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Sample records for chromatography mutation analysis

  1. Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

    DEFF Research Database (Denmark)

    Bathum, Lise; Münster, Anna-Marie; Nybo, Mads

    2008-01-01

    diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense......BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise......, giving a precise diagnosis and subsequently a better risk estimation....

  2. Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

    Directory of Open Access Journals (Sweden)

    Pei-Chin Lin

    2014-04-01

    Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

  3. Discordant diagnoses obtained by different approaches in antithrombin mutation analysis

    DEFF Research Database (Denmark)

    Feddersen, Søren; Nybo, Mads

    2014-01-01

    OBJECTIVES: In hereditary antithrombin (AT) deficiency it is important to determine the underlying mutation since the future risk of thromboembolism varies considerably between mutations. DNA investigations are in general thought of as flawless and irrevocable, but the diagnostic approach can...... be critical. We therefore investigated mutation results in the AT gene, SERPINC1, with two different approaches. DESIGN AND METHODS: Sixteen patients referred to the Centre for Thrombosis and Haemostasis, Odense University Hospital, with biochemical indications of AT deficiency, but with a negative denaturing...... high-performance liquid chromatography (DHPLC) mutation screening (routine approach until recently) were included. As an alternative mutation analysis, direct sequencing of all exons and exon-intron boundaries without pre-selection by DHPLC was performed. RESULTS: Out of sixteen patients...

  4. Ion Chromatography Applications in Wastewater Analysis

    Directory of Open Access Journals (Sweden)

    Rajmund Michalski

    2018-02-01

    Full Text Available Wastewater analysis is an important area in analytical and environmental chemistry. It can be performed with both the classic wet methods and instrumental techniques. The development of new methods, and modification of the existing ones, constitute a major task for researchers. Ion chromatography plays a predominant role in ion determinations with the instrumental methods. It offers several advantages over the conventional methods, such as simultaneous determinations of alkali and alkaline earth cations and ammonia. Ammonium ions cannot be determined by spectroscopic methods. Ion chromatography has been accepted world-wide as a reference method for analyzing anions and cations in water and wastewater due to the fact that it enables the replacement of several individual wet chemistry methods for common ions with one instrumental technique. The following article describes the principles of ion chromatography, such as stationary phases, eluents, detectors, and sample preparation methods. Moreover, the applications of ion chromatography in wastewater analyses and international standards are presented.

  5. Chromatography.

    Science.gov (United States)

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  6. Quantitative analysis of deuterium by gas chromatography

    International Nuclear Information System (INIS)

    Isomura, Shohei; Kaetsu, Hayato

    1981-01-01

    An analytical method for the determination of deuterium concentration in water and hydrogen gas by gas chromatography is described. HD and D 2 in a hydrogen gas sample were separated from H 2 by a column packed with Molecular Sieve 13X, using extra pure hydrogen gas as carrier. A thermal conductivity detector was used. Concentrations of deuterium were determined by comparison with standard samples. The error inherent to the present method was less a 1% on the basis of the calibration curves obtained with the standard samples. The average time required for the analysis was about 3 minutes. (author)

  7. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff J

    2003-12-01

    Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

  9. Chromium Speciation Analysis by Ion Chromatography Coupled ...

    African Journals Online (AJOL)

    Two methods coupling ion chromatography with inductively coupled plasma - optical emission spectroscopy (ICP-OES) were developed for the simultaneous separation and determination of Cr(III) and Cr(VI) species. In the first method, anion chromatography with sodium bicarbonate/carbonate solution as the eluent was ...

  10. Analysis of pollutants in air and water using gas chromatography and headspace gas chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Stenner, H.

    1980-01-01

    The combination 'personal sampling' with headspace gas chromatography to determine traces of formaldehyde, phenol and benzene in air is investigated in this work, with the aim of developing maximum workplace concentration values (MWL values). Further possible applications of gas chromatography in trace analysis in the environmentally protected area. The analysis of chromium in waste waters (Cr III as acetyl acetonate complex) is investigated as further possible application, whereby optimum conditions are obtained. A modified flame ionization detector was used to increase the detection sensitivity.

  11. Gas Chromatography-Mass Spectrometric Analysis and Insecticidal ...

    African Journals Online (AJOL)

    HP

    Original Research Article. Gas Chromatography-Mass Spectrometric Analysis and ... into a natural fumigant/insecticide for the control of stored product insects. Keywords: Mallotus ..... stability as well as reduce cost. ACKNOWLEDGEMENT.

  12. Gas Chromatography-Mass Spectrometric Analysis of Nematicidal ...

    African Journals Online (AJOL)

    Gas Chromatography-Mass Spectrometric Analysis of Nematicidal Essential Oil of Valeriana ... Tropical Journal of Pharmaceutical Research ... have a potential to be developed to natural nematicides for the control of cereal cyst nematodes.

  13. Ultra-high Performance Liquid Chromatography in Steroid Analysis

    OpenAIRE

    Salonen, Fanny

    2017-01-01

    The latest version of liquid chromatography is ultra-high performance (or pressure) chromatography (UHPLC). In the technique, short and narrow-bore columns with particle sizes below 3 µm are used. The extremely high pressure used results in very short analysis times, excellent separation, and good resolution. This makes UHPLC a good choice for steroidal analysis. Steroids are a highly interesting area of study; they can be recognized as biomarkers for several diseases and are a relevant topic...

  14. Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

    Science.gov (United States)

    Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

    2017-02-01

    Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

  15. Liquid chromatography - mass spectrometry analysis of pharmaceuticals

    International Nuclear Information System (INIS)

    Macasek, F.

    2003-01-01

    The drugs represent mostly non-volatile and thermally labile solutes, often available only in small amounts like it is in case of radiopharmaceuticals. Therefor, the favourable separation techniques for such compounds are HPLC, capillary electrophoresis and also TLC 1. Liquid chromatography with mass spectrometric detector (LC/MS) is especially powerful for their microanalysis. Mass spectrometry separating the ions in high vacuum was presumably used as detector for gas chromatography effluent but the on-line coupling with liquid eluant flow 0.1-1 mL/min is far more challenging. New types of ion sources were constructed for simultaneous removal of solvent and ionisation of solutes at atmospheric pressure (API). At present, a relatively wide choice of successfully designed commercial equipment is available either for small organic molecules and larger biomolecules (Perkin-Elmer, Agilent, Jeol, Bruker Daltonics, ThermoQuest, Shimadzu). The features of the LC/MS systems are presented. LC/MS as a new quality control tool for [F-18]fluorodeoxyglucose (FDG) radiopharmaceutical, which has became the most spread radiopharmaceutical for positron emission tomography (PET), was proposed. Other applications of the LC/MS are reviewed. (author)

  16. Supercritical fluid chromatography for lipid analysis in foodstuffs.

    Science.gov (United States)

    Donato, Paola; Inferrera, Veronica; Sciarrone, Danilo; Mondello, Luigi

    2017-01-01

    The task of lipid analysis has always challenged separation scientists, and new techniques in chromatography were often developed for the separation of lipids; however, no single technique or methodology is yet capable of affording a comprehensive screening of all lipid species and classes. This review acquaints the role of supercritical fluid chromatography within the field of lipid analysis, from the early developed capillary separations based on pure CO 2 , to the most recent techniques employing packed columns under subcritical conditions, including the niche multidimensional techniques using supercritical fluids in at least one of the separation dimensions. A short history of supercritical fluid chromatography will be introduced first, from its early popularity in the late 1980s, to the sudden fall and oblivion until the last decade, experiencing a regain of interest within the chromatographic community. Afterwards, the subject of lipid nomenclature and classification will be briefly dealt with, before discussing the main applications of supercritical fluid chromatography for food analysis, according to the specific class of lipids. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Phenylketonuria mutation analysis in Northern Ireland: A rapid stepwise approach

    Energy Technology Data Exchange (ETDEWEB)

    Zschocke, J.; Graham, C.A.; Nevin, N.C. [Queen`s Univ., Belfast (Australia)] [and others

    1995-12-01

    We present a multistep approach for the rapid analysis of phenylketonuria (PKU) mutations. In the first step, three common mutations and a polymorphic short tandem repeat (STR) system are rapidly analyzed with a fluorescent multiplex assay. In the second step, minihaplotypes combining STR and VNTR data are used to determine rare mutations likely to be present in an investigated patient, which are then confirmed by restriction enzyme analysis. The remaining mutations are analyzed with denaturant gradient-gel electrophoresis and sequencing. The first two steps together identify both mutations in 90%-95% of PKU patients, and results can be obtained within 2 d. We have investigated 121 Northern Irish families with hyperphenylalaninemia, including virtually all patients born since 1972, and have found 34 different mutations on 241 of the 242 mutant alleles. Three mutations (R408W, 165T, and F39L) account for 57.5% of mutations, while 14 mutations occur with a frequency of 1%-6%. The present analysis system is efficient and inexpensive and is particularly well suited to routine mutation analysis in a diagnostic setting. 19 refs., 5 tabs.

  18. Analysis of time of death of prenatally lethal Steeloid mutations

    International Nuclear Information System (INIS)

    Rinchik, E.M.; Cummings, C.C.; Bangham, J.W.; Hunsicker, P.R.; Phipps, E.L.; Stelzner, K.F.

    1987-01-01

    Deletion mutations have been extremely useful in initiating the functional and molecular dissections of regions of the mouse genome. For the d-se and c regions, for example, it was observed that radiation mutations carrying lethal factors separable, by complementation analysis, from the primary d, se, or c mutation itself, could often be associated at both the genetic and molecular levels with multilocus chromosomal deletions. Since many of the Oak Ridge Sld mutations arose in radiation mutagenesis experiments, a substantial number may carry chromosomal deletions that involve the Sl locus in chromosome 10. Because of the great value of deletion mutations for the genetic and molecular analysis of chromosomal regions and complex genetic loci, they have initiated a series of experiments designed to test whether radiation-induced Sld mutations carry other lethal factors, in addition to the lethality caused by severe alleles of the Sl locus itself, as one prescreen for identifying Sld's that are caused by deletions

  19. Mutational analysis of the HGO gene in Finnish alkaptonuria patients

    Science.gov (United States)

    de Bernabe, D. B.-V.; Peterson, P.; Luopajarvi, K.; Matintalo, P.; Alho, A.; Konttinen, Y.; Krohn, K.; de Cordoba, S. R.; Ranki, A.

    1999-01-01

    Alkaptonuria (AKU), the prototypic inborn error of metabolism, has recently been shown to be caused by loss of function mutations in the homogentisate-1,2-dioxygenase gene (HGO). So far 17 mutations have been characterised in AKU patients of different ethnic origin. We describe three novel mutations (R58fs, R330S, and H371R) and one common AKU mutation (M368V), detected by mutational and polymorphism analysis of the HGO gene in five Finnish AKU pedigrees. The three novel AKU mutations are most likely specific for the Finnish population and have originated recently.


Keywords: alkaptonuria; homogentisate-1,2-dioxygenase; Finland PMID:10594001

  20. Principles of Micellar Electrokinetic Capillary Chromatography Applied in Pharmaceutical Analysis

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    Árpád Gyéresi

    2013-02-01

    Full Text Available Since its introduction capillary electrophoresis has shown great potential in areas where electrophoretic techniques have rarely been used before, including here the analysis of pharmaceutical substances. The large majority of pharmaceutical substances are neutral from electrophoretic point of view, consequently separations by the classic capillary zone electrophoresis; where separation is based on the differences between the own electrophoretic mobilities of the analytes; are hard to achieve. Micellar electrokinetic capillary chromatography, a hybrid method that combines chromatographic and electrophoretic separation principles, extends the applicability of capillary electrophoretic methods to neutral analytes. In micellar electrokinetic capillary chromatography, surfactants are added to the buffer solution in concentration above their critical micellar concentrations, consequently micelles are formed; micelles that undergo electrophoretic migration like any other charged particle. The separation is based on the differential partitioning of an analyte between the two-phase system: the mobile aqueous phase and micellar pseudostationary phase. The present paper aims to summarize the basic aspects regarding separation principles and practical applications of micellar electrokinetic capillary chromatography, with particular attention to those relevant in pharmaceutical analysis.

  1. Simultaneous analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography.

    Science.gov (United States)

    Taguchi, Kaori; Fukusaki, Eiichiro; Bamba, Takeshi

    2014-10-03

    Chromatography techniques usually use a single state in the mobile phase, such as liquid, gas, or supercritical fluid. Chromatographers manage one of these techniques for their purpose but are sometimes required to use multiple methods, or even worse, multiple techniques when the target compounds have a wide range of chemical properties. To overcome this challenge, we developed a single method covering a diverse compound range by means of a "unified" chromatography which completely bridges supercritical fluid chromatography and liquid chromatography. In our method, the phase state was continuously changed in the following order; supercritical, subcritical and liquid. Moreover, the gradient of the mobile phase starting at almost 100% CO2 was replaced with 100% methanol at the end completely. As a result, this approach achieved further extension of the polarity range of the mobile phase in a single run, and successfully enabled the simultaneous analysis of fat- and water-soluble vitamins with a wide logP range of -2.11 to 10.12. Furthermore, the 17 vitamins were exceptionally separated in 4min. Our results indicated that the use of dense CO2 and the replacement of CO2 by methanol are practical approaches in unified chromatography covering diverse compounds. Additionally, this is a first report to apply the novel approach to unified chromatography, and can open another door for diverse compound analysis in a single chromatographic technique with single injection, single column and single system. Copyright © 2014. Published by Elsevier B.V.

  2. Establishment of analysis method for methane detection by gas chromatography

    Science.gov (United States)

    Liu, Xinyuan; Yang, Jie; Ye, Tianyi; Han, Zeyu

    2018-02-01

    The study focused on the establishment of analysis method for methane determination by gas chromatography. Methane was detected by hydrogen flame ionization detector, and the quantitative relationship was determined by working curve of y=2041.2x+2187 with correlation coefficient of 0.9979. The relative standard deviation of 2.60-6.33% and the recovery rate of 96.36%∼105.89% were obtained during the parallel determination of standard gas. This method was not quite suitable for biogas content analysis because methane content in biogas would be over the measurement range in this method.

  3. Mutational Analysis of Merkel Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Erstad, Derek J. [Department of Surgery, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114 (United States); Cusack, James C. Jr., E-mail: jcusack@mgh.harvard.edu [Division of Surgical Oncology, Harvard Medical School, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114 (United States)

    2014-10-17

    Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine malignancy that is associated with a poor prognosis. The pathogenesis of MCC is not well understood, and despite a recent plethora of mutational analyses, we have yet to find a set of signature mutations implicated in the majority of cases. Mutations, including TP53, Retinoblastoma and PIK3CA, have been documented in subsets of patients. Other mechanisms are also likely at play, including infection with the Merkel cell polyomavirus in a subset of patients, dysregulated immune surveillance, epigenetic alterations, aberrant protein expression, posttranslational modifications and microRNAs. In this review, we summarize what is known about MCC genetic mutations and chromosomal abnormalities, and their clinical significance. We also examine aberrant protein function and microRNA expression, and discuss the therapeutic and prognostic implications of these findings. Multiple clinical trials designed to selectively target overexpressed oncogenes in MCC are currently underway, though most are still in early phases. As we accumulate more molecular data on MCC, we will be better able to understand its pathogenic mechanisms, develop libraries of targeted therapies, and define molecular prognostic signatures to enhance our clinicopathologic knowledge.

