipsychotic drug, in rat plasma, bile, and urine. Rat plasma, bile, or urine samples were processed by liquid–liquid extraction and injected onto an LC-MS system for the quantification of lurasidone and ziprasidone (an internal standard). Lurasidone and ziprasidone were separated from endogenous substances using a Gemini C6-Phenyl column with mixture of acetonitrile and 0.1 % formic acid (80:20, v/v ) as the mobile phase. Quantification was performed using the selected ion monitoring mode at m/z 493 for lurasidone and m/z 413 for the IS. The detector response was specific and linear for lurasidone in the concentration range 5–5,000 ng mL −1 The intra- and inter-day accuracy and precision of the method were determined to be within the acceptable criteria for assay validation guidelines. In addition, lurasidone was stable under a variety of processing and handling conditions. Lurasidone concentrations could be readily measured in rat plasma, bile, and urine samples up to 24 h after an intravenous or oral administration, suggesting that the assay can be used in pharmacokinetic studies of lurasidone in rats. Content Type Journal Article Category Original Pages 1117-1128 DOI 10.1007/s10337-012-2294-5 Authors Yoon-Jee Chae, Department of Pharmaceutics, College of Pharmacy, Seoul National University, Gwanak 599, Gwanak-ro, Gwanak-gu, Seoul, 151-742 Republic of Korea Tae-Sung Koo, Graduate School of New Drug Discovery and Development, Chungnam National University, Yuseong, Daejeon, 305-764 Republic of Korea Kyeong-Ryoon Lee, Korea Research Institute of Chemical Technology, 141 Gajeongro, Yuseong, Daejeon, 305-343 Republic of Korea Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893 Journal Volume Volume 75 Journal Issue Volume 75, Numbers 19-20

Beschreibung:    Three alkoxyl-functionalized ionic liquids, 1-(3-triethoxysilyl propyl)-3-methyl imidazolium hexafluorophosphate (TESPMIM[PF 6 ]), 1-(3-triethoxysilyl propyl)-3-methyl imidazolium tetrafluoroborate (TESPMIM[BF 4 ]), and 1-(3-triethoxysilyl propyl)-3-methyl imidazolium bis(trifluoromethanesulfonyl)imide (TESPMIM[N(SO 2 CF 3 ) 2 ]), were synthesized and used as selective coating materials to prepare chemically bonded ionic-liquid-based organic–inorganic hybrid solid-phase microextraction (SPME) fibers by sol–gel technology. A possible mechanism of the sol–gel process is proposed, and the successful binding of ionic liquids to the formed silica substrate was confirmed by Fourier-transform infrared spectroscopy (FT-IR). These ionic-liquid-based sol–gel coatings have porous surface structure, high thermal stability, strong solvent resistance, wide pH application range, good coating preparation reproducibility, special selectivity, and extraction efficiency for both polar and nonpolar compounds, such as phenolic environmental estrogens, fatty acids, aromatic amines, alcohols, phthalate esters, and polycyclic aromatic hydrocarbons. The TESPMIM[PF 6 ]- and TESPMIM[BF 4 ]-coated fibers have much lower thermal stability (to 300 and 285 °C, respectively) than TESPMIM[N(SO 2 CF 3 ) 2 ]-coated fiber (to 454 °C). However, the selectivity of these two fibers is higher towards strong polar analytes, while lower towards medium polar or nonpolar analytes compared with TESPMIM[N(SO 2 CF 3 ) 2 ]-based fiber. This could be explained by the fact that different counteranions in ionic liquid structures have different steric hindrance, nucleophilicity, hydrophobicity, and ability to form hydrogen bonds, resulting in significant difference in the characteristics of the ionic-liquid-based SPME fibers, such as the surface morphology, thermal stability, selective extraction ability, etc. This work demonstrates that the performance of the ionic-liquid-based coatings can be simply tuned by changing the counteranions incorporated into the ionic liquid structures. Content Type Journal Article Category Original Pages 1-13 DOI 10.1007/s10337-012-2323-4 Authors Jianjun Shu, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070 China Chan Li, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070 China Mingming Liu, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070 China Hanlan Liu, College of Science, Huazhong Agricultural University, Wuhan, 430070 China Xionghan Feng, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070 China Wenfeng Tan, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070 China Fan Liu, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070 China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    In clinical medicine, urine creatinine concentration is an important marker in the evaluation of renal function and muscular dysfunctions. Herein, we reported a novel method for rapid determination of creatinine in urine by microchip electrophoresis with light-emitting diode induced fluorescence detection. Creatinine was derivatized by fluorescein isothiocyanate, and then quantitatively detected by the developed microchip LED induced fluorescence detection system. The excitation and emission wavelengths were 490 and 523 nm, respectively. The urine samples were analyzed after centrifuge and filtration. A baseline separation was obtained in 〈30 s using 10 mM borate buffer (pH 9.0, containing 45 mM sodium dodecylsulfate), with separation voltage of 1.5 kV. Good linearity was obtained ( r 2  = 0.9978) in the concentration range of 10.0–2.00 × 10 3  μM, and the limit of detection was 2.87 μM ( S/N  = 3). The recovery was 96.0–107 %, and the interday precision was 〈4.5 % ( n  = 6). To validate assay results, we compared the present method with the Jaffe’s colorimetric assay by measuring real urine samples. The method was reliable, sensitive, high-speed, low-cost and suitable for the routine analysis of creatinine in biofluids. Content Type Journal Article Category Original Pages 1-7 DOI 10.1007/s10337-012-2324-3 Authors Shuping Wang, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou, 510006 People’s Republic of China Xinchun Li, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou, 510006 People’s Republic of China Jianping Yang, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou, 510006 People’s Republic of China Xiujuan Yang, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou, 510006 People’s Republic of China Fenghua Hou, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou, 510006 People’s Republic of China Zuanguang Chen, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou, 510006 People’s Republic of China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Mike S. Lee: Mass Spectrometry Handbook Content Type Journal Article Category Book Review Pages 1-2 DOI 10.1007/s10337-012-2325-2 Authors Colin F. Poole, Wayne State University, Detroit, MI, USA Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    CE offers the possibility of fast, cheap and reproducible separations for active compounds of Chinese herbs. Lotusine ( 1 ), liensinine ( 2 ), isoliensinine ( 3 ) and neferine ( 4 ) were the major bioactive alkaloids in lotus plumule, which was used as an important Chinese herb. In this paper, simultaneous separation of 1 – 4 in lotus plumule by nonaqueous CZE has been achieved within 11 min by use of buffer consisting of 80 mM sodium acetate and 40 mM ammonium acetate methanol solution of pH 5.4. Analysis of the four alkaloids in ten plumule samples of Nelumbo nucifera was conducted. Limits of detection (LOD) of 1 – 4 by UV absorbance at 203 nm were achieved in the range of 1.5–2.8 μg mL −1 . Reproducibility (percent RSD) of the analysis by CZE were 2.33, 2.31, 0.78 and 2.58 %, respectively ( n  = 5). Content Type Journal Article Category Original Pages 1-6 DOI 10.1007/s10337-012-2312-7 Authors Guangming Yang, National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing, 210046 China Weidong Li, National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing, 210046 China Yang Pan, National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing, 210046 China Xia Tu, Department of Bio-Pharmacy and Laboratory of Medical Fungi and Phyto-Biotech, Nanjing University of Chinese Medicine, Nanjing, 210029 China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: A number of mouse models for spinal muscular atrophy (SMA) have been genetically engineered to recapitulate the severity of human SMA by using a targeted null mutation at the mouse Smn1 locus coupled with the transgenic addition of varying copy numbers of human SMN2 genes. Although this approach has been useful in modeling severe SMA and very mild SMA, a mouse model of the intermediate form of the disease would provide an additional research tool amenable for drug discovery. In addition, many of the previously engineered SMA strains are multi-allelic by design, containing a combination of transgenes and targeted mutations in the homozygous state, making further genetic manipulation difficult. A new genetic engineering approach was developed whereby variable numbers of SMN2 sequences were incorporated directly into the murine Smn1 locus. Using combinations of these alleles, we generated an allelic series of SMA mouse strains harboring no, one, two, three, four, five, six or eight copies of SMN2. We report here the characterization of SMA mutants in this series that displayed a range in disease severity from embryonic lethal to viable with mild neuromuscular deficits.