  4. Mutational Analysis of Merkel Cell Carcinoma

    International Nuclear Information System (INIS)

    Erstad, Derek J.; Cusack, James C. Jr.

    2014-01-01

    Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine malignancy that is associated with a poor prognosis. The pathogenesis of MCC is not well understood, and despite a recent plethora of mutational analyses, we have yet to find a set of signature mutations implicated in the majority of cases. Mutations, including TP53, Retinoblastoma and PIK3CA, have been documented in subsets of patients. Other mechanisms are also likely at play, including infection with the Merkel cell polyomavirus in a subset of patients, dysregulated immune surveillance, epigenetic alterations, aberrant protein expression, posttranslational modifications and microRNAs. In this review, we summarize what is known about MCC genetic mutations and chromosomal abnormalities, and their clinical significance. We also examine aberrant protein function and microRNA expression, and discuss the therapeutic and prognostic implications of these findings. Multiple clinical trials designed to selectively target overexpressed oncogenes in MCC are currently underway, though most are still in early phases. As we accumulate more molecular data on MCC, we will be better able to understand its pathogenic mechanisms, develop libraries of targeted therapies, and define molecular prognostic signatures to enhance our clinicopathologic knowledge

  5. Group type analysis of asphalt by column liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, C.; Yang, J.; Xue, Y.; Li, Y. [Chinese Academy of Science, Taiyuan (China)

    2008-07-01

    An improved analysis method for characterization of asphalt was established. The method is based on column chromatography technique. The asphalts were separated into four groups: saturates, aromatics, resins, and asphaltenes, quantitatively. About 0.1 g of sample was required in each analysis. About 20 mL of n-heptanes was used to separate out saturates first. Then about 35 mL of n-heptanes/dichloromethane (.5, v/v) mixture was used to separate out aromatics. About 30 mL of dichloromethane/tetrahydrofuran (1/3, v/v) mixture was used to separate out resin. The quality of the separation was confirmed by infrared spectra (IR) and {sup 1}H NMR analysis. The model compounds, tetracosan for saturates, dibenz(o)anthracen for aromatics, and acetanilide for resins were used for verification. The IR and {sup 1}H NMR analysis of the prepared fractions from the column liquid chromatography were in good agreement that of pure reagents.

  6. IDH Mutation Analysis in Ewing Sarcoma Family Tumors

    Directory of Open Access Journals (Sweden)

    Ki Yong Na

    2015-05-01

    Full Text Available Background: Isocitrate dehydrogenase (IDH catalyzes the oxidative decarboxylation of isocitrate to yield α-ketoglutarate (α-KG with production of reduced nicotinamide adenine dinucleotide (NADH. Dysfunctional IDH leads to reduced production of α-KG and NADH and increased production of 2-hydroxyglutarate, an oncometabolite. This results in increased oxidative damage and stabilization of hypoxia-inducible factor α, causing cells to be prone to tumorigenesis. Methods: This study investigated IDH mutations in 61 Ewing sarcoma family tumors (ESFTs, using a pentose nucleic acid clamping method and direct sequencing. Results: We identified four cases of ESFTs harboring IDH mutations. The number of IDH1 and IDH2 mutations was equal and the subtype of IDH mutations was variable. Clinicopathologic analysis according to IDH mutation status did not reveal significant results. Conclusions: This study is the first to report IDH mutations in ESFTs. The results indicate that ESFTs can harbor IDH mutations in previously known hot-spot regions, although their incidence is rare. Further validation with a larger case-based study would establish more reliable and significant data on prevalence rate and the biological significance of IDH mutations in ESFTs.

  7. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    Directory of Open Access Journals (Sweden)

    Amie J Radenbaugh

    Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

  8. Advances in silver ion chromatography for the analysis of fatty acids and triacylglycerols-2001 to 2011.

    Science.gov (United States)

    Momchilova, Svetlana M; Nikolova-Damyanova, Boryana M

    2012-01-01

    An effort is made to critically present the achievements in silver ion chromatography during the last decade. Novelties in columns, mobile-phase compositions and detectors are described. Recent applications of silver ion chromatography in the analysis of fatty acids and triacylglycerols are presented while stressing novel analytical strategies or new objects. The tendencies in the application of the method in complementary ways with reversed-phase chromatography, chiral chromatography and, especially, mass detection are outlined.

  9. Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics.

    Science.gov (United States)

    Kahsay, Getu; Song, Huiying; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin

    2014-01-01

    This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Supercritical fluid chromatography in drug analysis: a literature survey.

    Science.gov (United States)

    Salvador, A; Jaime, M A; Becerra, G; Guardia, M de L

    1996-08-01

    The applications of supercritical fluid chromatography to the analysis of drugs have been carefully revised from the literature compiled in the Analytical Abstracts until March 1994. Easy-to-read tables provide useful information about the state-of-the-art and possibilities offered by SFC in pharmaceutical analysis. The tables comprise extensive data about samples analyzed, pharmaceutical principles determined, solvents used and sample quantity injected, supercritical fluids and modifiers employed, injection system, instrumentation, experimental conditions for chromatographic separations (density, pressure, flow, temperature), characteristics of columns employed (type, support, length, diameter, particle film thickness, stationary phase), detectors, type of restrictors, and also some analytical features of the methods developed (such as retention time, resolution, sensitivity, limit of detection and relative standard deviation).

  11. Mutational analysis and clinical correlation of metastatic colorectal cancer.

    Science.gov (United States)

    Russo, Andrea L; Borger, Darrell R; Szymonifka, Jackie; Ryan, David P; Wo, Jennifer Y; Blaszkowsky, Lawrence S; Kwak, Eunice L; Allen, Jill N; Wadlow, Raymond C; Zhu, Andrew X; Murphy, Janet E; Faris, Jason E; Dias-Santagata, Dora; Haigis, Kevin M; Ellisen, Leif W; Iafrate, Anthony J; Hong, Theodore S

    2014-05-15

    Early identification of mutations may guide patients with metastatic colorectal cancer toward targeted therapies that may be life prolonging. The authors assessed tumor genotype correlations with clinical characteristics to determine whether mutational profiling can account for clinical similarities, differences, and outcomes. Under Institutional Review Board approval, 222 patients with metastatic colon adenocarcinoma (n = 158) and rectal adenocarcinoma (n = 64) who underwent clinical tumor genotyping were reviewed. Multiplexed tumor genotyping screened for >150 mutations across 15 commonly mutated cancer genes. The chi-square test was used to assess genotype frequency by tumor site and additional clinical characteristics. Cox multivariate analysis was used to assess the impact of genotype on overall survival. Broad-based tumor genotyping revealed clinical and anatomic differences that could be linked to gene mutations. NRAS mutations were associated with rectal cancer versus colon cancer (12.5% vs 0.6%; P colon cancer (13% vs 3%; P = .024) and older age (15.8% vs 4.6%; P = .006). TP53 mutations were associated with rectal cancer (30% vs 18%; P = .048), younger age (14% vs 28.7%; P = .007), and men (26.4% vs 14%; P = .03). Lung metastases were associated with PIK3CA mutations (23% vs 8.7%; P = .004). Only mutations in BRAF were independently associated with decreased overall survival (hazard ratio, 2.4; 95% confidence interval, 1.09-5.27; P = .029). The current study suggests that underlying molecular profiles can differ between colon and rectal cancers. Further investigation is warranted to assess whether the differences identified are important in determining the optimal treatment course for these patients. © 2014 American Cancer Society.

  12. [FANCA gene mutation analysis in Fanconi anemia patients].

    Science.gov (United States)

    Chen, Fei; Peng, Guang-Jie; Zhang, Kejian; Hu, Qun; Zhang, Liu-Qing; Liu, Ai-Guo

    2005-10-01

    To screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients. FANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing. FANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene. No functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

  13. A Novel Missense Mutation of Doublecortin: Mutation Analysis of Korean Patients with Subcortical Band Heterotopia

    Science.gov (United States)

    Kim, Myeong-Kyu; Park, Man-Seok; Kim, Byeong-Chae; Cho, Ki-Hyun; Kim, Young-Seon; Kim, Jin-Hee; Heo, Tag; Kim, Eun-Young

    2005-01-01

    The neuronal migration disorders, X-linked lissencephaly syndrome (XLIS) and subcortical band heterotopia (SBH), also called "double cortex", have been linked to missense, nonsense, aberrant splicing, deletion, and insertion mutations in doublecortin (DCX) in families and sporadic cases. Most DCX mutations identified to date are located in two evolutionarily conserved domains. We performed mutation analysis of DCX in two Korean patients with SBH. The SBH patients had mild to moderate developmental delays, drug-resistant generalized seizures, and diffuse thick SBH upon brain MRI. Sequence analysis of the DCX coding region in Patient 1 revealed a c.386 C>T change in exon 3. The sequence variation results in a serine to leucine amino acid change at position 129 (S129L), which has not been found in other family members of Patient 1 or in a large panel of 120 control X-chromosomes. We report here a novel c.386 C>T mutation of DCX that is responsible for SBH. PMID:16100463

  14. KIT mutation analysis in mast cell neoplasms

    DEFF Research Database (Denmark)

    Arock, M; Sotlar, K; Akin, C

    2015-01-01

    mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V...... can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should...... greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals....

  15. Analysis of sea water by difference chromatography. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Mangelsdorf, P.C. Jr.

    1977-02-01

    During the final period of this research contract the principal results obtained were: (a) the development of anion analysis by difference chromatography to the extent that SO/sub 4//sup =//Cl/sup -/ can be determined to better than 0.1 percent using an 0.5 ml seawater sample, (b) the determination of the ion-exchange cation complements of a variety of sediments in river water and in seawater, and (c) the discovery of a simple technique for the qualitative removal of NH/sub 4//sup +/ from seawater samples without altering the ratios of the other cations. This method supersedes the use of Cu-Chelex which has proved impossible to sustain.

  16. Mutational Analysis of the Rhodopsin Gene in Sector Retinitis Pigmentosa.

    Science.gov (United States)

    Napier, Maria L; Durga, Dash; Wolsley, Clive J; Chamney, Sarah; Alexander, Sharon; Brennan, Rosie; Simpson, David A; Silvestri, Giuliana; Willoughby, Colin E

    2015-01-01

    To determine the role of rhodopsin (RHO) gene mutations in patients with sector retinitis pigmentosa (RP) from Northern Ireland. A case series of sector RP in a tertiary ocular genetics clinic. Four patients with sector RP were recruited from the Royal Victoria Hospital (Belfast, Northern Ireland) and Altnagelvin Hospital (Londonderry, Northern Ireland) following informed consent. The diagnosis of sector RP was based on clinical examination, International Society for Clinical Electrophysiology of Vision (ISCEV) standard electrophysiology, and visual field analysis. DNA was extracted from peripheral blood leucocytes and the coding regions and adjacent flanking intronic sequences of the RHO gene were polymerase chain reaction (PCR) amplified and cycle sequenced. Rhodopsin mutational status. A heterozygous missense mutation in RHO (c.173C > T) resulting in a non-conservative substitution of threonine to methionine (p. Thr58Met) was identified in one patient and was absent from 360 control individuals. This non-conservative substitution (p.Thr58Met) replaces a highly evolutionary conserved polar hydrophilic threonine residue with a non-polar hydrophobic methionine residue at position 58 near the cytoplasmic border of helix A of RHO. The study identified a RHO gene mutation (p.Thr58Met) not previously reported in RP in a patient with sector RP. These findings outline the phenotypic variability associated with RHO mutations. It has been proposed that the regional effects of RHO mutations are likely to result from interplay between mutant alleles and other genetic, epigenetic and environmental factors.

  17. GPR143 gene mutation analysis in pediatric patients with albinism.

    Science.gov (United States)

    Trebušak Podkrajšek, Katarina; Stirn Kranjc, Branka; Hovnik, Tinka; Kovač, Jernej; Battelino, Tadej

    2012-09-01

    X-linked ocular albinism type 1 is difficult to differentiate clinically from other forms of albinism in young patients. X-linked ocular albinism type 1 is caused by mutations in the GPR143 gene, encoding melanosome specific G-protein coupled receptor. Patients typically present with moderately to severely reduced visual acuity, nystagmus, strabismus, photophobia, iris translucency, hypopigmentation of the retina, foveal hypoplasia and misrouting of optic nerve fibers at the chiasm. Following clinical ophthalmological evaluation, GPR143 gene mutational analyses were performed in a cohort of 15 pediatric male patients with clinical signs of albinism. Three different mutations in the GPR143 gene were identified in four patients, including a novel c.886G>A (p.Gly296Arg) mutation occurring "de novo" and a novel intronic c.360 + 5G>A mutation, identified in two related boys. Four patients with X-linked ocular albinism type 1 were identified from a cohort of 15 boys with clinical signs of albinism using mutation detection methods. Genetic analysis offers the possibility of early definitive diagnosis of ocular albinism type 1 in a significant portion of boys with clinical signs of albinism.

  18. [High-performance liquid-liquid chromatography in beverage analysis].

    Science.gov (United States)

    Bricout, J; Koziet, Y; de Carpentrie, B

    1978-01-01

    Liquid liquid chromatography was performed with columns packed with stationary phases chemically bonded to silica microparticules. These columns show a high efficiency and are used very easily. Flavouring compounds like aromatic aldehydes which have a low volatility were analyzed in brandy using a polar phase alkylnitrile. Sapid substances like amarogentin in Gentiana lutea or glyryrrhizin in Glycyrrhiza glabra were determined by reversed phase chromatography. Finally ionizable substances like synthetic dyes can be analyzed by paired ion chromatography witha non polar stationary phase.

  19. Lipidomic analysis of biological samples: Comparison of liquid chromatography, supercritical fluid chromatography and direct infusion mass spectrometry methods.

    Science.gov (United States)

    Lísa, Miroslav; Cífková, Eva; Khalikova, Maria; Ovčačíková, Magdaléna; Holčapek, Michal

    2017-11-24

    Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography - mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements

  20. Molecular analysis of radiation-induced mutations in vitro

    International Nuclear Information System (INIS)

    Kronenberg, A.