Beschreibung: Insulin resistance (IR) is a key determinant of type 2 diabetes (T2D) and other metabolic disorders. This genome-wide association study (GWAS) was designed to shed light on the genetic basis of fasting insulin (FI) and IR in 927 non-diabetic African Americans. 5 396 838 single-nucleotide polymorphisms (SNPs) were tested for associations with FI or IR with adjustments for age, sex, body mass index, hypertension status and first two principal components. Genotyped SNPs ( n = 12) with P 〈 5 x 10 –6 in African Americans were carried forward for de novo genotyping in 570 non-diabetic West Africans. We replicated SNPs in or near SC4MOL and TCERG1L in West Africans. The meta-analysis of 1497 African Americans and West Africans yielded genome-wide significant associations for SNPs in the SC4MOL gene: rs17046216 ( P = 1.7 x 10 –8 and 2.9 x 10 –8 for FI and IR, respectively); and near the TCERG1L gene with rs7077836 as the top scoring ( P = 7.5 x 10 –9 and 4.9 x 10 –10 for FI and IR, respectively). In silico replication in the MAGIC study ( n = 37 037) showed weak but significant association (adjusted P -value of 0.0097) for rs34602777 in the MYO5A gene. In addition, we replicated previous GWAS findings for IR and FI in Europeans for GCKR , and for variants in four T2D loci ( FTO , IRS1 , KLF14 and PPARG ) which exert their action via IR. In summary, variants in/near SC4MOL , and TCERG1L were associated with FI and IR in this cohort of African Americans and were replicated in West Africans. SC4MOL is under-expressed in an animal model of T2D and plays a key role in lipid biosynthesis, with implications for the regulation of energy metabolism, obesity and dyslipidemia. TCERG1L is associated with plasma adiponectin, a key modulator of obesity, inflammation, IR and diabetes.

Beschreibung: Loss of dystrophin protein due to mutations in the DMD gene causes Duchenne muscular dystrophy. Dystrophin loss also leads to the loss of the dystrophin glycoprotein complex (DGC) from the sarcolemma which contributes to the dystrophic phenotype. Tyrosine phosphorylation of dystroglycan has been identified as a possible signal to promote the proteasomal degradation of the DGC. In order to test the role of tyrosine phosphorylation of dystroglycan in the aetiology of DMD, we generated a knock-in mouse with a phenylalanine substitution at a key tyrosine phosphorylation site in dystroglycan, Y890. Dystroglycan knock-in mice ( Dag1 Y890F/Y890F ) had no overt phenotype. In order to examine the consequence of blocking dystroglycan phosphorylation on the aetiology of dystrophin-deficient muscular dystrophy, the Y890F mice were crossed with mdx mice an established model of muscular dystrophy. Dag1 Y890F/Y890F / mdx mice showed a significant improvement in several parameters of muscle pathophysiology associated with muscular dystrophy, including a reduction in centrally nucleated fibres, less Evans blue dye infiltration and lower serum creatine kinase levels. With the exception of dystrophin, other DGC components were restored to the sarcolemma including α-sarcoglycan, α-/β-dystroglycan and sarcospan. Furthermore, Dag1 Y890F/Y890F / mdx showed a significant resistance to muscle damage and force loss following repeated eccentric contractions when compared with mdx mice. While the Y890F substitution may prevent dystroglycan from proteasomal degradation, an increase in sarcolemmal plectin appeared to confer protection on Dag1 Y890F/Y890F / mdx mouse muscle. This new model confirms dystroglycan phosphorylation as an important pathway in the aetiology of DMD and provides novel targets for therapeutic intervention.

Beschreibung: Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array. Recent studies have proposed that FSHD pathology is caused by the misexpression of the DUX4 (double homeobox 4) gene resulting in production of a pathogenic protein, DUX4-FL, which has been detected in FSHD, but not in unaffected control myogenic cells and muscle tissue. Here, we report the analysis of DUX4 mRNA and protein expression in a much larger collection of myogenic cells and muscle biopsies derived from biceps and deltoid muscles of FSHD affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adult FSHD subjects yet to show clinical manifestations of the disease in the assayed muscles. In addition, we report DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology and indicate that quantitative modifiers of DUX4-fl expression and/or function and family genetic background are determinants of FSHD muscle disease progression.

Beschreibung:    Phospholipids have been shown to cause matrix effects particularly in liquid chromatography–mass spectrometry (LC–MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC–MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep–wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021–3.268 μmol L −1 ; R 2  = 0.9983–0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2–23.5 nmol L −1 and the LOQs of 15.4–70.5 nmol L −1 for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples. Content Type Journal Article Category Original Pages 1-13 DOI 10.1007/s10337-012-2330-5 Authors Merisa Moriarty, Department of Chemistry, PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research), Cork Institute of Technology, Cork, Ireland Aoife Lee, Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland Brendan O’Connell, Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland Mary Lehane, Department of Chemistry, PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research), Cork Institute of Technology, Cork, Ireland Helen Keeley, Child and Adolescent Mental Health Services, Health Service Executive, South, North Cork Area and the National Suicide Research Foundation, Cork, Ireland Ambrose Furey, Team Elucidate and PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research) research groups, Cork Institute of Technology, Cork, Ireland Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Graphene, a novel class of carbon nanostructure, possesses an ultra-high specific surface area (theoretical value 2,630 m 2  g −1 ), and both sides of the planar sheets of graphene are available for molecule adsorption. Graphene has already been used for preconcentration, extraction, and electrochemical selective determination. In this study, we used graphene to clean up pigments in cucumber for analysis, and measured eight pyrethroid model analytes using GC with electron capture detection (ECD). The recoveries of the 8 pyrethroids were 75–116 % with RSDs below 10 %, and LOQs ranged from 2.5 to 10 μg kg −1 . Comparative studies showed that graphene was superior to graphitized carbon black for the purification of pigments. We also investigated the ability of graphene to clean up spinach. A promising new adsorbent for pesticide residue analysis was developed. Graphene has significant potential as an effective adsorbent of pigments. Content Type Journal Article Category Original Pages 1-7 DOI 10.1007/s10337-012-2299-0 Authors Xiaoli Wu, College of Science, China Agricultural University, Beijing, 100193 People’s Republic of China Hongyan Zhang, College of Science, China Agricultural University, Beijing, 100193 People’s Republic of China Lixuan Meng, College of Science, China Agricultural University, Beijing, 100193 People’s Republic of China Xiaotong Liu, College of Science, China Agricultural University, Beijing, 100193 People’s Republic of China Yongqiang Ma, College of Science, China Agricultural University, Beijing, 100193 People’s Republic of China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A highly sensitive and rapid method was developed that involves capillary electrophoresis for separation and determination of the stereoisomeric impurity of folinic acid diastereomers. In this method, vancomycin was used as the chiral selector, and a solution of poly(dimethylacrylamide) (PDMA) was prepared for dynamic coating of the capillary wall to minimize the adsorption of vancomycin. This method was optimized for six factors including concentrations of the organic modifier and vancomycin, pH and concentration of the background electrolyte, column temperature, and separation voltage. The following conditions were established: 100 mM Tris-phosphate buffer (pH 6.0) containing 1.0 mM vancomycin and 5 % acetonitrile at 30 °C, and −15 kV applied voltage on the PDMA dynamically coated capillary. Preliminary validation was performed with the determination of limit of quantification and detection, accuracy, precision, and linearity. Under our optimized method, the folinic acid diastereomers were baseline-separated within 7.5 min, and a (6 S ,2′ S )-calcium folinate sample with 0.08 % stereoisomeric impurity was determined. Content Type Journal Article Category Short Communication Pages 1-5 DOI 10.1007/s10337-012-2304-7 Authors Zhaoyan Wang, School of Pharmacy, Lanzhou University, Lanzhou, 730000 China Changjun Mu, School of Life Science, Lanzhou University, Lanzhou, 730000 China Jingwu Kang, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032 China Zhide Hu, School of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000 China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A simple and sensitive method for the determination of free aliphatic amines using 10-phenyl-acridone-2-sulfonyl chloride (PASC) as a labeling reagent by high-performance liquid chromatography with fluorescence detection and online mass spectrometry identification (HPLC-FLD-MS) has been developed. Derivatization conditions including reagent concentration, buffer pH, reaction time and temperature were optimized. PASC reacted with aliphatic amines at 50 °C for 4 min in aqueous acetonitrile (ACN) in the presence of sodiumtetraborate–NaOH buffer (0.10 mol L −1 , pH 9.0) to give high yields of PASC-amine derivatives. Derivatives exhibited intense fluorescence with an excitation maximum at λ ex 265 nm and an emission maximum at λ em 418 nm. The separation of derivatives was performed by a reversed-phase Hypersil BDS C8 column in combination with a gradient elution. The identification of derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion detection mode. Excellent linear responses were observed with the correlation coefficients of larger than 0.9997, and detection limits (at a signal-to-noise of 3:1) were from 3.0 to 24.3 fmol. Comparing with 10-ethyl-acridine-2-sulfonyl chloride (EASC), PASC exhibited more intense fluorescence and ultraviolet absorbance. The proposed method is sensitive and reproducible for the determination of aliphatic amines from water and soil samples. Content Type Journal Article Category Original Pages 1-10 DOI 10.1007/s10337-012-2298-1 Authors Shujing Ning, Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, 273156 China Jinmao You, Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, 273156 China Zhiwei Sun, Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, 273156 China Shijuan Zhang, Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, 810008 China Zhongyin Ji, Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, 810008 China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Abnormal presence of autophagic vacuoles is evident in brains of patients with Parkinson's disease (PD), in contrast to the rare detection of autophagosomes in a normal brain. However, the actual cause and pathological significance of these observations remain unknown. Here, we demonstrate a role for mitochondrial metabolism in the regulation of the autophagy-lysosomal pathway in ex vivo and in vitro models of PD. We show that transferring mitochondria from PD patients into cells previously depleted of mitochondrial DNA is sufficient to reproduce the alterations in the autophagic system observed in PD patient brains. Although the initial steps of this pathway are not compromised, there is an increased accumulation of autophagosomes associated with a defective autophagic activity. We prove that this functional decline was originated from a deficient mobilization of autophagosomes from their site of formation toward lysosomes due to disruption in microtubule-dependent trafficking. This contributed directly to a decreased proteolytic flux of α-synuclein and other autophagic substrates. Our results lend strong support for a direct impact of mitochondria in autophagy as defective autophagic clearance ability secondary to impaired microtubule trafficking is driven by dysfunctional mitochondria. We uncover mitochondria and mitochondria-dependent intracellular traffic as main players in the regulation of autophagy in PD.

Beschreibung: Resistin is a polypeptide hormone that was reported to be associated with insulin resistance, inflammation and risk of type 2 diabetes and cardiovascular disease. We conducted a genome-wide association (GWA) study on circulating resistin levels in individuals of European ancestry drawn from the two independent studies: the Nurses' Health Study ( n = 1590) and the Health, Aging and Body Composition Study ( n = 1658). Single-nucleotide polymorphisms (SNPs) identified in the GWA analysis were replicated in an independent cohort of Europeans: the Gargano Family Study ( n = 659). We confirmed the association with a previously known locus, the RETN gene (19p13.2), and identified two novel loci near the TYW3/CRYZ gene (1p31) and the NDST4 gene (4q25), associated with resistin levels at a genome-wide significant level, best represented by SNP rs3931020 ( P = 6.37 x 10 –12 ) and SNP rs13144478 ( P = 6.19 x 10 –18 ), respectively. Gene expression quantitative trait loci analyses showed a significant cis association between the SNP rs3931020 and CRYZ gene expression levels ( P = 3.68 x 10 –7 ). We also found that both of these two SNPs were significantly associated with resistin gene ( RETN ) mRNA levels in white blood cells from 68 subjects with type 2 diabetes (both P = 0.02). In addition, the resistin-rising allele of the TYW3/CRYZ SNP rs3931020, but not the NDST4 SNP rs13144478, showed a consistent association with increased coronary heart disease risk [odds ratio = 1.18 (95% CI, 1.03–1.34); P = 0.01]. Our results suggest that genetic variants in TYW3/CRYZ and NDST4 loci may be involved in the regulation of circulating resistin levels. More studies are needed to verify the associations of the SNP rs13144478 with NDST4 gene expression and resistin-related disease.

Beschreibung: SERPINA3 (Serpin peptidase inhibitor clade A member 3), also known as a1-antichymotrypsin, is a serine protease inhibitor involved in a wide range of biological processes. Recently, it has been shown to be up-regulated in human placental diseases in association with a hypomethylation of the 5' region of the gene. In the present study, we show that the promoter of SERPINA3 is transcriptionally activated by three transcription factors (TFs) (SP1, MZF1 and ZBTB7B), the level of induction being dependent on the rs1884082 single nucleotide polymorphism (SNP) located inside the promoter, the T allele being consistently induced to a higher level than the G, with or without added TFs. When the promoter was methylated, the response to ZBTB7B was allele specific (the G allele was strongly induced, while the T allele was strongly down-regulated). We propose an adaptive model to explain the interest of such a regulation for placental function and homeostasis. Overexpression of SERPINA3 in JEG-3 cells, a trophoblast cell model, decreased cell adhesion to the extracellular matrix and to neighboring cells, but protects them from apoptosis, suggesting a way by which this factor could be deleterious at high doses. In addition, we show in different human populations that the T allele appears to predispose to Intra Uterine Growth Restriction (IUGR), while a G allele at a second SNP located in the second exon (rs4634) increases the risk of preeclampsia. Our results provide mechanistic views inside the involvement of SERPINA3 in placental diseases, through its regulation by a combination of epigenetic, genetic and TF-mediated regulations.

Beschreibung: Apoptosis, or programmed cell death, is a cellular pathway involved in normal cell turnover, developmental tissue remodeling, embryonic development, cellular homeostasis maintenance and chemical-induced cell death. Caspases are a family of intracellular proteases that play a key role in apoptosis. Aberrant activation of caspases has been implicated in human diseases. In particular, numerous findings implicate Caspase-6 (Casp6) in neurodegenerative diseases, including Alzheimer disease (AD) and Huntington disease (HD), highlighting the need for a deeper understanding of Casp6 biology and its role in brain development. The use of targeted caspase-deficient mice has been instrumental for studying the involvement of caspases in apoptosis. The goal of this study was to perform an in-depth neuroanatomical and behavioral characterization of constitutive Casp6-deficient ( Casp6 –/–) mice in order to understand the physiological function of Casp6 in brain development, structure and function. We demonstrate that Casp6 –/– neurons are protected against excitotoxicity, nerve growth factor deprivation and myelin-induced axonal degeneration. Furthermore, Casp6-deficient mice show an age-dependent increase in cortical and striatal volume. In addition, these mice show a hypoactive phenotype and display learning deficits. The age-dependent behavioral and region-specific neuroanatomical changes observed in the Casp6 –/– mice suggest that Casp6 deficiency has a more pronounced effect in brain regions that are involved in neurodegenerative diseases, such as the striatum in HD and the cortex in AD.

Beschreibung: In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α M β 2 -mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fib 390-396A ) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α M β 2 binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α M β 2 blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α M β 2 interactions may provide a novel strategy for DMD treatment.