    1996-01-01

    This review will focus on the nature of specific locus mutations detected in mammalian cells exposed in vitro to different types of ionizing radiations. Ionizing radiation has been shown to produce a wide variety of heritable alterations in DNA. These range from single base pair substitutions to stable loss or translocation of large portions of whole chromosomes. Data will be reviewed for certain test systems that reveal different mutation spectra. Techniques for the analysis of molecular alterations include applications of the polymerase chain reaction, some of which may be coupled with DNA sequence analysis, and a variety of hybridization-based techniques. The complexity of large scale rearrangements is approached with cytogenetic techniques including high resolution banding and various applications of the fluorescence in situ hybridization (FISH) technique. Radiation-induced mutant frequencies and mutation spectra are a function of the linkage constraints on the recovery of viable mutants for a given locus and test system. 44 refs

  1. Trends in the analysis of natural gas by capillary gas chromatography

    NARCIS (Netherlands)

    Cramers, C.A.M.G.; Rossum, van G.J.

    1986-01-01

    The importance of correct determination of physical and chemical properties of natural gas is evident. The calculation of calorific value or hydrocarbon dew point requires detailed analysis as can be provided by gas chromatography. Analysis by gas chromatography is a necessary complement to direct

  2. Knowledge-based analysis of functional impacts of mutations in ...

    Indian Academy of Sciences (India)

    Knowledge-based analysis of functional impacts of mutations in microRNA seed regions. Supplementary figure 1. Summary of predicted miRNA targets from ... All naturally occurred SNPs in seed regions of human miRNAs. The information of the columns is given in the second sheet. Hihly expressed miRNAs are ...

  3. Analysis of short-chain acids from anaerobic bacteria by high-performance liquid chromatography.

    OpenAIRE

    Guerrant, G O; Lambert, M A; Moss, C W

    1982-01-01

    A standard mixture of 25 short-chain fatty acids was resolved by high-performance liquid chromatography, using an Aminex HPX-87 column. The acids produced in culture media by anaerobic bacteria were analyzed by high-performance liquid chromatography after extraction with ether and reextraction into a small volume of 0.1 N NaOH. The presence of fumaric acid in culture extracts of Peptostreptococcus anaerobius was confirmed by gas chromatography-mass spectrometry analysis of the trapped eluent ...

  4. [Mutation analysis of seven patients with Waardenburg syndrome].

    Science.gov (United States)

    Hao, Ziqi; Zhou, Yongan; Li, Pengli; Zhang, Quanbin; Li, Jiao; Wang, Pengfei; Li, Xiangshao; Feng, Yong

    2016-06-01

    To perform genetic analysis for 7 patients with Waardenburg syndrome. Potential mutation of MITF, PAX3, SOX10 and SNAI2 genes was screened by polymerase chain reaction and direct sequencing. Functions of non-synonymous polymorphisms were predicted with PolyPhen2 software. Seven mutations, including c.649-651delAGA (p.R217del), c.72delG (p.G24fs), c.185T>C (p.M62T), c.118C>T (p.Q40X), c.422T>C (p.L141P), c.640C>T (p.R214X) and c.28G>T(p.G43V), were detected in the patients. Among these, four mutations of the PAX3 gene (c.72delG, c.185T>C, c.118C>T and c.128G>T) and one SOX10 gene mutation (c.422T>C) were not reported previously. Three non-synonymous SNPs (c.185T>C, c.128G>T and c.422T>C) were predicted as harmful. Genetic mutations have been detected in all patients with Waardenburg syndrome.

  5. Analysis of APC mutation in human ameloblastoma and clinical significance.

    Science.gov (United States)

    Li, Ning; Liu, Bing; Sui, Chengguang; Jiang, Youhong

    2016-01-01

    As a highly conserved signaling pathway, Wnt/β-catenin signal transduction pathway plays an important role in many processes. Either in the occurrence or development of tumor, activation of this pathway takes an important place. APC inhibits Wnt/β-catenin pathway to regulate cell proliferation and differentiation. This study aimed to investigate the function of cancer suppressor gene. PCR amplification and sequencing method was used to analyze APC mutations of human clinical specimens. The pathological specimens were collected for PCR and clear electrophoretic bands were obtained after electrophoresis. The gene sequence obtained after purification and sequencing analysis was compared with the known APC gene sequence (NM_000038.5). Base mutations at APC 1543 (T → C), APC-4564 (G → A), APC-5353 (T → G), APC-5550 (T → A) and APC-5969 (G → A) locus existed in 22 (27.5 %), 12 (15 %), 5 (6.25 %), 13 (16.25 %) and 12 patients (15 %), respectively. Gene mutations existed in ameloblastoma, and the mutation loci were 1543 locus (T → C), 4564 locus (G → A), 5353 locus (T → G), 5550 locus (T → A) and 5969 locus (G → A) 15 %, respectively. APC mutation plays a certain role in monitoring the tumor malignant degree as it may indicate the transition process of ameloblastoma malignant phenotype.

  6. Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

    Science.gov (United States)

    Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

    2018-03-09

    The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

  7. The Use of Gas Chromatography for Biogas Analysis

    Science.gov (United States)

    Andersen, Amanda; Seeley, John; Aurandt, Jennifer

    2010-04-01

    Energy from natural gas accounts for 24 percent of energy consumed in the US. Natural gas is a robust form of energy which is rich in methane content and is low in impurities. This quality suggests that it is a very clean and safe gas; it can be used in providing heat, a source for cooking, and in powering vehicles. The downside is that it is a non-renewable resource. On the contrary, methane rich gas that is produced by the breakdown of organic material in an anaerobic environment, called biogas, is a renewable energy source. This research focuses on the gas analysis portion of the creation of the anaerobic digestion and verification laboratory where content and forensic analysis of biogas is performed. Gas Chromatography is implemented as the optimal analytical tool for quantifying the components of the biogas including methane, carbon dioxide, hydrogen sulfide and siloxanes. In addition, the problems associated with the undesirable components are discussed. Anaerobic digestion of primary sludge has consistently produced about 55 percent methane; future goals of this research include studying different substrates to increase the methane yield and decrease levels of impurities in the gas.

  8. A mutational analysis of Caenorhabditis elegans in space

    International Nuclear Information System (INIS)

    Zhao Yang; Lai, Kenneth; Cheung, Iris; Youds, Jillian; Tarailo, Maja; Tarailo, Sanja; Rose, Ann

    2006-01-01

    The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight

  9. A mutational analysis of Caenorhabditis elegans in space

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yang [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Lai, Kenneth [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Cheung, Iris [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Youds, Jillian [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Tarailo, Maja [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Tarailo, Sanja [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Rose, Ann [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada)]. E-mail: arose@gene.nce.ubc.ca

    2006-10-10

    The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.

  10. Use and practice of achiral and chiral supercritical fluid chromatography in pharmaceutical analysis and purification.

    Science.gov (United States)

    Lemasson, Elise; Bertin, Sophie; West, Caroline

    2016-01-01

    The interest of pharmaceutical companies for complementary high-performance chromatographic tools to assess a product's purity or enhance this purity is on the rise. The high-throughput capability and economic benefits of supercritical fluid chromatography, but also the "green" aspect of CO2 as the principal solvent, render supercritical fluid chromatography very attractive for a wide range of pharmaceutical applications. The recent reintroduction of new robust instruments dedicated to supercritical fluid chromatography and the progress in stationary phase technology have also greatly benefited supercritical fluid chromatography. Additionally, it was shown several times that supercritical fluid chromatography could be orthogonal to reversed-phase high-performance liquid chromatography and could efficiently compete with it. Supercritical fluid chromatography is an adequate tool for small molecules of pharmaceutical interest: synthetic intermediates, active pharmaceutical ingredients, impurities, or degradation products. In this review, we first discuss about general chromatographic conditions for supercritical fluid chromatography analysis to better suit compounds of pharmaceutical interest. We also discuss about the use of achiral and chiral supercritical fluid chromatography for analytical purposes and the recent applications in these areas. The use of preparative supercritical fluid chromatography by pharmaceutical companies is also covered. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Risk of malignant paraganglioma in SDHB-mutation and SDHD-mutation carriers: a systematic review and meta-analysis

    NARCIS (Netherlands)

    van Hulsteijn, L.T.; Dekkers, O.M.; Hes, F.J.; Smit, J.W.A.; Corssmit, E.P.

    2012-01-01

    The main objective of this study was to perform a systematic review and meta-analysis on the risk of developing malignant paraganglioma (PGL) in SDHB-mutation and SDHD-mutation carriers. PubMed, EMBASE, Web of Science, COCHRANE and Academic Search Premier (2000-August 2011) and references of key

  12. Gas Chromatography-Mass Spectrometric Analysis and Insecticidal ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research ... apelta aerial parts was analyzed by gas chromatography/mass spectrometric (GC/MS) to determine its composition. ... into a natural fumigant/insecticide for the control of stored product insects.

  13. Nuclearization of ionic chromatography system for fission products analysis

    International Nuclear Information System (INIS)

    Dimeglio, Remi

    1996-06-01

    The accident at Tchernobyl in 1986 had entailed the release in the atmosphere of different products coming from the splitting of the fuel. It is to better understand, and also to warn this type of catastrophe that the CEA (Commissariat a L'Energie Atomique) develops many programs of researches, aiming to characterize these fission products and to study their mechanisms of relaxation. Thus, the LESC (Laboratoire d'Etude de la Surete du Combustible) takes part, since several years, in many nuclear safety experiences, and in particular to the project PHEBUS PF, that is a reconstitution, in reduced scale, of an accident entailing the fusion of the reactor core. The aim of the researches that have been led during this training period was to the nuclearization of an HPIC (High Performance Ion Chromatography) system, dedicated to the analysis of the PHEBUS PF fission products analysis. The first step was to develop HPIC lines already settled, so as to reduce the quantity of wastes. Indeed, those one are very difficult to process in a radioactive area. For this purpose, we have implanted a column cationic more effective, so as to decrease analysis times, and, by there even, the quantity of sewage generated. We have equally replaced, on lines cationic and anionic, the system of suppression of the eluent conductivity, to make it thriftier in fluid. But the radioactive products characterization necessitates that all analyses are led within a special box with gloves. The second step of the project was therefore to adapt the system to this type of cell, and to its automation. It has been necessary to modify the system of sample injection, the system of detection, and to put in place a supplementary box with gloves, connected by sieve to the first, for the active products dilution. (author) [fr

  14. Ion Chromatography-on-a-chip for Water Quality Analysis

    Science.gov (United States)

    Kidd, R. D.; Noell, A.; Kazarians, G.; Aubrey, A. D.; Scianmarello, N.; Tai, Y.-C.

    2015-01-01

    We report progress towards developing a Micro-Electro-Mechanical Systems (MEMS)- based ion chromatograph (IC) for crewed spacecraft water analysis. This IC-chip is an offshoot of a NASA-funded effort to produce a high performance liquid chromatograph (HPLC)-chip. This HPLC-chip system would require a desalting (i.e. ion chromatography) step. The complete HPLC instrument consists of the Jet Propulsion Labortory's (JPL's) quadrupole ion trap mass spectrometer integrated with a state-of-the-art MEMS liquid chromatograph (LC) system developed by the California Institute of Technology's (Caltech's) Micromachining Laboratory. The IC version of the chip consist of an electrolysis-based injector, a separation column, two electrolysis pumps for gradient generation, mixer, and a built-in conductivity detector. The HPLC version of the chip also includes a nanospray tip. The low instrument mass, coupled with its high analytical capabilities, makes the LC chip ideally suitable for wide range of applications such as trace contaminant, inorganic analytical science and, when coupled to a mass spectrometer, a macromolecular detection system for either crewed space exploration vehicles or robotic planetary missions.

  15. Quantitative analysis of soil chromatography. I. Water and radionuclide transport

    Energy Technology Data Exchange (ETDEWEB)

    Reeves, M.; Francis, C.W.; Duguid, J.O.

    1977-12-01

    Soil chromatography has been used successfully to evaluate relative mobilities of pesticides and nuclides in soils. Its major advantage over the commonly used suspension technique is that it more accurately simulates field conditions. Under such conditions the number of potential exchange sites is limited both by the structure of the soil matrix and by the manner in which the carrier fluid moves through this structure. The major limitation of the chromatographic method, however, has been its qualitative nature. This document represents an effort to counter this objection. A theoretical basis is specified for the transport both of the carrier eluting fluid and of the dissolved constituent. A computer program based on this theory is developed which optimizes the fit of theoretical data to experimental data by automatically adjusting the transport parameters, one of which is the distribution coefficient k/sub d/. This analysis procedure thus constitutes an integral part of the soil chromatographic method, by means of which mobilities of nuclides and other dissolved constituents in soils may be quantified.

  16. Quantitative analysis of soil chromatography. I. Water and radionuclide transport

    International Nuclear Information System (INIS)

    Reeves, M.; Francis, C.W.; Duguid, J.O.

    1977-12-01

    Soil chromatography has been used successfully to evaluate relative mobilities of pesticides and nuclides in soils. Its major advantage over the commonly used suspension technique is that it more accurately simulates field conditions. Under such conditions the number of potential exchange sites is limited both by the structure of the soil matrix and by the manner in which the carrier fluid moves through this structure. The major limitation of the chromatographic method, however, has been its qualitative nature. This document represents an effort to counter this objection. A theoretical basis is specified for the transport both of the carrier eluting fluid and of the dissolved constituent. A computer program based on this theory is developed which optimizes the fit of theoretical data to experimental data by automatically adjusting the transport parameters, one of which is the distribution coefficient k/sub d/. This analysis procedure thus constitutes an integral part of the soil chromatographic method, by means of which mobilities of nuclides and other dissolved constituents in soils may be quantified

  17. Potential application of gas chromatography to the analysis of hydrogen isotopes

    International Nuclear Information System (INIS)

    Warner, D.K.; Sprague, R.E.; Bohl, D.R.

    1976-01-01

    Gas chromatography is used at Mound Laboratory for the analysis of hydrogen isotopic impurities in gas mixtures. This instrumentation was used to study the applicability of the gas chromatography technique to the determination of the major components of hydrogen isotopic gas mixtures. The results of this study, including chromatograms and precision data, are presented

  18. Plasma chromatography

    International Nuclear Information System (INIS)

    Anon.

    1984-01-01

    This book examines the fundamental theory and various applications of ion mobility spectroscopy. Plasma chromatography developed from research on the diffusion and mobility of ions. Topics considered include instrument design and description (e.g., performance, spectral interpretation, sample handling, mass spectrometry), the role of ion mobility in plasma chromatography (e.g., kinetic theory of ion transport), atmospheric pressure ionization (e.g., rate equations), the characterization of isomers by plasma chromatography (e.g., molecular ion characteristics, polynuclear aromatics), plasma chromatography as a gas chromatographic detection method (e.g., qualitative analysis, continuous mobility monitoring, quantitative analysis), the analysis of toxic vapors by plasma chromatography (e.g., plasma chromatograph calibration, instrument control and data processing), the analysis of semiconductor devices and microelectronic packages by plasma chromatography/mass spectroscopy (e.g., analysis of organic surface contaminants, analysis of water in sealed electronic packages), and instrument design and automation (hardware, software)

  19. [Column chromatography purification and analysis of biodiesel by transesterification].

    Science.gov (United States)

    Liu, Yang; Yi, Huai-feng; Chen, Yu; Wu, Yu-long; Yang, Ming-de; Chen, Zeng; Tong, Jun-mao

    2012-02-01

    In the present paper, crude biodiesel prepared with sorbifolia oil as raw material by transesterification was purified by column chromatography, then the composition of biodiesel was analyzed by gas chromatography, FTIR, GC-MS and 1H NMR. Column chromatography can separate the crude biodiesel into two fractions: petroleum ether eluted fraction (A1) and methanol eluted fraction (A2). Petroleum ether eluted fraction was mainly biodiesel fraction, which was produced from sorbifolia oil by transesterification, including methyl linoleate, methyl cis-9-octadecenoate and so on; methanol eluted fraction was mainly glycerol fraction, which came from the side reaction of transesterification. The results show that the purity of refined biodiesel increased from 77.51% to 93.872, and the product recovery rate reached up to 91.04% after the purification by column chromatography. The results obtained by FTIR and 1H NMR further showed that the column chromatography can effectively improve the purity of biodiesel. This paper provides a basis for industrialization of purification of biodiesel.

  20. Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates.

    Science.gov (United States)

    Adam, Frédérick; Thiébaut, Didier; Bertoncini, Fabrice; Courtiade, Marion; Hennion, Marie-Claire

    2010-02-19

    This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GCxGC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC x GC columns sets (twin-GCxGC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GCxGC analysis of both saturated and unsaturated fractions. The benefits of SFC-twin-GC x GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC x GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GCxGC run, and for the first time, an extended PIONA analysis of diesel samples is presented. Copyright 2009 Elsevier B.V. All rights reserved.

  1. Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates

    NARCIS (Netherlands)

    Moerdijk-Poortvliet, Tanja C. W.; Schierbeek, Henk; Houtekamer, Marco; van Engeland, Tom; Derrien, Delphine; Stal, Lucas J.; Boschker, Henricus T. S.

    2015-01-01

    We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ(13)C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although

  2. Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates

    NARCIS (Netherlands)

    Moerdijk-Poortvliet, T.C.W.; Schierbeek, H.; Houtekamer, M.; van Engeland, T.; Derrien, D.; Stal, L.J.; Boschker, H.T.S.

    2015-01-01

    We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of d13C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although

  3. Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates

    NARCIS (Netherlands)

    Moerdijk-Poortvliet, T.C.W.; Schierbeek, H.; Houtekamer, M.; van Engeland, T.; Derrien, D.; Stal, L.J.; Boschker, H.T.S.

    2015-01-01

    Rationale: We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ13C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence,

  4. Analysis of moniliformin in maize plants using hydrophilic interaction chromatography

    DEFF Research Database (Denmark)

    Sørensen, Jens Laurids; Nielsen, Kristian Fog; Thrane, Ulf

    2007-01-01

    A novel HPLC method was developed for detection of the Fusarium mycotoxin, moniliformin in whole maize plants. The method is based on hydrophilic interaction chromatography (HILIC) on a ZIC zwitterion column combined with diode array detection and negative electrospray mass spectrometry (ESI...

  5. Gas Chromatography-Mass Spectrometric Analysis of Nematicidal ...

    African Journals Online (AJOL)

    Purpose: To investigate the chemical composition and nematicidal activity of the essential oil of. Valeriana amurensis ... Methods: The essential oil of V. amurensis roots was obtained by hydrodistillation and analyzed by gas chromatography (GC) ..... Aβ1-40 and Caspase-3 in Alzheimer's disease model rat's brain. J Chin ...

  6. Liquid chromatography and liquid chromatography-mass spectrometry analysis of donepezil degradation products

    Directory of Open Access Journals (Sweden)

    Mladenović Aleksandar R.

    2015-01-01

    Full Text Available This study describes the investigation of degradation products of donepezil (DP using stability indicating RP-HPLC method for determination of donepezil, which is a centrally acting reversible acetylcholinesterase inhibitor. In order to investigate the stability of drug and formed degradation products, a forced degradation study of drug sample and finished product under different forced degradation conditions has been conducted. Donepezil hydrochloride and donepezil tablets were subjected to stress degradation conditions recommended by International Conference on Harmonization (ICH. Donepezil hydrochloride solutions were subjected to acid and alkali hydrolysis, chemical oxidation and thermal degradation. Significant degradation was observed under alkali hydrolysis and oxidative degradation conditions. Additional degradation products were observed under the conditions of oxidative degradation. The degradation products observed during forced degradation studies were monitored using the high performance liquid chromatography (HPLC method developed. The parent method was modified in order to obtain LC-MS compatible method which was used to identify the degradation products from forced degradation samples using high resolution mass spectrometry. The mass spectrum provided the precise mass from which derived molecular formula of drug substance and degradation products formed and proved the specificity of the method unambiguously. [Projekat Ministarstva nauke Republike Srbije, br. 172013

  7. Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography.

    Science.gov (United States)

    Venkatramani, C J; Al-Sayah, Mohammad; Li, Guannan; Goel, Meenakshi; Girotti, James; Zang, Lisa; Wigman, Larry; Yehl, Peter; Chetwyn, Nik

    2016-02-01

    A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC-SFC in pharmaceutical analysis is presented. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Mutational analysis of GALT gene in Greek patients with galactosaemia: identification of two novel mutations and clinical evaluation.

    Science.gov (United States)

    Schulpis, Kleopatra H; Thodi, Georgia; Iakovou, Konstantinos; Chatzidaki, Maria; Dotsikas, Yannis; Molou, Elina; Triantafylli, Olga; Loukas, Yannis L

    2017-10-01

    Classical galactosaemia is an inborn error of metabolism due to the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). The aim of the study was to identify the underlying mutations in Greek patients with GALT deficiency and evaluate their psychomotor and speech development. Patients with GALT deficiency (n = 17) were picked up through neonatal screening. Mutational analysis was conducted via Sanger sequencing, while in silico analysis was used in the cases of novel missense mutations. Psychomotor speech development tests were utilized for the clinical evaluation of the patients. Eleven different mutations in the GALT gene were detected in the patient cohort, including two novel ones. The most frequent mutation was p.Q188R (c.563 A > G). As for the novel mutations, p.M298I (c.894 G > A) was identified in four out of 32 independent alleles, while p.P115S (c.343 C > T) was identified once. Psychomotor evaluation revealed that most of the patients were found in the borderline area (Peabody test), while only two had speech delay problems. The WISK test revealed three patients at borderline limits and two were at lower than normal limits. The mutational spectrum of the GALT gene in Greek patients is presented for the first time. The mutation p.Q188R is the most frequent among Greek patients. Two novel mutations were identified and their potential pathogenicity was estimated. Regarding the phenotypic characteristics, psychomotor disturbances and speech delay were mainly observed among GALT-deficient patients.

  9. Application of ion chromatography in pharmaceutical and drug analysis.

    Science.gov (United States)

    Jenke, Dennis

    2011-08-01

    Ion chromatography (IC) has developed and matured into an important analytical methodology in a number of diverse applications and industries, including pharmaceuticals. This manuscript provides a review of IC applications for the determinations of active and inactive ingredients, excipients, degradation products, and impurities relevant to pharmaceutical analyses and thus serves as a resource for investigators looking for insights into the use of the IC methodology in this field of application.

  10. Liquid Chromatography for Analysis of Metformin in Myrmeleon sp.

    OpenAIRE

    Afidatul Muadifah; Hermin Sulistyarti; Sasangka Prasetyawan

    2017-01-01

    Myrmeleon sp is a typical of insect larva which has been used in Indonesia for diabetes treatment. However, there is no sufficient scientific report explaining the bioactive compounds in this insect. Based on our preliminary research, this insect contained metformin, i.e. one of bioactive compounds for the treatment of type-2 diabetes. Therefore, this study is focused on the development of separation technique using high performance liquid chromatography (HPLC) on a reverse phase C-18 column ...

  11. Haplotype analysis suggest that the MLH1 c.2059C > T mutation is a Swedish founder mutation.

    Science.gov (United States)

    von Salomé, Jenny; Liu, Tao; Keihäs, Markku; Morak, Moni; Holinski-Feder, Elke; Berry, Ian R; Moilanen, Jukka S; Baert-Desurmont, Stéphanie; Lindblom, Annika; Lagerstedt-Robinson, Kristina

    2017-12-29

    Lynch syndrome (LS) predisposes to a spectrum of cancers and increases the lifetime risk of developing colorectal- or endometrial cancer to over 50%. Lynch syndrome is dominantly inherited and is caused by defects in DNA mismatch-repair genes MLH1, MSH2, MSH6 or PMS2, with the vast majority detected in MLH1 and MSH2. Recurrent LS-associated variants observed in apparently unrelated individuals, have either arisen de novo in different families due to mutation hotspots, or are inherited from a founder (a common ancestor) that lived several generations back. There are variants that recur in some populations while also acting as founders in other ethnic groups. Testing for founder mutations can facilitate molecular diagnosis of Lynch Syndrome more efficiently and more cost effective than screening for all possible mutations. Here we report a study of the missense mutation MLH1 c.2059C > T (p.Arg687Trp), a potential founder mutation identified in eight Swedish families and one Finnish family with Swedish ancestors. Haplotype analysis confirmed that the Finnish and Swedish families shared a haplotype of between 0.9 and 2.8 Mb. While MLH1 c.2059C > T exists worldwide, the Swedish haplotype was not found among mutation carriers from Germany or France, which indicates a common founder in the Swedish population. The geographic distribution of MLH1 c.2059C > T in Sweden suggests a single, ancient mutational event in the northern part of Sweden.

  12. Single base pair mutation analysis by PNA directed PCR clamping

    DEFF Research Database (Denmark)

    Ørum, H.; Nielsen, P.E.; Egholm, M.

    1993-01-01

    A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity...... allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers....

  13. Preoperative RAS Mutational Analysis Is of Great Value in Predicting Follicular Variant of Papillary Thyroid Carcinoma

    Directory of Open Access Journals (Sweden)

    Tae Sook Hwang

    2015-01-01

    Full Text Available Follicular variant of papillary thyroid carcinoma (FVPTC, particularly the encapsulated subtype, often causes a diagnostic dilemma. We reconfirmed the molecular profiles in a large number of FVPTCs and investigated the efficacy of the preoperative mutational analysis in indeterminate thyroid nodules. BRAF V600E/K601E and RAS mutational analysis was performed on 187 FVPTCs. Of these, 132 (70.6% had a point mutation in one of the BRAF V600E (n=57, BRAF K601E (n=11, or RAS (n=64 genes. All mutations were mutually exclusive. The most common RAS mutations were at NRAS codon 61. FNA aspirates from 564 indeterminate nodules were prospectively tested for BRAF and RAS mutation and the surgical outcome was correlated with the mutational status. Fifty-seven and 47 cases were positive for BRAF and RAS mutation, respectively. Twenty-seven RAS-positive patients underwent surgery and all except one patient had FVPTC. The PPV and accuracy of RAS mutational analysis for predicting FVPTC were 96% and 84%, respectively. BRAF or RAS mutations were present in more than two-thirds of FVPTCs and these were mutually exclusive. BRAF mutational analysis followed by N, H, and KRAS codon 61 mutational analysis in indeterminate thyroid nodules would streamline the management of patients with malignancies, mostly FVPTC.

  14. Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Bindslev-Jensen, Carsten

    2014-01-01

    The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order...... to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case-series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect...... the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation...

  15. Application of Pyrolysis - Gas Chromatography/Mass Spectrometry in Failure Analysis in the Automotive Industry

    OpenAIRE

    Kusch, Peter (Dr.)

    2015-01-01

    This book chapter describes application examples of gas chromatography/mass spectrometry and pyrolysis – gas chromatography/mass spectrometry in failure analysis for the identification of chemical materials like mineral oils and nitrile rubber gaskets. Furthermore, failure cases demanding identification of polymers/copolymers in fouling on the compressor wall of a car air conditioner and identification of fouling on the surface of a bearing race from the automotive industry are demonstr...

  16. [Observation and analysis on mutation of routine STR locus].

    Science.gov (United States)

    Li, Qiu-yang; Feng, Wei-jun; Yang, Qin-gen

    2005-05-01

    To observe and analyze the characteristic of mutation at STR locus. 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.

  17. A parylene-based dual channel microelectrophoresis system for rapid mutation detection via heteroduplex analysis

    NARCIS (Netherlands)

    Sukas, S.; Erson, Ayse Elif; Sert, Cuneyt; Kulah, Haluk

    2008-01-01

    A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 μm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing

  18. Application of ion chromatography in the analysis of metals

    International Nuclear Information System (INIS)

    Doepke, T.; Braun, N.; Wuensch, G.

    1992-01-01

    Methods for the determination of chloride in molybdenum, tungsten, niobium and tantalum and of phosphorus in molybdenum and tungsten are presented. After oxidative digestion the analytes are separated from the matrix and accumulated in a small volume of liquid. Unsuppressed ion chromatography serves as the final determination method. The trace-matrix-separation and enrichment of chloride is largely independent of the kind of matrix. The procedure is therefore also applicable to concentrated solutions of various salts. A closed system ensures chloride blanks around 0.2 ppm and detection limits in the higher ppb range. A modification allows an enrichment of bromide and the simultaneous determination of chloride and bromide. (orig.) [de

  19. Analysis of artificial fireplace logs by high temperature gas chromatography.

    Science.gov (United States)

    Kuk, Raymond J

    2002-11-01

    High temperature gas chromatography is used to analyze the wax of artificial fireplace logs (firelogs). Firelogs from several different manufacturers are studied and compared. This study shows that the wax within a single firelog is homogeneous and that the wax is also uniform throughout a multi-firelog package. Different brands are shown to have different wax compositions. Firelogs of the same brand, but purchased in different locations, also have different wax compositions. With this information it may be possible to associate an unknown firelog sample to a known sample, but a definitive statement of the origin cannot be made.

  20. Pachyonychia congenita: Report of two cases and mutation analysis

    Directory of Open Access Journals (Sweden)

    Jia-Ming Yeh

    2012-09-01

    Full Text Available Pachyonychia congenita (PC comprises a group of rare autosomal dominant genetic disorders that involve ectodermal dysplasia. It is characterized by hypertrophic nail dystrophy, focal palmoplantar keratoderma, follicular keratoses, and oral leukokeratosis. Historically, PC has been subdivided into two subtypes, PC-1 or PC-2, on the basis of clinical presentation. However, differential diagnosis based on clinical grounds, especially in young and/or not fully penetrant patients, can be difficult. In addition, clinical analysis of the large case series has shown that there is considerable phenotypic overlap between these two subtypes recently. Based on the advent of molecular genetics and the identification of the genes causing PC, more specific nomenclature has been adopted. Therefore, diagnosis at the molecular level is useful and important to confirm the clinical impression. In this report, we describe two typical cases of PC with mutation analysis revealed a small deletion (514_516delACC, Asn172del and a point mutation (487 G > A, GAG → AAG, Glu163Lys in the KRT6A gene.