Beschreibung: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder recognized in fragile X premutation carriers. Using Drosophila , we previously identified elongated non-coding CGG repeats in FMR1 allele as the pathogenic cause of FXTAS. Here, we use this same FXTAS Drosophila model to conduct a chemical screen that reveals small molecules that can ameliorate the toxic effects of fragile X premutation ribo-CGG (rCGG) repeats, among them several known phospholipase A 2 (PLA 2 ) inhibitors. We show that specific inhibition of PLA 2 activity could mitigate the neuronal deficits caused by fragile X premutation rCGG repeats, including lethality and locomotion deficits. Furthermore, through a genetic screen, we identified a PLA 2 Drosophila ortholog that specifically modulates rCGG repeat-mediated neuronal toxicity. Our results demonstrate the utility of Drosophila models for unbiased small molecule screens and point to PLA 2 as a possible therapeutic target to treat FXTAS.

Beschreibung:    A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10 μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5 → 152.2 ( m / z ) for lobeglitazone and 446.1 → 321.2 ( m / z ) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000 ng mL −1 for plasma and 0.2–250 ng mL −1 for urine ( r 2  ≥ 0.9996). The mean percent extraction recovery of lobeglitazone was 90.8 % for plasma and 87.3 % for urine, while the recoveries of the IS were greater than 86.4 % for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3 % (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5 %, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized. Content Type Journal Article Category Short Communication Pages 1-7 DOI 10.1007/s10337-012-2238-0 Authors Bora Kim, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Hyun-Suk Shin, Analytical Chemistry Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea Jung-Ryul Kim, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Kyung-Soo Lim, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Seo Hyun Yoon, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Kyung-Sang Yu, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Sang-Goo Shin, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea In-Jin Jang, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Joo-Youn Cho, Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, 101 Daehangno, Jongno-gu, Seoul, 110-744 Republic of Korea Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Expanded bed adsorption (EBA) is a practical method for the separation of nanoparticulates. In order to analysis the local hydrodynamic and adsorption behavior of nanoparticle (NP)-based biological feedstock, a modified Nano Biotechnology Group EBA column with a 26-mm inner diameter was used to withdraw liquid from different axial positions of the column. Fabricated egg albumin (EA) NPs with an average size of 70 nm were employed as a model system and viral size/charge mimic to assess the relationship between hydrodynamic and adsorption performance of NPs at the different column regions. The effects of influential factors, including flow velocity and initial concentration of NPs, on NP hydrodynamic behavior and adsorption kinetics along the bed height were investigated. NP hydrodynamic studies confirmed that non-uniform behavior dominated the system and a decreasing trend of liquid mixing/dispersion with increase of bed height was observed in this column. The results demonstrated an increase in the mixing/dispersion at certain bed heights with the increase in both the velocity and feed initial concentration. Breakthrough curves were measured at various column points to determine the adsorption performance [dynamic binding capacity (DBC) and yield] in different bed positions/zones. Yield and DBC of NPs were improved along the bed height, whereas liquid velocity had the opposite effect. Increasing the initial concentration of NPs enhanced only the DBC. Separation of EA NPs under optimal conditions was 87 %, which is an excellent result for a one-pass frontal chromatography method. Content Type Journal Article Category Original Pages 1-12 DOI 10.1007/s10337-012-2235-3 Authors Mohsen Jahanshahi, Nanotechnology Research Institute, Faculty of Chemical Engineering, Babol University of Technology, P.O. Box 484, Babol, Iran Mohammad Taghi Hamed Mosavian, Faculty of Chemical Engineering, Ferdowsi University of Mashhad, Mashhad, Iran Elham Sadat Taheri Otaghsara, Nanotechnology Research Institute, Faculty of Chemical Engineering, Babol University of Technology, P.O. Box 484, Babol, Iran Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A liquid chromatographic tandem mass spectrometric (LC–MS–MS) method for the determination of five chemical coccidiostats (decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine) and five ionophore coccidiostats (maduramicin, monensin, narasin, salinomycin, and semduramicin) in yoghurt, kefir, and sour cream is presented. Lasalocid, the sixth ionophore listed in 124/2009/EC was not included because of its extremely dissimilar behavior during sample preparation. Main steps of the method include extraction with acetonitrile, centrifugation, clean-up on Oasis HLB solid phase extraction cartridge, evaporation under nitrogen stream, and LC–MS–MS determination. Selectivity, linearity, sensitivity, accuracy, repeatability, within-laboratory reproducibility, limit of determination, and limit of quantitation were determined during the validation procedure. The method proved to be applicable for both qualitative and quantitative determination of the ten above-mentioned target compounds. In our in-house fermentation experiments, milk fortified with coccidiostats was fermented to get yoghurt, kefir, and sour cream. Our results show that the coccidiostat content did not change significantly during fermentation for any of the target compounds. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2236-2 Authors Szilárd Nász, Joint Research and Training Laboratory on Separation Techniques, Eötvös Loránd University, Pázmány Péter sétány 1/A, Budapest, 1117 Hungary Etelka M. Károlyné, Alföldi Tej Kft, Seregélyesi út 127, Székesfehérvár, 8000 Hungary Tamás Rikker, Wessling International Research and Educational Center, Fóti út 56, Budapest, 1047 Hungary Zsuzsanna Eke, Joint Research and Training Laboratory on Separation Techniques, Eötvös Loránd University, Pázmány Péter sétány 1/A, Budapest, 1117 Hungary Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    The use of the large retention index database for identification and filtering of false positive hits in GC–MS analysis of the ylang-ylang essential oil is illustrated. Differences between experimental retention indices and database values of retention indices of candidate compounds provide additional constraints on the list of candidates for a target compound. Over 100 components of ylang-ylang essential oil (total grade) were identified. The main components, with concentrations more than 4 %, are β-caryophyllene, germacrene D, benzyl benzoate, linalool, geranyl acetate, α- (E,E) -farnesene and isobornyl acetate. Content Type Journal Article Category Short Communication Pages 1-8 DOI 10.1007/s10337-012-2231-7 Authors V. I. Babushok, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA N. R. Andriamaharavo, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    The objective of current investigation was to study the degradation behavior of l -DOPA under different conditions by high performance liquid chromatography (HPLC), and to develop and validate a stability-indicating HPLC method. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision and specificity. Oxidation was found to occur in alkaline and to some extent in thermal conditions, while the drug was stable when incubated at acidic conditions and under photolytic stress. The oxidation of l -DOPA was observed to follow first-order kinetics. The degradation rate constants and half-life were calculated. The cytotoxicity and enzymatic degradation of l -DOPA was examined using the human intestinal epithelial Caco-2 cells. The drug was rapidly decarboxylated by aromatic amino acid decarboxylase to dopamine. The conversion of l -DOPA to dopamine was dose- and time-dependent. Content Type Journal Article Category Original Pages 1-10 DOI 10.1007/s10337-012-2229-1 Authors Yong Zhi Zhou, Drug Delivery Research Unit, School of Pharmacy, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand Raid G. Alany, Drug Delivery Research Unit, School of Pharmacy, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand Victor Chuang, School of Pharmacy, Faculty of Health Sciences, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987, Perth, WA 6845, Australia Jingyuan Wen, Drug Delivery Research Unit, School of Pharmacy, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS n ) was developed for screening of potential anti-osteoporosis agents in E. koreanum . Four compounds identified as epimedin A, epimedin B, epimedin C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H -tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS n is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2232-6 Authors Rui Wang, State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 People’s Republic of China Jian-Guang Luo, State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 People’s Republic of China Ling-Yi Kong, State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 People’s Republic of China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    A simple method is described for efficient isolation of compounds having an antibacterial effect. Two thyme ( Thymus vulgaris ) essential oils, obtained from the market, were chosen as prospective materials likely to feature several bioactive components when examined by thin layer chromatography coupled with direct bioautography as a screening method. The newly developed infusion overpressured layer chromatographic separation method coupled with direct bioautography assured that only the active components were isolated by means of overrun overpressured layer chromatography (OPLC) with on-line detection and fractionation. Each of the 5 collected fractions represented one of the five antimicrobial essential oil components designated at the screening. The purity and the activity of the fractions were confirmed with chromatography coupled with various detection methods (UV, vanillin–sulphuric acid reagent, direct bioautography). The antibacterial components were identified with GC–MS as thymol, carvacrol, (−)-linalool, diethyl-phthalate, and α-terpineol. The oil component diethyl-phthalate is an artificial compound, used as a plasticizer or detergent base in the industry. Our results support that exploiting its flexibility and the possible hyphenations, OPLC is especially attractive for isolation of antimicrobial components from various matrixes. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2233-5 Authors Ágnes M. Móricz, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Herman O. Str. 15, Budapest, 1022 Hungary Péter G. Ott, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Herman O. Str. 15, Budapest, 1022 Hungary Andrea Böszörményi, Institute of Pharmacognosy, Faculty of Pharmacy, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary Éva Lemberkovics, Institute of Pharmacognosy, Faculty of Pharmacy, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary Emil Mincsovics, OPLC-NIT Engineering Co., Ltd, Budapest Andor Str. 60, Budapest, 1119 Hungary Ernő Tyihák, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Herman O. Str. 15, Budapest, 1022 Hungary Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene implicated in major mental illnesses, such as schizophrenia, depression, bipolar disorder and autism, but the link between this protein and the pathology of these diseases remains unclear. Recently, DISC1 has been demonstrated to form insoluble protein aggregates in vitro and in human post-mortem brain tissue but the cellular dynamics of these DISC1 aggregates and their effects on neuronal function are unknown. Using a combination of biochemistry and live cell confocal and video microscopy, we characterize the properties of DISC1 aggregates and their effects on cellular function. We demonstrate that DISC1 protein aggregates are recruited to the aggresome and degraded there by the autophagic pathway. We show that there is a compromised exchange between DISC1 in aggresomes and the cytosolic DISC1 pool, and that the large DISC1 aggregates, which can also co-recruit endogenous soluble DISC1, exhibit altered trafficking. Moreover, we demonstrate that large DISC1 aggregates have a pathological effect in neurons by causing the disruption of intracellular transport of key organellar cargo, such as mitochondria. These data, therefore, show that DISC1 is recruited to aggresomes with negative effects on neuronal function, and suggests a novel DISC1-based mechanism for neuronal pathology.