  1. Doping control analysis of anabolic steroids in equine urine by gas chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Wong, April S Y; Leung, Gary N W; Leung, David K K; Wan, Terence S M

    2017-09-01

    Anabolic steroids are banned substances in equine sports. Gas chromatography-mass spectrometry (GC-MS) has been the traditional technique for doping control analysis of anabolic steroids in biological samples. Although liquid chromatography-mass spectrometry (LC/MS) has become an important technique in doping control, the detection of saturated hydroxysteroids by LC-MS remains a problem due to their low ionization efficiency under electrospray. The recent development in fast-scanning gas-chromatography-triple-quadrupole mass spectrometry (GC-MS/MS) has provided a better alternative with a significant reduction in chemical noise by means of selective reaction monitoring. Herein, we present a sensitive and selective method for the screening of over 50 anabolic steroids in equine urine using gas chromatography-tandem mass spectrometry (GC-MS/MS). Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Chromatography Analysis of Jatropha Curcas L Oil Raw and Refined

    International Nuclear Information System (INIS)

    Chitue de Assuncao Nascimento, Juliano; Lafargue Perez, Francisco; Diaz Velazquez, Manuel

    2013-01-01

    The chromatography of gases is one of the most versatile methods utilized in the chemical laboratory, which permits the determinations of the percentages and types of fatty acids present in vegetable oils. The objective of this work was to carry out a comparative study of the composition of the fatty acids, of the mechanically extracted oil from the seed of the Jatropha curcas L plant in the raw and refined state, using the data obtained by gas chromatography. The fatty acids of the oil were determined as methylic esteres. The analyst was carried out in a gas chromatographer, with a detector of ionization by way of flame (FID), Agilent 7890A (Agilent, USA) coupled to HP Pentium 4 computer, with a data processing program. The temperatures of the injector and of the detector were 250 °C and 260 °C respectively. The data obtained chromatographically of the oil of Jatropha curcas L, raw and refined indicates that the refinery process did not vary the composition of the fatty acid, because there are no significant differences between them. Also, the results obtained by other investigators, showed that the predominant fatty acids obtained from the oil of the Jatropha curcas L were palmitic and stearic in the saturated fatty acids, and oleic and linoleic in the unsaturated fatty acids

  3. Chromatography analysis of Jatropha Curcas L oil raw and refined

    International Nuclear Information System (INIS)

    Chitue de Assuncao Nascimento, Juliano; Lafargue Perez, Francisco; Diaz Velazquez, Manuel

    2013-01-01

    The chromatography of gases is one of the most versatile methods utilized in the chemical laboratory, which permits the determinations of the percentages and types of fatty acids present in vegetable oils. The objective of this work was to carry out a comparative study of the composition of the fatty acids, of the mechanically extracted oil from the seed of the Jatropha curcas L plant in the raw and refined state, using the data obtained by gas chromatography. The fatty acids of the oil were determined as methylic esteres. The analyst was carried out in a gas chromatographer, with a detector of ionization by way of flame (FID), Agilent 7890A (Agilent, USA) coupled to HP Pentium 4 computer, with a data processing program. The temperatures of the injector and of the detector were 250℃ and 260℃ respectively. The data obtained chromatographically of the oil of Jatropha curcas L, raw and refined indicates that the refinery process did not vary the composition of the fatty acid, because there are no significant differences between them. Also, the results obtained by other investigators, showed that the predominant fatty acids obtained from the oil of the Jatropha curcas L were palmitic and stearic in the saturated fatty acids, and oleic and linoleic in the unsaturated fatty acids

  4. Application of micellar electrokinetic capillary chromatography for routine analysis of different materials

    Directory of Open Access Journals (Sweden)

    Injac Rade

    2008-01-01

    Full Text Available Micellar electrokinetic capillary chromatography (MEKC has become a popular mode among the several capillary electro-migration techniques. Most drug analysis can be performed by using MEKC because of its wide applicability. Separation of very complex mixtures, determination of drugs in the biological materials, etc., can be successfully achieved by MEKC. This review surveys typical applications of MEKC analysis. Recent advances in MEKC, especially with solid-phase extraction and large-volume sample stacking, are described. Modes of electrokinetic chromatography including MEKC, a separation theory of MEKC, environmental friendly analysis, and selectivity manipulation in MEKC are also briefly mentioned.

  5. Impact of comprehensive two-dimensional gas chromatography with mass spectrometry on food analysis.

    Science.gov (United States)

    Tranchida, Peter Q; Purcaro, Giorgia; Maimone, Mariarosa; Mondello, Luigi

    2016-01-01

    Comprehensive two-dimensional gas chromatography with mass spectrometry has been on the separation-science scene for about 15 years. This three-dimensional method has made a great positive impact on various fields of research, and among these that related to food analysis is certainly at the forefront. The present critical review is based on the use of comprehensive two-dimensional gas chromatography with mass spectrometry in the untargeted (general qualitative profiling and fingerprinting) and targeted analysis of food volatiles; attention is focused not only on its potential in such applications, but also on how recent advances in comprehensive two-dimensional gas chromatography with mass spectrometry will potentially be important for food analysis. Additionally, emphasis is devoted to the many instances in which straightforward gas chromatography with mass spectrometry is a sufficiently-powerful analytical tool. Finally, possible future scenarios in the comprehensive two-dimensional gas chromatography with mass spectrometry food analysis field are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Hydrophilic interaction liquid chromatography in the speciation analysis of selenium.

    Science.gov (United States)

    Sentkowska, Aleksandra; Pyrzynska, Krystyna

    2018-02-01

    The hydrophilic interaction liquid chromatography (HILIC) coupled to mass spectrometry was employed to study retention behavior of selected selenium compounds using two different HILIC stationary phases: silica and zwitterionic. Two organic solvents - acetonitrile and methanol - were compared as a component of mobile phase. Separation parameters such as a content of organic modifier, the eluent pH and inorganic buffer concentration were investigated. Based on all observations, methanol seems to be beneficial for the separation of studied compounds. The optimal HILIC separation method involved silica column and eluent composed of 85% MeOH and CH 3 COONH 4 (8 mM, pH 7) was compared to RP method in terms of time of the single run, the separation efficiency and limit of detection. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Analysis of penicillin G in milk by liquid chromatography.

    Science.gov (United States)

    Boison, J O; Keng, L J; MacNeil, J D

    1994-01-01

    A liquid chromatographic (LC) method that was previously developed for penicillin G residues in animal tissues has been adapted to milk and milk products. After protein precipitation with sodium tungstate, samples are applied to a C18 solid-phase extraction cartridge, from which penicillin is eluted, derivatized with 1,2,4-triazole-mercuric chloride solution, and analyzed by isocratic liquid chromatography (LC) on a C18 column with UV detection at 325 nm. Quantitation is done with reference to penicillin V as an internal standard. Penicillin G recoveries were determined to be > 70% on standards fortified at 3-60 ppb. Accuracy approached 100% using the penicillin V internal standard. The detection limit for penicillin G residues was 3 ppb in fluid milk. Samples may be confirmed by thermospray/LC at concentrations approaching the detection limit of the UV method.

  8. Improvement of recovery and repeatability in liquid chromatography-mass spectrometry analysis of peptides

    NARCIS (Netherlands)

    van Midwoud, Paul M.; Rieux, Laurent; Bischoff, Rainer; Verpoorte, Elisabeth; Niederlander, Harm A. G.

    2007-01-01

    Poor repeatability of peak areas is a problem frequently encountered in peptide analysis with nanoLiquid Chromatography coupled on-line with Mass Spectrometry (nanoLC-MS). As a result, quantitative analysis will be seriously hampered unless the observed variability can be corrected in some way.

  9. Systematic Analysis of Splice-Site-Creating Mutations in Cancer

    Directory of Open Access Journals (Sweden)

    Reyka G. Jayasinghe

    2018-04-01

    Full Text Available Summary: For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. : Jayasinghe et al. identify nearly 2,000 splice-site-creating mutations (SCMs from over 8,000 tumor samples across 33 cancer types. They provide a more accurate interpretation of previously mis-annotated mutations, highlighting the importance of integrating data types to understand the functional and the clinical implications of splicing mutations in human disease. Keywords: splicing, RNA, mutations of clinical relevance

  10. Quantitative analysis of abused drugs in physiological fluids by gas chromatography/chemical ionization mass spectrometry

    International Nuclear Information System (INIS)

    Foltz, R.L.

    1978-01-01

    Methods have been developed for quantitative analysis of commonly abused drugs in physiological fluids using gas chromatography/chemical ionization mass spectrometry. The methods are being evaluated in volunteer analytical and toxicological laboratories, and analytical manuals describing the methods are being prepared. The specific drug and metabolites included in this program are: Δ 9 -tetrahydrocannabinol, methadone, phencyclidine, methaqualone, morphine, amphetamine, methamphetamine, mescaline, 2,5-dimethoxy-4-methyl amphetamine, cocaine, benzoylecgonine, diazepam, and N-desmethyldiazepam. The current analytical methods utilize relatively conventional instrumentation and procedures, and are capable of measuring drug concentrations as low as 1 ng/ml. Various newer techniques such as sample clean-up by high performance liquid chromatography, separation by glass capillary chromatography, and ionization by negative ion chemical ionization are being investigated with respect to their potential for achieving higher sensitivity and specificity, as well as their ability to facilitate simultaneous analysis of more than one drug and metabolite. (Auth.)

  11. Application of gas chromatography to analysis of spirit-based alcoholic beverages.

    Science.gov (United States)

    Wiśniewska, Paulina; Śliwińska, Magdalena; Dymerski, Tomasz; Wardencki, Waldemar; Namieśnik, Jacek

    2015-01-01

    Spirit-based beverages are alcoholic drinks; their production processes are dependent on the type and origin of raw materials. The composition of this complex matrix is difficult to analyze, and scientists commonly choose gas chromatography techniques for this reason. With a wide selection of extraction methods and detectors it is possible to provide qualitative and quantitative analysis for many chemical compounds with various functional groups. This article describes different types of gas chromatography techniques and their most commonly used associated extraction techniques (e.g., LLE, SPME, SPE, SFE, and SBME) and detectors (MS, TOFMS, FID, ECD, NPD, AED, O or EPD). Additionally, brief characteristics of internationally popular spirit-based beverages and application of gas chromatography to the analysis of selected alcoholic drinks are presented.

  12. Anion analysis in uranium more concentrates by ion chromatography

    International Nuclear Information System (INIS)

    Badaut, V.

    2009-01-01

    In the present exploratory study, the applicability of anionic impurities or attributing nuclear material to a certain chemical process or origin has been investigated. Anions (e.g., nitrate, sulphate, fluoride, chloride) originate from acids or salt solutions that are used for processing of solutions containing uranium or plutonium. The study focuses on uranium ore concentrates ('yellow cakes') originating from different mines. Uranium is mined from different types of ore body and depending on the type of rock, different chemical processes for leaching, dissolving and precipitating the uranium need to be applied. Consequently, the anionic patterns observed in he products of these processes (the 'ore concentrates') are different. The concentrations of different anionic species were measured by ion chromatography using conductivity detection. The results show clear differences of anion concentrations and patterns between samples from different uranium mines. Besides this, differences between sampling campaigns n a same mine were also observed indicating that the uranium ore is not homogeneous in a mine. These within-mine variations, however, were smaller than the between-mine variations. (author)

  13. High resolution melting analysis: a rapid and accurate method to detect CALR mutations.

    Directory of Open Access Journals (Sweden)

    Cristina Bilbao-Sieyro

    Full Text Available The recent discovery of CALR mutations in essential thrombocythemia (ET and primary myelofibrosis (PMF patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN. We tested the feasibility of high-resolution melting (HRM as a screening method for rapid detection of CALR mutations.CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET.Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34, 14% of persistent thrombocytosis suggestive of MPN (3/21 and none of the secondary thrombocytosis (0/98. Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%.This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations.

  14. Phytochemical analysis of ethanolic extract of Dichrostachys Cinerea W and Arn leaves by a thin layer chromatography, high performance thin layer chromatography and column chromatography

    OpenAIRE

    M Vijayalakshmi; K Periyanayagam; K Kavitha; K Akilandeshwari

    2013-01-01

    Background: The leaves of Dichrostachys cinerea are used as laxative, diuretic, painkiller. It is also used in the treatment of gonorrhoea, boils, oedema, gout, veneral diseases and nasopharyngeal affections, etc. Materials and Methods: The Phytochemical investigation of ethanolic extract of D. cinerea leaves were performed by standard chemical tests, thin layer chromatography (TLC) by using various solvent systems, and by high performance liquid chromatography (HPTLC). Two compounds were...

  15. Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

    Science.gov (United States)

    Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

    2009-06-01

    Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

  16. The application of a linear algebra to the analysis of mutation rates.

    Science.gov (United States)

    Jones, M E; Thomas, S M; Clarke, K

    1999-07-07

    Cells and bacteria growing in culture are subject to mutation, and as this mutation is the ultimate substrate for selection and evolution, the factors controlling the mutation rate are of some interest. The mutational event is not observed directly, but is inferred from the phenotype of the original mutant or of its descendants; the rate of mutation is inferred from the number of such mutant phenotypes. Such inference presumes a knowledge of the probability distribution for the size of a clone arising from a single mutation. We develop a mathematical formulation that assists in the design and analysis of experiments which investigate mutation rates and mutant clone size distribution, and we use it to analyse data for which the classical Luria-Delbrück clone-size distribution must be rejected. Copyright 1999 Academic Press.

  17. DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Mahmoud, J.; Fossett, N.G.; Arbour-Reily, P.; McDaniel, M.; Tucker, A.; Chang, S.H.; Lee, W.R.

    1991-01-01

    The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and 'point mutations'. Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the breakpoint with one of the repeats deleted

  18. OGG1 Mutations and Risk of Female Breast Cancer: Meta-Analysis and Experimental Data

    Directory of Open Access Journals (Sweden)

    Kashif Ali

    2015-01-01

    Full Text Available In first part of this study association between OGG1 polymorphisms and breast cancer susceptibility was explored by meta-analysis. Second part of the study involved 925 subjects, used for mutational analysis of OGG1 gene using PCR-SSCP and sequencing. Fifteen mutations were observed, which included five intronic mutations, four splice site mutations, two 3′UTR mutations, three missense mutations, and a nonsense mutation. Significantly (pG and 3′UTR variant g.9798848G>A. Among intronic mutations, highest (~15 fold increase in breast cancer risk was associated with g.9793680G>A (p<0.009. Similarly ~14-fold increased risk was associated with Val159Gly (p<0.01, ~17-fold with Gly221Arg (p<0.005, and ~18-fold with Ser326Cys (p<0.004 in breast cancer patients compared with controls, whereas analysis of nonsense mutation showed that ~13-fold (p<0.01 increased breast cancer risk was associated with Trp375STOP in patients compared to controls. In conclusion, a significant association was observed between OGG1 germ line mutations and breast cancer risk. These findings provide evidence that OGG1 may prove to be a good candidate of better diagnosis, treatment, and prevention of breast cancer.

  19. Mutational analysis of PHEX, FGF23, DMP1, SLC34A3 and CLCN5 in patients with hypophosphatemic rickets

    DEFF Research Database (Denmark)

    Beck-Nielsen, Signe; Brixen, Kim; Gram, Jeppe

    2012-01-01

    This study aimed to identify the underlying genetic mutation in patients with hypophosphatemic rickets (HR). Genomic DNA was analysed for mutations in PHEX, FGF23 and CLCN5 by polymerase chain reaction (PCR) followed by denaturing high-performance liquid chromatography (dHPLC). Bi-directional seq...... identification rate of gene mutations causing HR, including 12 novel PHEX and one novel DMP1 mutation.Journal of Human Genetics advance online publication, 14 June 2012; doi:10.1038/jhg.2012.56....

  20. [Analysis of microalbuminuria with immunonephelometry and high performance liquid chromatography. Evaluation of new criteria].

    Science.gov (United States)

    Markó, Lajos; Molnár, Gergo Attila; Wagner, Zoltán; Koszegi, Tamás; Matus, Zoltán; Mohás, Márton; Kuzma, Mónika; Szijártó, István András; Wittmann, István

    2008-01-13

    Evaluation study, the rate of microalbuminuria positivity among the immunonephelometrically negative patients decreased to 14.5% by high performance liquid chromatography and the decrease in the number of microalbuminuria positive cases by high performance liquid chromatography could be observed mainly in the diabetic and hypertensive group (49% vs. 7.5%), while slighter decrease could be observed in the non-diabetic hypertensive group (37% vs. 26.5%). Applying the traditional criteria, the strongest predictor was the male gender by the logistic regression analysis. In 28% of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established using high performance liquid chromatography. Almost in one-third of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established by high performance liquid chromatography for which diagnosis three constitutive urine examinations are still needed. New criteria determined by the Heart Outcomes Prevention Evaluation study can be used neither in case of diabetic and hypertensive patients, nor in the case of non-diabetic hypertensive patients. The gender as the most important predictor of microalbuminuria cannot be ignored.

  1. Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography-electrospray ionization mass spectrometry

    NARCIS (Netherlands)

    Coulier, L.; Bas, R.; Jespersen, S.; Verheij, E.; Werf, M.J. van der; Hankemeier, T.

    2006-01-01

    We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides,

  2. Trace analysis in the food and beverage industry by capillary gas chromatography: system performance and maintenance.

    Science.gov (United States)

    Hayes, M A

    1988-04-01

    Gas chromatography (GC) is the most widely used analytical technique in the food and beverage industry. This paper addresses the problems of sample preparation and system maintenance to ensure the most sensitive, durable, and efficient results for trace analysis by GC in this industry.

  3. Comprehensive two-dimensional gas chromatography for the analysis of organohalogenated micro-contaminants

    NARCIS (Netherlands)

    Korytar, P.; Haglund, P.; Boer, de J.; Brinkman, U.A.Th.

    2006-01-01

    We explain the principles of comprehensive two-dimensional gas chromatography (GC × GC), and discuss key instrumental aspects - with emphasis on column combinations and mass spectrometric detection. As the main item of interest, we review the potential of GC × GC for the analysis of

  4. High Performance Liquid Chromatography of Some Analgesic Compounds: An Instrumental Analysis Experiment.

    Science.gov (United States)

    Haddad, Paul; And Others

    1983-01-01

    Background information, procedures, and results are provided for an experiment demonstrating techniques of solvent selection, gradient elution, pH control, and ion-pairing in the analysis of an analgesic mixture using reversed-phase liquid chromatography on an octadecylsilane column. Although developed using sophisticated/expensive equipment, less…

  5. Gas chromatography/sniffing port analysis evaluated for aroma release from rehydrated French beans (Phaseolus vulgaris).

    NARCIS (Netherlands)

    Ruth, van S.M.; Roozen, J.P.; Cozijnsen, J.L.

    1996-01-01

    The technique of gas chromatography/sniffing port analysis was evaluated for studying the release of aroma compounds from rehydrated diced French beans. The aroma compounds 2-—methylbutanal and hexanal were released at a constant rate over time. An identical selection of odour active compounds was

  6. Quantitative analysis of target components by comprehensive two-dimensional gas chromatography

    NARCIS (Netherlands)

    Mispelaar, V.G. van; Tas, A.C.; Smilde, A.K.; Schoenmakers, P.J.; Asten, A.C. van

    2003-01-01

    Quantitative analysis using comprehensive two-dimensional (2D) gas chromatography (GC) is still rarely reported. This is largely due to a lack of suitable software. The objective of the present study is to generate quantitative results from a large GC x GC data set, consisting of 32 chromatograms.

  7. Two-Dimensional Liquid Chromatography Analysis of Polystyrene/Polybutadiene Block Copolymers.

    Science.gov (United States)

    Lee, Sanghoon; Choi, Heejae; Chang, Taihyun; Staal, Bastiaan

    2018-05-15

    A detailed characterization of a commercial polystyrene/polybutadiene block copolymer material (Styrolux) was carried out using two-dimensional liquid chromatography (2D-LC). The Styrolux is prepared by statistical linking reaction of two different polystyrene- block-polybutadienyl anion precursors with a multivalent linking agent. Therefore, it is a mixture of a number of branched block copolymers different in molecular weight, composition, and chain architecture. While individual LC analysis, including size exclusion chromatography, interaction chromatography, or liquid chromatography at critical condition, is not good enough to resolve all the polymer species, 2D-LC separations coupling two chromatography methods were able to resolve all polymer species present in the sample; at least 13 block copolymer species and a homopolystyrene blended. Four different 2D-LC analyses combining a different pair of two LC methods provide their characteristic separation results. The separation characteristics of the 2D-LC separations are compared to elucidate the elution characteristics of the block copolymer species.

  8. Application in pesticide analysis: Liquid chromatography - A review of the state of science for biomarker discovery and identification

    Science.gov (United States)

    Book Chapter 18, titled Application in pesticide analysis: Liquid chromatography - A review of the state of science for biomarker discovery and identification, will be published in the book titled High Performance Liquid Chromatography in Pesticide Residue Analysis (Part of the C...

  9. Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives

    Science.gov (United States)

    Edelmann, Mariola J.

    2011-01-01

    Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide. PMID:22174558

  10. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R. [Ohio State Univ., Columbus, OH (United States); Moxley, R.T. [Univ. of Rochester Medical Center, NY (United States)

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  11. ANALYSIS AND IDENTIFICATION SPIKING CHEMICAL COMPOUNDS RELATED TO CHEMICAL WEAPON CONVENTION IN UNKNOWN WATER SAMPLES USING GAS CHROMATOGRAPHY AND GAS CHROMATOGRAPHY ELECTRON IONIZATION MASS SPECTROMETRY

    Directory of Open Access Journals (Sweden)

    Harry Budiman

    2010-06-01

    Full Text Available The identification and analysis of chemical warfare agents and their degradation products is one of important component for the implementation of the convention. Nowadays, the analytical method for determination chemical warfare agent and their degradation products has been developing and improving. In order to get the sufficient analytical data as recommended by OPCW especially in Proficiency Testing, the spiking chemical compounds related to Chemical Weapon Convention in unknown water sample were determined using two different techniques such as gas chromatography and gas chromatography electron-impact ionization mass spectrometry. Neutral organic extraction, pH 11 organic extraction, cation exchanged-methylation, triethylamine/methanol-silylation were performed to extract the chemical warfare agents from the sample, before analyzing with gas chromatography. The identification of chemical warfare agents was carried out by comparing the mass spectrum of chemicals with mass spectrum reference from the OPCW Central Analytical Database (OCAD library while the retention indices calculation obtained from gas chromatography analysis was used to get the confirmation and supported data of  the chemical warfare agents. Diisopropyl methylphosphonate, 2,2-diphenyl-2-hydroacetic acid and 3-quinuclidinol were found in unknown water sample. Those chemicals were classified in schedule 2 as precursor or reactant of chemical weapons compound in schedule list of Chemical Weapon Convention.   Keywords: gas chromatography, mass spectrometry, retention indices, OCAD library, chemical warfare agents

  12. Speciation analysis of cobalt in foods by high-performance liquid chromatography and neutron activation analysis

    International Nuclear Information System (INIS)

    Muto, Toshio; Koyama, Motoko

    1994-01-01

    A combined method by coupling high-performance liquid chromatography (HPLC, as a separation method) with neutron activation analysis (as a detection method) have been applied to the speciation analysis of cobalt in daily foods (e.g. egg, fish and milk). Cobalt species including free cobalt, vitamin B 12 and protein-bound cobalt were separated with a preparative HPLC and a centrifuge. Subsequently, the determination of cobalt in the separated species was made by neutron activation analysis. The results showed that the content of the total cobalt in the foods was found to lie in the range 0.4-11ng/g(0.4-11ppb) based on wet weight. The compositions of free cobalt, vitamin B 12 and protein-bound cobalt were ranged 16-43%, 55-73%, 2.3-17%, respectively. These experimental evidences suggest that the combination of HPLC and neutron activation analysis is expected to be a useful tool for speciation analysis of trace elements in biological as well as environmental materials. (author)

  13. Analysis of radioactive mixed hazardous waste using derivatization gas chromatography/mass spectrometry, liquid chromatography, and liquid chromatography/mass spectrometry

    International Nuclear Information System (INIS)

    Campbell, J.A.; Lerner, B.D.; Bean, R.M.; Grant, K.E.; Lucke, R.B.; Mong, G.M.; Clauss, S.A.

    1994-08-01

    Six samples of core segments from Tank 101-SY were analyzed for chelators, chelator fragments, and several carboxylic acids by derivatization gas chromatography/mass spectrometry. The major components detected were ethylenediaminetetraacetic acid, nitroso-iminodiacetic acid, nitrilotriacetic acid, citric acid, succinic acid, and ethylenediaminetriacetic acid. The chelator of highest concentration was ethylenediaminetetraacetic acid in all six samples analyzed. Liquid chromatography was used to quantitate low molecular weight acids including oxalic, formic, glycolic, and acetic acids, which are present in the waste as acid salts. From 23 to 61% of the total organic carbon in the samples analyzed was accounted for by these acids

  14. Analysis of human serum by liquid chromatography-mass spectrometry: improved sample preparation and data analysis.

    Science.gov (United States)

    Govorukhina, N I; Reijmers, T H; Nyangoma, S O; van der Zee, A G J; Jansen, R C; Bischoff, R

    2006-07-07

    Discovery of biomarkers is a fast developing field in proteomics research. Liquid chromatography coupled on line to mass spectrometry (LC-MS) has become a powerful method for the sensitive detection, quantification and identification of proteins and peptides in biological fluids like serum. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomics studies. To perform future comparative analyses of samples from a serum bank of cervical cancer patients in a longitudinal and cross-sectional manner, methodology based on the depletion of high-abundance proteins followed by tryptic digestion and LC-MS has been developed. Two sample preparation methods were tested in terms of their efficiency to deplete high-abundance serum proteins and how they affect the repeatability of the LC-MS data sets. The first method comprised depletion of human serum albumin (HSA) on a dye ligand chromatographic and immunoglobulin G (IgG) on an immobilized Protein A support followed by tryptic digestion, fractionation by cation-exchange chromatography, trapping on a C18 column and reversed-phase LC-MS. The second method included depletion of the six most abundant serum proteins based on multiple immunoaffinity chromatography followed by tryptic digestion, trapping on a C18 column and reversed-phase LC-MS. Repeatability of the overall procedures was evaluated in terms of retention time and peak area for a selected number of endogenous peptides showing that the second method, besides being less time consuming, gave more repeatable results (retention time: <0.1% RSD; peak area: <30% RSD). Application of an LC-MS component detection algorithm followed by principal component analysis (PCA) enabled discrimination of serum samples that were spiked with horse heart cytochrome C from non-spiked serum and the detection of a concentration trend, which correlated to the amount of spiked horse heart

  15. DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

    Science.gov (United States)

    Lenato, Gennaro M; Lastella, Patrizia; Di Giacomo, Marilena C; Resta, Nicoletta; Suppressa, Patrizia; Pasculli, Giovanna; Sabbà, Carlo; Guanti, Ginevra

    2006-02-01

    Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation. 2006 Wiley-Liss, Inc.

  16. Toxic Compounds Analysis With High Performance Liquid Chromatography Detected By Electro Chemical Detector (Ecd)

    OpenAIRE

    Hideharu Shintaniq

    2014-01-01

    The principal area of application of high performance liquid chromatography-electrochemical detector (HPLC-ECD) has been in the analysis of naturally-occurring analytes, such as catecholamines, and pharmaceuticals in biological samples, HPLC-ECD has also applied to the analysis of pesticides and other analytes of interest to the toxicologist. In this paper, toxic area is described. In these, ammatoxins, aromatic amine, nitro-compounds, algal toxins, fungal toxins, pesticides, veterinary drug ...

  17. Polymer Analysis by Liquid Chromatography/Electrospray Ionization Time-of-Flight Mass Spectrometry.

    Science.gov (United States)

    Nielen, M W; Buijtenhuijs, F A

    1999-05-01

    Hyphenation of liquid chromatography (LC) techniques with electrospray ionization (ESI) orthogonal acceleration time-of-flight (oa-TOF) mass spectrometry (MS) provides both MS-based structural information and LC-based quantitative data in polymer analysis. In one experimental setup, three different LC modes are interfaced with MS:  size-exclusion chromatography (SEC/MS), gradient polymer elution chromatography (GPEC/MS), and liquid chromatography at the critical point of adsorption (LCCC/MS). In SEC/MS, both absolute mass calibration of the SEC column based on the polymer itself and determination of monomers and end groups from the mass spectra are achieved. GPEC/MS shows detailed chemical heterogeneity of the polymer and the chemical composition distribution within oligomer groups. In LCCC/MS, the retention behavior is primarily governed by chemical heterogeneities, such as different end group functionalities, and quantitative end group calculations can be easily made. The potential of these methods and the benefit of time-of-flight analyzers in polymer analysis are discussed using SEC/MS of a polydisperse poly(methyl methacrylate) sample, GPEC/MS of dipropoxylated bisphenol A/adipic acid polyester resin, LCCC/MS of alkylated poly(ethylene glycol), and LCCC/MS of terephthalic acid/neopentyl glycol polyester resin.

  18. Immunoaffinity chromatography for the sample pretreatment of Taxus plant and cell extracts prior to analysis of taxanes by high-performance liquid chromatography

    NARCIS (Netherlands)

    Theodoridis, G; Haasnoot, W; Schilt, R; Jaziri, M; Diallo, B; Papadoyannis, IN; de Jong, GJ; Cazemier, G.