Beschreibung: Abnormalities in Z-disc proteins cause hypertrophic (HCM), dilated (DCM) and/or restrictive cardiomyopathy (RCM), but disease-causing mechanisms are not fully understood. Myopalladin (MYPN) is a Z-disc protein expressed in striated muscle and functions as a structural, signaling and gene expression regulating molecule in response to muscle stress. MYPN was genetically screened in 900 patients with HCM, DCM and RCM, and disease-causing mechanisms were investigated using comparative immunohistochemical analysis of the patient myocardium and neonatal rat cardiomyocytes expressing mutant MYPN. Cardiac-restricted transgenic (Tg) mice were generated and protein–protein interactions were evaluated. Two nonsense and 13 missense MYPN variants were identified in subjects with DCM, HCM and RCM with the average cardiomyopathy prevalence of 1.66%. Functional studies were performed on two variants (Q529X and Y20C) associated with variable clinical phenotypes. Humans carrying the Y20C-MYPN variant developed HCM or DCM, whereas Q529X-MYPN was found in familial RCM. Disturbed myofibrillogenesis with disruption of α-actinin2, desmin and cardiac ankyrin repeat protein (CARP) was evident in rat cardiomyocytes expressing MYPN Q529X . Cardiac-restricted MYPN Y20C Tg mice developed HCM and disrupted intercalated discs, with disturbed expression of desmin, desmoplakin, connexin43 and vinculin being evident. Failed nuclear translocation and reduced binding of Y20C-MYPN to CARP were demonstrated using in vitro and in vivo systems. MYPN mutations cause various forms of cardiomyopathy via different protein–protein interactions. Q529X-MYPN causes RCM via disturbed myofibrillogenesis, whereas Y20C-MYPN perturbs MYPN nuclear shuttling and leads to abnormal assembly of terminal Z-disc within the cardiac transitional junction and intercalated disc.

Beschreibung: In addition to the genetic constitution inherited by an organism, the developmental trajectory and resulting mature phenotype are also determined by mechanisms acting during critical windows in early life that influence and establish stable patterns of gene expression. This is the crux of the developmental origins of health and disease hypothesis that suggests undernutrition during gestation and infancy predisposes to ill health in later life. The hypothesis that periconceptional maternal micronutrient supplementation might affect fetal genome-wide methylation within gene promoters was explored in cord blood samples from offspring of Gambian women enrolled into a unique randomized, double blind controlled trial. Significant changes in the epigenome in cord blood DNA samples were further explored in a subset of offspring at 9 months. Gender-specific changes related to periconceptional nutritional supplementation were identified in cord blood DNA samples, some of which showed persistent changes in infant blood DNA samples. Significant effects of periconceptional micronutrient supplementation were also observed in postnatal samples which were not evident in cord blood. In this Gambian population, the increased death rate of individuals born in nutritionally poor seasons has been related to infection and it is of interest that we identified differential methylation at genes associated with defence against infection and immune response. Although the sample size was relatively small, these pilot data suggest that periconceptional nutrition in humans is an important determinant of newborn whole genome methylation patterns but may also influence postnatal developmental patterns of gene promoter methylation linking early with disease risk.

Beschreibung: Genome-wide association studies have identified susceptibility loci for esophageal squamous cell carcinoma (ESCC). We conducted a meta-analysis of all single-nucleotide polymorphisms (SNPs) that showed nominally significant P -values in two previously published genome-wide scans that included a total of 2961 ESCC cases and 3400 controls. The meta-analysis revealed five SNPs at 2q33 with P 〈 5 x 10 –8 , and the strongest signal was rs13016963, with a combined odds ratio (95% confidence interval) of 1.29 (1.19–1.40) and P = 7.63 x 10 –10 . An imputation analysis of 4304 SNPs at 2q33 suggested a single association signal, and the strongest imputed SNP associations were similar to those from the genotyped SNPs. We conducted an ancestral recombination graph analysis with 53 SNPs to identify one or more haplotypes that harbor the variants directly responsible for the detected association signal. This showed that the five SNPs exist in a single haplotype along with 45 imputed SNPs in strong linkage disequilibrium, and the strongest candidate was rs10201587, one of the genotyped SNPs. Our meta-analysis found genome-wide significant SNPs at 2q33 that map to the CASP8 / ALS2CR12/TRAK2 gene region. Variants in CASP8 have been extensively studied across a spectrum of cancers with mixed results. The locus we identified appears to be distinct from the widely studied rs3834129 and rs1045485 SNPs in CASP8 . Future studies of esophageal and other cancers should focus on comprehensive sequencing of this 2q33 locus and functional analysis of rs13016963 and rs10201587 and other strongly correlated variants.