    2002-01-01

    The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked

  19. BRCA1 and BRCA2 germline mutation analysis among Indian ...

    Indian Academy of Sciences (India)

    Prakash

    specific association between BRCA1 or BRCA2 mutations with cancer type was seen, ... Materials and methods ..... KS is a Wellcome Trust International Senior Research .... of BRCA1/2 associated breast cancer: a systematic qualitative.

  20. Analysis of lignans in Magnoliae Flos by turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry.

    Science.gov (United States)

    Zhou, Xuan; Chen, Cen; Ye, Xiaolan; Song, Fenyun; Fan, Guorong; Wu, Fuhai

    2016-04-01

    In this study, a method coupling turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry was developed for analyzing the lignans in Magnoliae Flos. By the online pretreatment of turbulent flow chromatography solid-phase extraction, the impurities removal and analytes concentration were automatically processed, and the lignans were separated rapidly and well. Seven lignans of Magnoliae Flos including epieudesmin, magnolin, 1-irioresinol-B-dimethyl ether, epi-magnolin, fargesin aschantin, and demethoxyaschantin were identified by comparing their retention behavior, UV spectra, and mass spectra with those of reference substances or literature data. The developed method was validated, and the good results showed that the method was not only automatic and rapid, but also accurate and reliable. The turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry method holds a high potential to become an effective method for the quality control of lignans in Magnoliae Flos and a useful tool for the analysis of other complex mixtures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Multi-center analysis of glucocerebrosidase mutations in Parkinson disease

    Science.gov (United States)

    Sidransky, Ellen; Nalls, Michael A.; Aasly, Jan O.; Aharon-Peretz, Judith; Annesi, Grazia; Barbosa, Egberto Reis; Bar-Shira, Anat; Berg, Daniela; Bras, Jose; Brice, Alexis; Chen, Chiung-Mei; Clark, Lorraine N.; Condroyer, Christel; De Marco, Elvira Valeria; Dürr, Alexandra; Eblan, Michael J.; Fahn, Stanley; Farrer, Matthew; Fung, Hon-Chung; Gan-Or, Ziv; Gasser, Thomas; Gershoni-Baruch, Ruth; Giladi, Nir; Griffith, Alida; Gurevich, Tanya; Januario, Cristina; Kropp, Peter; Lang, Anthony E.; Lee-Chen, Guey-Jen; Lesage, Suzanne; Marder, Karen; Mata, Ignacio F.; Mirelman, Anat; Mitsui, Jun; Mizuta, Ikuko; Nicoletti, Giuseppe; Oliveira, Catarina; Ottman, Ruth; Orr-Urtreger, Avi; Pereira, Lygia V.; Quattrone, Aldo; Rogaeva, Ekaterina; Rolfs, Arndt; Rosenbaum, Hanna; Rozenberg, Roberto; Samii, Ali; Samaddar, Ted; Schulte, Claudia; Sharma, Manu; Singleton, Andrew; Spitz, Mariana; Tan, Eng-King; Tayebi, Nahid; Toda, Tatsushi; Troiano, André; Tsuji, Shoji; Wittstock, Matthias; Wolfsberg, Tyra G.; Wu, Yih-Ru; Zabetian, Cyrus P.; Zhao, Yi; Ziegler, Shira G.

    2010-01-01

    Background Recent studies indicate an increased frequency of mutations in the gene for Gaucher disease, glucocerebrosidase (GBA), among patients with Parkinson disease. An international collaborative study was conducted to ascertain the frequency of GBA mutations in ethnically diverse patients with Parkinson disease. Methods Sixteen centers participated, including five from the Americas, six from Europe, two from Israel and three from Asia. Each received a standard DNA panel to compare genotyping results. Genotypes and phenotypic data from patients and controls were analyzed using multivariate logistic regression models and the Mantel Haenszel procedure to estimate odds ratios (ORs) across studies. The sample included 5691 patients (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews). Results All 16 centers could detect GBA mutations, L444P and N370S, and the two were found in 15.3% of Ashkenazi patients with Parkinson disease (ORs = 4.95 for L444P and 5.62 for N370S), and in 3.2% of non-Ashkenazi patients (ORs = 9.68 for L444P and 3.30 for N370S). GBA was sequenced in 1642 non-Ashkenazi subjects, yielding a frequency of 6.9% for all mutations, demonstrate that limited mutation screens miss half the mutant alleles. The presence of any GBA mutation was associated with an OR of 5.43 across studies. Clinically, although phenotypes varied, subjects with a GBA mutation presented earlier, and were more likely to have affected relatives and atypical manifestations. Conclusion Data collected from sixteen centers demonstrate that there is a strong association between GBA mutations and Parkinson disease. PMID:19846850

  2. Systematic Analysis of Splice-Site-Creating Mutations in Cancer.

    Science.gov (United States)

    Jayasinghe, Reyka G; Cao, Song; Gao, Qingsong; Wendl, Michael C; Vo, Nam Sy; Reynolds, Sheila M; Zhao, Yanyan; Climente-González, Héctor; Chai, Shengjie; Wang, Fang; Varghese, Rajees; Huang, Mo; Liang, Wen-Wei; Wyczalkowski, Matthew A; Sengupta, Sohini; Li, Zhi; Payne, Samuel H; Fenyö, David; Miner, Jeffrey H; Walter, Matthew J; Vincent, Benjamin; Eyras, Eduardo; Chen, Ken; Shmulevich, Ilya; Chen, Feng; Ding, Li

    2018-04-03

    For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Meta-analysis of Neuroblastomas Reveals a Skewed ALK Mutation Spectrum in Tumors with MYCN Amplification

    NARCIS (Netherlands)

    de Brouwer, Sara; de Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M.; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; van Maerken, Tom; de Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H.; Schramm, Alexander; van den Broecke, Caroline; Vermeulen, Joëlle; van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib N.; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

    2010-01-01

    Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic

  4. Evaluation of three gas chromatography and two direct mass spectrometry techniques for aroma analysis of dried red bell peppers

    NARCIS (Netherlands)

    Ruth, van S.M.; Boscaini, E.; Mayr, D.; Pugh, J.; Posthumus, M.A.

    2003-01-01

    Three gas chromatography methods and two direct mass spectrometry techniques were compared for the analysis of the aroma of rehydrated diced red bell peppers. Gas chromatography methods included systems with olfactometry detection (GC-O), flame ionisation detection (GC-FID) and mass spectrometry

  5. Ion chromatography for the precise analysis of chloride and sodium in sweat for the diagnosis of cystic fibrosis

    NARCIS (Netherlands)

    Doorn, J.; Storteboom, T. T. R.; Mulder, A. M.; de Jong, W. H. A.; Rottier, B. L.; Kema, I. P.

    BACKGROUND: Measurement of chloride in sweat is an essential part of the diagnostic algorithm for cystic fibrosis. The lack in sensitivity and reproducibility of current methods led us to develop an ion chromatography/high-performance liquid chromatography (IC/HPLC) method, suitable for the analysis

  6. Quantitative analysis of pharmaceuticals in biological fluids using high-performance liquid chromatography coupled to mass spectrometry: a review.

    Science.gov (United States)

    Plumb, R S; Dear, G J; Mallett, D N; Higton, D M; Pleasance, S; Biddlecombe, R A

    2001-01-01

    1. The development of bio-analysis of drug molecules over the last 10 years is reviewed, focusing on advances in sample preparation, liquid chromatography and detection. 2. Developments have led to improvements in detection sensitivity, enhancements in specificity and increased capacity. 3. Emerging technologies such as monolithic column chromatography and miniaturized chip-based systems are discussed.

  7. Liquid chromatography mass spectrometry for analysis of microbial metabolites

    DEFF Research Database (Denmark)

    Klitgaard, Andreas

    to human health. Because of this, methods for detection and analysis of these compounds are vital. Estimates suggest that there are around 1.5 million different fungal species on Earth, dwarfing the number of plants estimated to 300,000, meaning that there potentially are many more interesting compounds...... is of large commercial interest for production of the bioactive compounds of the future. One part of my study focused on identification and elucidation of the biosynthesis of a nonribosomal peptide (NRP) nidulanin A from Aspergillus nidulans. Although the study was successful several analogs were......Filamentous fungi serve a very important role in Nature where they break down organic matter, releasing nutrients that can be used by other organisms. Fungi and other microorganisms also produce a wide array of bioactive compounds, the secondary metabolites( SMs), used for such diverse roles...

  8. Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

    Directory of Open Access Journals (Sweden)

    Fernández-Rodríguez Juana

    2012-03-01

    Full Text Available Abstract Background Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. Methods The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. Results This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes in a set of controls (138 women and 146 men did not detect seven of them. Conclusions Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.

  9. Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

    International Nuclear Information System (INIS)

    Fernández-Rodríguez, Juana; Schindler, Detlev; Capellá, Gabriel; Brunet, Joan; Lázaro, Conxi; Pujana, Miguel Angel; Quiles, Francisco; Blanco, Ignacio; Teulé, Alex; Feliubadaló, Lídia; Valle, Jesús del; Salinas, Mónica; Izquierdo, Àngel; Darder, Esther

    2012-01-01

    Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them. Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease

  10. Mutational Analysis of Oculocutaneous Albinism: A Compact Review

    Science.gov (United States)

    Kamaraj, Balu

    2014-01-01

    Oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by either complete lack of or a reduction of melanin biosynthesis in the melanocytes. The OCA1A is the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3, and OCA4 show some pigment accumulation over time. Mutations in TYR, OCA2, TYRP1, and SLC45A2 are mainly responsible for causing oculocutaneous albinism. Recently, two new genes SLC24A5 and C10orf11 are identified that are responsible to cause OCA6 and OCA7, respectively. Also a locus has been mapped to the human chromosome 4q24 region which is responsible for genetic cause of OCA5. In this paper, we summarized the clinical and molecular features of OCA genes. Further, we reviewed the screening of pathological mutations of OCA genes and its molecular mechanism of the protein upon mutation by in silico approach. We also reviewed TYR (T373K, N371Y, M370T, and P313R), OCA2 (R305W), TYRP1 (R326H and R356Q) mutations and their structural consequences at molecular level. It is observed that the pathological genetic mutations and their structural and functional significance of OCA genes will aid in development of personalized medicine for albinism patients. PMID:25093188

  11. Simplifying the detection of MUTYH mutations by high resolution melting analysis

    International Nuclear Information System (INIS)

    López-Villar, Isabel; Martínez-López, Joaquín; Ayala, Rosa; Wesselink, Jan; Morillas, Juan Diego; López, Elena; Marín, José Carlos; Díaz-Tasende, José; González, Sara; Robles, Luis

    2010-01-01

    MUTYH-associated polyposis (MAP) is a disorder caused by bi-allelic germline MUTYH mutation, characterized by multiple colorectal adenomas. In order to identify mutations in MUTYH gene we applied High Resolution Melting (HRM) genotyping. HRM analysis is extensively employed as a scanning method for the detection of heterozygous mutations. Therefore, we applied HRM to show effectiveness in detecting homozygous mutations for these clinically important and frequent patients. In this study, we analyzed phenotype and genotype data from 82 patients, with multiple (>= 10) synchronous (19/82) or metachronous (63/82) adenomas and negative APC study (except one case). Analysis was performed by HRM-PCR and direct sequencing, in order to identify mutations in MUTYH exons 7, 12 and 13, where the most prevalent mutations are located. In monoallelic mutation carriers, we evaluated entire MUTYH gene in search of another possible alteration. HRM-PCR was performed with strict conditions in several rounds: the first one to discriminate the heteroduplex patterns and homoduplex patterns and the next ones, in order to refine and confirm parameters. The genotypes obtained were correlated to phenotypic features (number of adenomas (synchronous or metachronous), colorectal cancer (CRC) and family history). MUTYH germline mutations were found in 15.8% (13/82) of patients. The hot spots, Y179C (exon 7) and G396D (exon 13), were readily identified and other mutations were also detected. Each mutation had a reproducible melting profile by HRM, both heterozygous mutations and homozygous mutations. In our study of 82 patients, biallelic mutation is associated with being a carrier of ≥10 synchronous polyps (p = 0.05) and there is no association between biallelic mutation and CRC (p = 0.39) nor family history (p = 0.63). G338H non-pathogenic polymorphism (exon 12) was found in 23.1% (19/82) of patients. In all cases there was concordance between HRM (first and subsequent rounds) and sequencing

  12. [Study on the analysis of organogermanium compounds by ion chromatography].

    Science.gov (United States)

    Chen, Q; Mou, S; Hou, X; Ni, Z

    1997-05-01

    A new high performance ion exchange chromatographic method for separation and determination of three organogermanium compounds beta-carboxyethylgermanium sesquioxide (I), beta-(alpha-methyl) carboxyethylgermanium sesquioxide (II) and di-(beta-carboxyethyl) germanium hydroxide (III) has been developed. A Dionex DX-300 Ion Chromatograph equipped with a Dionex PED-II pulsed electrochemical detector (conductivity mode), a Dionex AMMS-1 anion micromembrane suppressor, and a Dionex ACI advanced computer interface coupled with AI-450 chromatographic software was employed. The separation was achieved by using a Dionex IonPac AS4A-SC column as analytical column, sodium tetraborate solution as eluent, and sulfuric acid solution as regenerant. For reducing run time, a gradient program was chosen. The detection limits (S/N = 3, expressed as germanium) for the three compounds were 0.038mg/L (I), 0.035mg/L (II) and 0.025mg/L (III), respectively. The method has been applied to the analysis of two tonic oral drinks, and the average recoveries for the three compounds ranged from 95%-101%. The results obtained were in agreement with those of hydride generation atomic fluorescence spectrometry (HG-AFS).