Beschreibung: Aneurysmal subarachnoid hemorrhage (aSAH) is the most serious subtype of stroke. Genetic factors have been known to play an important role in the development of intracranial aneurysm (IA), some of which further progress to subarachnoid hemorrhage (SAH). In this study, we conducted a genome-wide association study (GWAS) to identify common genetic variants that are associated with the risk of IA, using 1383 aSAH subjects and 5484 control individuals in the Japanese population. We selected 36 single-nucleotide polymorphisms (SNPs) that showed suggestive association ( P 〈 1 x 10 –4 ) in the GWAS as well as additional 7 SNPs that were previously reported to be associated with IA, and further genotyped an additional set of 1048 IA cases and 7212 controls. We identified an SNP, rs6842241, near EDNRA at chromosome 4q31.22 (combined P -value = 9.58 x 10 –9 ; odds ratio = 1.25), which was found to be significantly associated with IA. Additionally, we successfully replicated and validated rs10757272 on CDKN2BAS at chromosome 9p21.3 (combined P -value = 1.55 x 10 –7 ; odds ratio = 1.21) to be significantly associated with IA as previously reported. Furthermore, we performed functional analysis with the associated genetic variants on EDNRA , and identified two alleles of rs6841581 that have different binding affinities to a nuclear protein(s). The transcriptional activity of the susceptible allele of this variant was significantly lower than the other, suggesting that this functional variant might affect the expression of EDNRA and subsequently result in the IA susceptibility. Identification of genetic variants on EDNRA is of clinical significance probably due to its role in vessel hemodynamic stress. Our findings should contribute to a better understanding of physiopathology of IA.

Beschreibung: Response to Letter to the Editor Regarding: Comparison of Several Methods of Chromatographic Baseline Removal with a New Approach Based on Quantile Regression Content Type Journal Article Category Letter to the Editor Pages 315-316 DOI 10.1007/s10337-012-2191-y Authors Ł. Komsta, Department of Medicinal Chemistry, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893 Journal Volume Volume 75 Journal Issue Volume 75, Numbers 5-6

Beschreibung:    The retention behavior of melamine (MEL), ammeline (AMN), ammelide, and cyanuric acid on various hydrophilic interaction liquid chromatography (HILIC) stationary phases was studied. Using fully optimized electrospray conditions and ammonium formate buffer pH 4/acetonitrile (20:80 v/v) as a mobile phase, precursor and product ions for each compound were identified. For the LC separation study, the effect of the following parameters was investigated: the type of buffer, the effect of the pH of ammonium formate buffer, the dilution percentage of the standard solutions in the vial, the stability of the standard solutions, and the column equilibration time. The retention and separation of melamine and its hydrolysis products on several HILIC columns was also investigated and retention models were proposed. Retention mechanisms were discussed for all the compounds. When the percentage of acetonitrile in the mobile phase was increased, the retention times of ammelide, ammeline and melamine were shifted to higher values, while the retention time of cyanuric acid did not change significantly. The separation of compounds with isobaric transitions (MEL, AMN) was achieved on four HILIC columns, namely TSKgel Amide-80, Luna HILIC, XBridge HILIC, and ZIC-HILIC (at either 10/90 or 15/85 ammonium formate buffer pH 4/ACN). Content Type Journal Article Category Original Pages 1-11 DOI 10.1007/s10337-012-2228-2 Authors Maroula G. Kokotou, Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece Nikolaos S. Thomaidis, Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Malondialdehyde (MDA) is an end-product of lipid peroxidation and a side product of thromboxane A 2 synthesis. Moreover, it is not only a frequently measured biomarker of oxidative stress, but its high reactivity and toxicity underline the fact that this molecule is more than “just” a biomarker. Additionally, MDA was proven to be a mutagenic substance. Having said this, it is evident that there is a major interest in the highly selective and sensitive analysis of this molecule in various matrices. In this review, we will provide a brief overview of the most recent developments and techniques for the liquid chromatography (LC) and gas chromatography (GC)-based analysis of MDA in different matrices. While the 2-thiobarbituric acid assay still is the most prominent methodology for determining MDA, several advanced techniques have evolved, including GC–MS(MS), LC–MS(MS) as well as several derivatization-based strategies. Content Type Journal Article Category Review Pages 1-8 DOI 10.1007/s10337-012-2237-1 Authors Martin Giera, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center (LUMC), Albinusdreef 2, 2300 RC Leiden, The Netherlands Henk Lingeman, BioMolecular Analysis, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Wilfried M. A. Niessen, BioMolecular Analysis, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    Acepromazine maleate (Sedalin ® ) was administered orally to six thoroughbred horses at a dose of 0.15 mg kg −1 . Urine and blood samples were collected up to 412 h post-administration. Plasma and urine were hydrolysed; plasma samples were then processed using liquid–liquid extraction and urine samples using solid-phase extraction. A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification for acepromazine of 10 pg mL −1 in plasma and 100 pg mL −1 in urine. Acepromazine, hydroxyethylpromazine, hydroxyacepromazine, hydroxyethylpromazine sulphoxide, hydroxyethylhydroxypromazine, dihydroxyacepromazine and dihydroxyhydroxyethylpromazine were detected in the post-administration samples. The parent drug and its metabolites were identified using a combination of UPLC–MS/MS and accurate mass measurement. Separation of the structural isomers hydroxyethylpromazine sulphoxide and hydroxyethylhydroxypromazine was another significant outcome of this work and demonstrated the advantages to be gained from investing in chromatographic method development. Content Type Journal Article Category Original Pages 1-9 DOI 10.1007/s10337-012-2234-4 Authors M. E. Wieder, HFL Sport Science, Cambridgeshire, UK B. P. Gray, HFL Sport Science, Cambridgeshire, UK P. R. Brown, HFL Sport Science, Cambridgeshire, UK S. Hudson, HFL Sport Science, Cambridgeshire, UK C. M. Pearce, HFL Sport Science, Cambridgeshire, UK S. W. Paine, British Horseracing Authority, London, UK L. Hillyer, British Horseracing Authority, London, UK Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung:    High-performance liquid chromatography (HPLC), over-pressured-layer chromatography (OPLC) and thin-layer chromatography (TLC) techniques with micellar mobile phases were proposed to evaluate the lipophilicity of 21 newly synthesized 1,2,4-triazoles, compounds of potential importance in medicine or agriculture as fungicides. Micellar parameters log  k m were compared with extrapolated R M 0 values determined from reversed-phase (RP) TLC experimental data obtained on RP-8 stationary phases as well as with log  P values (Alog  Ps , AClog  P , Alog  P , Mlog  P , KowWin, xlog  P 2 and xlog  P 3) calculated from molecular structures of solutes tested. The results obtained by applying principal component analysis (PCA) and linear regression showed considerable similarity between partition and retention parameters as alternative lipophilicity descriptors, and indicated micellar chromatography as a suitable technique to study lipophilic properties of organic substances. In micellar HPLC, RP-8e column (Purospher) was applied, whereas in OPLC and TLC, RP-CN plates were applied, which was the novelty of this study and allowed the use of micellar effluents in planar chromatography measurements. Content Type Journal Article Category Original Pages 1-8 DOI 10.1007/s10337-012-2227-3 Authors Małgorzata Janicka, Department of Physical Chemistry, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland Katarzyna Stępnik, Department of Physical Chemistry, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland Anna Pachuta-Stec, Department of Organic Chemistry, Faculty of Pharmacy, Medical University, 6 Staszica St, 20-081 Lublin, Poland Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893

Beschreibung: The leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of autosomal-dominant Parkinson disease (PD). Mitochondrial dysfunction represents a critical event in the pathogenesis of PD. We demonstrated that wild-type (WT) LRRK2 expression caused mitochondrial fragmentation along with increased mitochondrial dynamin-like protein (DLP1, also known as DRP1), a fission protein, which was further exacerbated by expression of PD-associated mutants (R1441C or G2019S) in both SH-SY5Y and differentiated primary cortical neurons. We also found that LRRK2 interacted with DLP1, and LRRK2–DLP1 interaction was enhanced by PD-associated mutations that probably results in increased mitochondrial DLP1 levels. Co-expression of dominant-negative DLP1 K38A or WT Mfn2 blocked LRRK2-induced mitochondrial fragmentation, mitochondrial dysfunction and neuronal toxicity. Importantly, mitochondrial fragmentation and dysfunction were not observed in cells expressing either GTP-binding deficient mutant LRRK2 K1347A or kinase-dead mutant D1994A which has minimal interaction with DLP1 and did not increase the mitochondrial DLP1 level. We concluded that LRRK2 regulates mitochondrial dynamics by increasing mitochondrial DLP1 through its direct interaction with DLP1, and LRRK2 kinase activity plays a critical role in this process.

Beschreibung: FANCM is the most highly conserved protein within the Fanconi anaemia (FA) tumour suppressor pathway. However, although FANCM contains a helicase domain with translocase activity, this is not required for its role in activating the FA pathway. Instead, we show here that FANCM translocaseactivity is essential for promoting replication fork stability. We demonstrate that cells expressing translocase-defective FANCM show altered global replication dynamics due to increased accumulation of stalled forks that subsequently degenerate into DNA double-strand breaks, leading to ATM activation, CTBP-interacting protein (CTIP)-dependent end resection and homologous recombination repair. Accordingly, abrogation of ATM or CTIP function in FANCM-deficient cells results in decreased cell survival. We also found that FANCM translocase activity protects cells from accumulating 53BP1-OPT domains, which mark lesions resulting from problems arising during replication. Taken together, these data show that FANCM plays an essential role in maintaining chromosomal integrity by promoting the recovery of stalled replication forks and hence preventing tumourigenesis.

Beschreibung: There are numerous genes for which loss-of-function mutations do not produce apparent phenotypes even though statistically significant quantitative changes to biological pathways are observed. To evaluate the biological meaning of small effects is challenging. Bardet–Biedl syndrome (BBS) is a heterogeneous autosomal recessive disorder characterized by obesity, retinopathy, polydactyly, renal malformations, learning disabilities and hypogenitalism, as well as secondary phenotypes including diabetes and hypertension. BBS knockout mice recapitulate most human phenotypes including obesity, retinal degeneration and male infertility. However, BBS knockout mice do not develop polydacyly. Here we showed that the loss of BBS genes in mice result in accumulation of Smoothened and Patched 1 in cilia and have a decreased Shh response. Knockout of Bbs7 combined with a hypomorphic Ift88 allele (orpk as a model for Shh dysfuction) results in embryonic lethality with e12.5 embryos having exencephaly, pericardial edema, cleft palate and abnormal limb development, phenotypes not observed in Bbs7 –/– mice . Our results indicate that BBS genes modulate Shh pathway activity and interact genetically with the intraflagellar transport (IFT) pathway to play a role in mammalian development. This study illustrates an effective approach to appreciate the biological significance of a small effect.

Beschreibung: A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca 2+ ]c); elevated [Ca 2+ ]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca 2+ ]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca 2+ ]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca 2+ insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca 2+ homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.

Beschreibung: Recombination plays a fundamental role in meiosis. Non-exchange gene conversion (non-crossover, NCO) may facilitate homologue pairing, while reciprocal crossover (CO) physically connects homologues so they orientate appropriately on the meiotic spindle. In males, X–Y homologous pairing and exchange occurs within the two pseudoautosomal regions (PARs) together comprising 〈5% of the human sex chromosomes. Successful meiosis depends on an obligatory CO within PAR1, while the nature and role of exchange within PAR2 is unclear. Here, we describe the identification and characterization of a typical ~1 kb wide recombination hotspot within PAR2. We find that both COs and NCOs are strongly modulated in trans by the presumed chromatin remodelling protein PRDM9, and in cis by a single nucleotide polymorphism (SNP) located at the hotspot centre that appears to influence recombination initiation and which causes biased gene conversion in SNP heterozygotes. This, the largest survey to date of human NCOs reveals for the first time substantial inter-individual variation in the NCO:CO ratio. Although the extent of biased transmission at the central marker in COs is similar across men, it is highly variable among NCO recombinants. This suggests that cis -effects are mediated not only through recombination initiation frequencies varying between haplotypes but also through subsequent processing, with the potential to significantly intensify meiotic drive of hotspot-suppressing alleles. The NCO:CO ratio and extent of transmission distortion among NCOs appear to be inter-related, suggesting the existence of two NCO pathways in humans.

Beschreibung: Recent genome-wide association studies (GWAS) identified a number of prostate cancer (PC) susceptibility loci, but most of their functional significances are not elucidated. Through our previous GWAS for PC in a Japanese population and subsequent resequencing and fine mapping, we here identified that IRX4 (Iroquois homeobox 4) , coding Iroquois homeobox 4, is a causative gene of the PC susceptibility locus (rs12653946) at chromosome 5p15. IRX4 is expressed specifically in the prostate and heart, and quantitative expression analysis revealed a significant association between the genotype of rs12653946 and IRX4 expression in normal prostate tissues. Knockdown of IRX4 in PC cells enhanced their growth and IRX4 overexpression in PC cells suppressed their growth, indicating the functional association of IRX4 with PC and its tumor suppressive effect. Immunoprecipitation confirmed its protein–protein interaction to vitamin D receptor (VDR), and we found a significant interaction between IRX4 and VDR in their reciprocal transcriptional regulation. These findings indicate that the PC-susceptibility locus represented by rs12653946 at 5p15 is likely to regulate IRX4 expression in prostate which could suppress PC growth by interacting with the VDR pathway, conferring to PC susceptibility.

Beschreibung: Recent genome-wide association studies (GWAS) have identified a number of novel genetic associations with complex human diseases. In spite of these successes, results from GWAS generally explain only a small proportion of disease heritability, an observation termed the ‘missing heritability problem’. Several sources for the missing heritability have been proposed, including the contribution of many common variants with small individual effect sizes, which cannot be reliably found using the standard GWAS approach. The goal of our study was to explore a complimentary approach, which combines GWAS results with functional data in order to identify novel genetic associations with small effect sizes. To do so, we conducted a GWAS for lymphocyte count, a physiologic quantitative trait associated with asthma, in 462 Hutterites. In parallel, we performed a genome-wide gene expression study in lymphoblastoid cell lines from 96 Hutterites. We found significant support for genetic associations using the GWAS data when we considered variants near the 193 genes whose expression levels across individuals were most correlated with lymphocyte counts. Interestingly, these variants are also enriched with signatures of an association with asthma susceptibility, an observation we were able to replicate. The associated loci include genes previously implicated in asthma susceptibility as well as novel candidate genes enriched for functions related to T cell receptor signaling and adenosine triphosphate synthesis. Our results, therefore, establish a new set of asthma susceptibility candidate genes. More generally, our observations support the notion that many loci of small effects influence variation in lymphocyte count and asthma susceptibility.

Beschreibung: A variety of conditions lead to anemia, which affects one-quarter of the world's population. Previous genome-wide association studies revealed a number of genetic polymorphisms significantly associated with plasma iron status. To evaluate the association of genetic variants in genes involved in iron delivery and hepcidin regulation pathways with the risk of iron-deficiency anemia (IDA), the following single nucleotide polymorphisms were genotyped in 2139 unrelated elderly Chinese women: rs3811647 ( TF ), rs7385804 ( TFR2 ), rs235756 ( BMP2 ), and rs855791(V736A) and rs4820268 ( TMPRSS6, encoding matriptase-2). We identified common variants in TMPRSS6 as being genetic risk factors for both iron deficiency (OR rs855791 = 1.55, P = 4.96 x 10 –8 ) and IDA (OR rs855791 = 1.78, P = 8.43 x 10 –9 ). TMPRSS6 polymorphisms were also associated with lower serum iron (SI) and hemoglobin levels, consistent with their associations to increased iron deficiency and anemia risk. Variants rs3811647 in TF and rs7385804 in TFR2 were associated with reduced SI, serum transferrin and transferrin saturation levels; however, these variants were not associated with iron deficiency or anemia risk. Our findings suggest that TF , TFR2 and TMPRSS6 polymorphisms are significantly associated with decreased iron status, but only variants in TMPRSS6 are genetic risk factors for iron deficiency and IDA.

Beschreibung: Pluripotent stem cells are derived from culture of early embryos or the germline and can be induced by reprogramming of somatic cells. Barriers to reprogramming that stabilize the differentiated state and have tumor suppression functions are expected to exist. However, we have a limited understanding of what such barriers might be. To find novel barriers to reprogramming to pluripotency, we compared the transcriptional profiles of the mouse germline with pluripotent and somatic cells, in vivo and in vitro . There is a remarkable global expression of the transcriptional program for pluripotency in primordial germ cells (PGCs). We identify parallels between PGC reprogramming to pluripotency and human germ cell tumorigenesis, including the loss of LATS2, a tumor suppressor kinase of the Hippo pathway. We show that knockdown of LATS2 increases the efficiency of induction of pluripotency in human cells. LATS2 RNAi, unlike p53 RNAi, specifically enhances the generation of fully reprogrammed iPS cells without accelerating cell proliferation. We further show that LATS2 represses reprogramming in human cells by post-transcriptionally antagonizing TAZ but not YAP, two downstream effectors of the Hippo pathway. These results reveal transcriptional parallels between germ cell transformation and the generation of iPS cells and indicate that the Hippo pathway constitutes a barrier to cellular reprogramming.

Beschreibung:    Several months ago, a paper by Komsta describing a method of baseline removal based on quantile regression for chromatographic datasets was published in this journal entitled “Comparison of Several Methods of Chromatographic Baseline Removal with a New Approach Based on Quantile Regression”. By comparing baseline removal methods based on polynomial, spline, loess and Whittaker smoother with the newly introduced method, Komsta concluded that the main advantage of this baseline removal method is visible better performance and short computational time. Unfortunately several wrong conclusions about the baseline removal methods based on Whittaker smoother contained in this paper. We feel that wrong conclusions about the baseline removal methods based on Whittaker smoother needs to be corrected so that it will not mislead the reader. Content Type Journal Article Category Letter to the Editor Pages 313-314 DOI 10.1007/s10337-012-2192-x Authors Zhi-Min Zhang, College of Chemistry and Chemical Engineering, Research Center of Modernization of Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Yi-Zeng Liang, College of Chemistry and Chemical Engineering, Research Center of Modernization of Chinese Medicines, Central South University, Changsha, 410083 People’s Republic of China Journal Chromatographia Online ISSN 1612-1112 Print ISSN 0009-5893 Journal Volume Volume 75 Journal Issue Volume 75, Numbers 5-6

Beschreibung: Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase ( DHODH )], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genée–Weidemann or postaxial acrofacial dysostosis) syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes ( CAD , UMPS ), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh , Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

Beschreibung: Restless legs syndrome (RLS), also known as Willis–Ekbom disease, is a sensory–motor neurological disorder with a circadian component. RLS is characterized by uncomfortable sensations in the extremities, generally at night or during sleep, which often leads to an uncontrollable urge to move them for relief. Recently, genomic studies identified single-nucleotide polymorphisms in BTBD9 , along with three other genes, as being associated with a higher risk of RLS. Little is known about the function of BTBD9 or its potential role in the pathophysiology of RLS. We therefore examined a line of Btbd9 mutant mice we recently generated for phenotypes similar to symptoms found in RLS patients. We observed that the Btbd9 mutant mice had motor restlessness, sensory alterations likely limited to the rest phase, and decreased sleep and increased wake times during the rest phase. Additionally, the Btbd9 mutant mice had altered serum iron levels and monoamine neurotransmitter systems. Furthermore, the sensory alterations in the Btbd9 mutant mice were relieved using ropinirole, a dopaminergic agonist widely used for RLS treatment. These results, taken together, suggest that the Btbd9 mutant mice model several characteristics similar to RLS and would therefore be the first genotypic mouse model of RLS. Furthermore, our data provide further evidence that BTBD9 is involved in RLS, and future studies of the Btbd9 mutant mice will help shine light on its role in the pathophysiology of RLS. Finally, our data argue for the utility of Btbd9 mutant mice to discover and screen novel therapeutics for RLS.

Beschreibung: Achondroplasia (ACH) and thanatophoric dysplasia (TD) are caused by gain-of-function mutations of fibroblast growth factor receptor 3 ( FGFR3 ) and they are the most common forms of dwarfism and lethal dwarfism, respectively. Currently, there are few effective treatments for ACH. For the neonatal lethality of TD patients, no practical effective therapies are available. We here showed that systemic intermittent PTH (1-34) injection can rescue the lethal phenotype of TD type II (TDII) mice and significantly alleviate the retarded skeleton development of ACH mice. PTH-treated ACH mice had longer naso-anal length than ACH control mice, and the bone lengths of humeri and tibiae were rescued to be comparable with those of wild-type control mice. Our study also found that the premature fusion of cranial synchondroses in ACH mice was partially corrected after the PTH (1-34) treatment, suggesting that the PTH treatment may rescue the progressive narrowing of neurocentral synchondroses that cannot be readily corrected by surgery. In addition, we found that the PTH treatment can improve the osteopenia and bone structure of ACH mice. The increased expression of PTHrP and down-regulated FGFR3 level may be responsible for the positive effects of PTH on bone phenotype of ACH and TDII mice.

Beschreibung: Patients affected by bipolar disorder (BD) frequently report abnormalities in sleep/wake cycles. In addition, they showed abnormal oscillating melatonin secretion, a key regulator of circadian rhythms and sleep patterns. The acetylserotonin O-methyltransferase (ASMT) is a key enzyme of the melatonin biosynthesis and has recently been associated with psychiatric disorders such as autism spectrum disorders and depression. In this paper, we analysed rare and common variants of ASMT in patients with BD and unaffected control subjects and performed functional analysis of these variants by assaying the ASMT activity in their B-lymphoblastoid cell lines. We sequenced the coding and the regulatory regions of the gene in a discovery sample of 345 patients with BD and 220 controls. We performed an association study on this discovery sample using common variants located in the promoter region and showed that rs4446909 was significantly associated with BD ( P = 0.01) and associated with a lower mRNA level ( P 〈 10 –4 ) and a lower enzymatic activity ( P 〈 0.05) of ASMT. A replication study and a meta-analysis using 480 independent patients with BD and 672 controls confirmed the significant association between rs4446909 and BD ( P = 0.002). These results correlate with the general lower ASMT enzymatic activity observed in patients with BD ( P = 0.001) compared with controls. Finally, several deleterious ASMT mutations identified in patients were associated with low ASMT activity ( P = 0.01). In this study, we determined how rare and common variations in ASMT might play a role in BD vulnerability and suggest a general role of melatonin as susceptibility factor for BD.

Beschreibung: Pantothenate kinase-associated neurodegeneration (PKAN) is a neurodegenerative disease belonging to the group of neurodegeneration with brain iron accumulation disorders. It is characterized by progressive impairments in movement, speech and cognition. The disease is inherited in a recessive manner due to mutations in the Pantothenate Kinase-2 ( PANK2 ) gene that encodes a mitochondrial protein involved in Coenzyme A synthesis. To investigate the link between a PANK2 gene defect and iron accumulation, we analyzed primary skin fibroblasts from three PKAN patients and three unaffected subjects. The oxidative status of the cells and their ability to respond to iron were analyzed in both basal and iron supplementation conditions. In basal conditions, PKAN fibroblasts show an increase in carbonylated proteins and altered expression of antioxidant enzymes with respect to the controls. After iron supplementation, the PKAN fibroblasts had a defective response to the additional iron. Under these conditions, ferritins were up-regulated and Transferrin Receptor 1 (TfR1) was down-regulated to a minor extent in patients compared with the controls. Analysis of iron regulatory proteins (IRPs) reveals that, with respect to the controls, PKAN fibroblasts have a reduced amount of membrane-associated mRNA-bound IRP1, which responds imperfectly to iron. This accounts for the defective expression of ferritin and TfR1 in patients' cells. The inaccurate quantity of these proteins produced a higher bioactive labile iron pool and consequently increased iron-dependent reactive oxygen species formation. Our results suggest that Pank2 deficiency promotes an increased oxidative status that is further enhanced by the addition of iron, potentially causing damage in cells.