  13. Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation

    Science.gov (United States)

    2017-01-01

    Polymeric nanoparticles have become indispensable in modern society with a wide array of applications ranging from waterborne coatings to drug-carrier-delivery systems. While a large range of techniques exist to determine a multitude of properties of these particles, relating physicochemical properties of the particle to the chemical structure of the intrinsic polymers is still challenging. A novel, highly orthogonal separation system based on comprehensive two-dimensional liquid chromatography (LC × LC) has been developed. The system combines hydrodynamic chromatography (HDC) in the first-dimension to separate the particles based on their size, with ultrahigh-performance size-exclusion chromatography (SEC) in the second dimension to separate the constituting polymer molecules according to their hydrodynamic radius for each of 80 to 100 separated fractions. A chip-based mixer is incorporated to transform the sample by dissolving the separated nanoparticles from the first-dimension online in tetrahydrofuran. The polymer bands are then focused using stationary-phase-assisted modulation to enhance sensitivity, and the water from the first-dimension eluent is largely eliminated to allow interaction-free SEC. Using the developed system, the combined two-dimensional distribution of the particle-size and the molecular-size of a mixture of various polystyrene (PS) and polyacrylate (PACR) nanoparticles has been obtained within 60 min. PMID:28745485

  14. Application of DNA chips in the analysis of gene mutation in HBV

    International Nuclear Information System (INIS)

    Wang Yongzhong; Ruan Lihua; Zhou Guoping; Wu Guoxiang; Chen Min

    2005-01-01

    Objective: To investigate the clinical applicability of DNA chips for analysis of gene mutation in HBV. Methods: Serum HBV DNA from 47 patients with viral hepatitis type B was amplified with PCR. Possible gene mutations were searched for in site 1896 of pre-C section, sites 1762,1764 of BCP section and sites 528, 552 of P section with DNA chip method based upon membrane coloration. Results: In the 32 patients without lamivudine treatment, the results were as follows: (1) 6 specimens with HBsAg + , HBeAg + , HBeAb - , no mutations observed. (2) 13 specimens with HBsAg + , HBeAg - , HBeAb + , mutations at site 1896, pre- C 4 cases, mutations at sites 1762,1764, BCP 11 cases. (3) 13 specimens with HBsAg + , HBeAg + , HBeAb + , mutations at site 1896 pre -C 4 cases, mutations at sites 1762,1764 BCP 13 cases. In the 15 patients after 48 weeks treatment with lamivudine but remained HBV DNA positive, mutations were observed at: site 1896 pre-C, 5 cases, sites 1762,1764 BCP, 6 cases, site 528 P section, 2 cases, site 552 P section, YVDD 4 cases, YIDD 7 cases. Conclusion: Mutations at sites 1896, 1762,1764 were more frequent in patients with HBeAb + and were related to the negative expression of HBeAg, Mutations at 1762,1764 BCP were closely related to the changes of HBeAg/HBeAb. P section mutations were only observed after lamivadine treatment and were related to resistance against the drug. DNA chip method based upon membrane coloration for detection of gene mutation was expedient and specific and worth popularization. (authors)

  15. BRAF, KIT, NRAS, GNAQ and GNA11 mutation analysis in cutaneous melanomas in Turkish population

    Directory of Open Access Journals (Sweden)

    Ismail Yilmaz

    2015-01-01

    Full Text Available Background: KIT and mitogen-activated protein kinase cascade are important for melanomagenesis. In the present study, we analyzed the frequency of BRAF, NRAS, KIT, GNAQ and GNA11 gene mutations and investigated their association with clinicopathological features of melanomas in Turkish population. Materials and Methods: Forty-seven primary cutaneous melanomas were included in our study. Sanger sequencing method was used for mutation analysis in all cases. Results: Mean age was 62.1 (29-101 years. Female:male ratio was 17:30. Among 47 melanomas, 14 (29.8% BRAF, 10 (21.3% NRAS, 4 (8.5% KIT and 1(2.1% GNAQ gene mutations were detected. Two of the KIT mutations were found in acral lentiginous melanoma (ALM. In the head and neck region, mutation frequency was significantly lower than in other locations (P = 0.035. The only GNAQ gene mutation (p.Q209L was detected in a melanoma arising from blue nevus located on the scalp. None of the melanomas harbored NRAS exon 2, KIT exon 13/17/18, GNAQ exon 4 and GNA11 exon 4/5 mutations. Overall mutation frequency did not show significant difference between metastatic (8/14, 57.1% and nonmetastatic (18/33, 54.5% patients. We did not observe any significant association between mutation status and gender or age of various patients. Conclusions: Our results support that BRAF and NRAS gene mutations are common in cutaneous melanomas. The activating mutations of KIT gene are rare and especially seen in ALM. GNAQ and GNA11 mutations are infrequent in cutaneous melanomas and may be associated only with melanomas arising from blue nevus.

  16. [Recent advances in analysis of petroleum geological samples by comprehensive two-dimensional gas chromatography].

    Science.gov (United States)

    Gao, Xuanbo; Chang, Zhenyang; Dai, Wei; Tong, Ting; Zhang, Wanfeng; He, Sheng; Zhu, Shukui

    2014-10-01

    Abundant geochemical information can be acquired by analyzing the chemical compositions of petroleum geological samples. The information obtained from the analysis provides scientifical evidences for petroleum exploration. However, these samples are complicated and can be easily influenced by physical (e. g. evaporation, emulsification, natural dispersion, dissolution and sorption), chemical (photodegradation) and biological (mainly microbial degradation) weathering processes. Therefore, it is very difficult to analyze the petroleum geological samples and they cannot be effectively separated by traditional gas chromatography/mass spectrometry. A newly developed separation technique, comprehensive two-dimensional gas chromatography (GC x GC), has unique advantages in complex sample analysis, and recently it has been applied to petroleum geological samples. This article mainly reviews the research progres- ses in the last five years, the main problems and the future research about GC x GC applied in the area of petroleum geology.

  17. Taste characteristics of Chinese bayberry juice characterized by sensory evaluation, chromatography analysis, and an electronic tongue.

    Science.gov (United States)

    Yu, Haiyan; Zhang, Yan; Zhao, Jie; Tian, Huaixiang

    2018-05-01

    To evaluate the taste characteristics of Chinese bayberry juice, four types of bayberry juice sourced from different origins and varieties were analysed using sensory evaluation, chromatography, spectroscopy analysis and an electronic tongue (E-tongue). Nine organic acids and three sugars were assessed using high performance liquid chromatography. Total polyphenols were measured by spectrophotometry. The overall taste profile was collected using the E-tongue. The four types of bayberry juice differed in the sensory attributes of sour, sweet, bitter, and astringent. The E-tongue responses combined with discriminant analysis were able to characterise the taste profiles of the juices. The relationships between the taste compounds and the sensory panel scores established by partial least squares showed that total polyphenols, quininic acid, maleic acid, fructose, citric acid, lactic acid, succinic acid and sucrose made significant contributions to the taste characteristics of the Chinese bayberry juice.

  18. Analysis of polymeric phenolics in red wines using different techniques combined with gel permeation chromatography fractionation.

    Science.gov (United States)

    Guadalupe, Zenaida; Soldevilla, Alberto; Sáenz-Navajas, María-Pilar; Ayestarán, Belén

    2006-04-21

    A multiple-step analytical method was developed to improve the analysis of polymeric phenolics in red wines. With a common initial step based on the fractionation of wine phenolics by gel permeation chromatography (GPC), different analytical techniques were used: high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-mass spectrometry (MS), capillary zone electrophoresis (CZE) and spectrophotometry. This method proved to be valid for analyzing different families of phenolic compounds, such as monomeric phenolics and their derivatives, polymeric pigments and proanthocyanidins. The analytical characteristics of fractionation by GPC were studied and the method was fully validated, yielding satisfactory statistical results. GPC fractionation substantially improved the analysis of polymeric pigments by CZE, in terms of response, repeatability and reproducibility. It also represented an improvement in the traditional vanillin assay used for proanthocyanidin (PA) quantification. Astringent proanthocyanidins were also analyzed using a simple combined method that allowed these compounds, for which only general indexes were available, to be quantified.

  19. Polymorphism and mutation analysis of genomic DNA on cancer

    International Nuclear Information System (INIS)

    Ohta, Tsutomu

    2003-01-01

    DNA repair is a universal process in living cells that maintains the structural integrity of chromosomal DNA molecules in face of damage. A deficiency in DNA damage repair is associated with an increased cancer risk by increasing a mutation frequency of cancer-related genes. Variation in DNA repair capacity may be genetically determined. Therefore, we searched single-nucleotide polymorphisms (SNPs) in major DNA repair genes. This led to the finding of 600 SNPs and mutations including many novel SNPs in Japanese population. Case-control studies to explore the contribution of the SNPs in DNA repair genes to the risk of lung cancer revealed that five SNPs are associated with lung carcinogenesis. One of these SNPs is found in RAD54L gene, which is involved in double-strand DNA repair. We analyzed and reported activities of Rad54L protein with SNP and mutations. (authors)

  20. Domain-restricted mutation analysis to identify novel driver events in human cancer

    Directory of Open Access Journals (Sweden)

    Sanket Desai

    2017-10-01

    Full Text Available Analysis of mutational spectra across various cancer types has given valuable insights into tumorigenesis. Different approaches have been used to identify novel drivers from the set of somatic mutations, including the methods which use sequence conservation, geometric localization and pathway information. Recent computational methods suggest use of protein domain information for analysis and understanding of the functional consequence of non-synonymous mutations. Similarly, evidence suggests recurrence at specific position in proteins is robust indicators of its functional impact. Building on this, we performed a systematic analysis of TCGA exome derived somatic mutations across 6089 PFAM domains and significantly mutated domains were identified using randomization approach. Multiple alignment of individual domain allowed us to prioritize for conserved residues mutated at analogous positions across different proteins in a statistically disciplined manner. In addition to the known frequently mutated genes, this analysis independently identifies low frequency Meprin and TRAF-Homology (MATH domain in Speckle Type BTB/POZ (SPOP protein, in prostate adenocarcinoma. Results from this analysis will help generate hypotheses about the downstream molecular mechanism resulting in cancer phenotypes.

  1. [Analysis of gene mutation in a Chinese family with Norrie disease].

    Science.gov (United States)

    Zhang, Tian-xiao; Zhao, Xiu-li; Hua, Rui; Zhang, Jin-song; Zhang, Xue

    2012-09-01

    To detect the pathogenic mutation in a Chinese family with Norrie disease. Clinical diagnosis was based on familial history, clinical sign and B ultrasonic examination. Peripheral blood samples were obtained from all available members in a Chinese family with Norrie disease. Genomic DNA was extracted from lymphocytes by the standard SDS-proteinase K-phenol/chloroform method. Two coding exons and all intron-exon boundaries of the NDP gene were PCR amplified using three pairs of primers and subjected to automatic DNA sequence. The causative mutation was confirmed by restriction enzyme analysis and genotyping analysis in all members. Sequence analysis of NDP gene revealed a missense mutation c.220C > T (p.Arg74Cys) in the proband and his mother. Further mutation identification by restriction enzyme analysis and genotyping analysis showed that the proband was homozygote of this mutation. His mother and other four unaffected members (III3, IV4, III5 and II2) were carriers of this mutation. The mutant amino acid located in the C-terminal cystine knot-like domain, which was critical motif for the structure and function of NDP. A NDP missense mutation was identified in a Chinese family with Norrie disease.

  2. Core-Shell Columns in High-Performance Liquid Chromatography: Food Analysis Applications

    OpenAIRE

    Preti, Raffaella

    2016-01-01

    The increased separation efficiency provided by the new technology of column packed with core-shell particles in high-performance liquid chromatography (HPLC) has resulted in their widespread diffusion in several analytical fields: from pharmaceutical, biological, environmental, and toxicological. The present paper presents their most recent applications in food analysis. Their use has proved to be particularly advantageous for the determination of compounds at trace levels or when a large am...

  3. RECENT ADVANCES IN ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE ANALYSIS OF TRADITIONAL CHINESE MEDICINE

    Science.gov (United States)

    Huang, Huilian; Liu, Min; Chen, Pei

    2014-01-01

    Traditional Chinese medicine has been widely used for the prevention and treatment of various diseases for thousands of years in China. Ultra-high performance liquid chromatography (UHPLC) is a relatively new technique offering new possibilities. This paper reviews recent developments in UHPLC in the separation and identification, fingerprinting, quantification, and metabolism of traditional Chinese medicine. Recently, the combination of UHPLC with MS has improved the efficiency of the analysis of these materials. PMID:25045170

  4. Analysis of Bacterial Vaginosis-Related Amines in Vaginal Fluid by Gas Chromatography and Mass Spectrometry

    OpenAIRE

    Wolrath, Helen; Forsum, Urban; Larsson, P. G.; Borén, Hans

    2001-01-01

    The presence of various amines in vaginal fluid from women with malodorous vaginal discharge has been reported before. The investigations have used several techniques to identify the amines. However, an optimized quantification, together with a sensitive analysis method in connection with a diagnostic procedure for vaginal discharge, including the syndrome of bacterial vaginosis, as defined by the accepted “gold standard,” has not been done before. We now report a sensitive gas chromatographi...

  5. High-performance liquid ion-pair chromatography in inorganic analysis

    International Nuclear Information System (INIS)

    Alimarin, I.P.; Basova, E.M.; Bol'shova, T.A.; Ivanov, V.M.

    1990-01-01

    In literature review for the recent 15 years theoretical foundations, regularities and mechanisms of ionized compound retention in reverse-phase ion-pair chromatography are considered, possibilities and prospects of its application in inorganic analysis being demonstrated. Analytic characteristics of the methods for the determination of inorganic anions (I - , IO 3 - , MoO 4 2- , etc.), as well as metals (Zr, Hf, V, Nb, Mo, W, Ru, lanthanides, etc.) in the form of chelates, are given

  6. Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

    Science.gov (United States)

    Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

    2001-10-30

    The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular

  7. Expression analysis revealing destabilizing mutations in phosphomannomutase 2 deficiency (PMM2-CDG): expression analysis of PMM2-CDG mutations.

    Science.gov (United States)

    Vega, Ana Isabel; Pérez-Cerdá, Celia; Abia, David; Gámez, Alejandra; Briones, Paz; Artuch, Rafael; Desviat, Lourdes R; Ugarte, Magdalena; Pérez, Belén

    2011-08-01

    Deficiency of phosphomannomutase (PMM2, MIM#601785) is the most common congenital disorder of glycosylation. Herein we report the genetic analysis of 22 Spanish PMM2 deficient patients and the functional analysis of 14 nucleotide changes in a prokaryotic expression system in order to elucidate their molecular pathogenesis. PMM2 activity assay revealed the presence of six protein changes with no enzymatic activities (p.R123Q, p.R141H, p.F157S, p.P184T, p.F207S and p.D209G) and seven mild protein changes with residual activities ranging from 16 to 54% (p.L32R, p.V44A p.D65Y, p.P113L p.T118S, p.T237M and p.C241S) and also one variant change with normal activity (p.E197A). The results obtained from Western blot analysis, degradation time courses of 11 protein changes and structural analysis of the PMM2 protein, suggest that the loss-of-function of most mutant proteins is based on their increased susceptibility to degradation or aggregation compared to the wild type protein, considering PMM2 deficiency as a conformational disease. We have identified exclusively catalytic protein change (p.D209G), catalytic protein changes affecting protein stability (p.R123Q and p.R141H), two protein changes disrupting the dimer interface (p.P113L and p.T118S) and several misfolding changes (p.L32R, p.V44A, p.D65Y, p.F157S, p.P184T, p.F207S, p.T237M and p.C241S). Our current work opens a promising therapeutic option using pharmacological chaperones to revert the effect of the characterized misfolding mutations identified in a wide range of PMM2 deficient patients.

  8. Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

    Science.gov (United States)

    Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

    2011-01-17

    Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  9. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  10. Detailed analysis of petroleum cuts by multidimensional gas chromatography; Analyse detaillee de coupes petrolieres par chromatographie en phase gazeuse multidimensionnelle

    Energy Technology Data Exchange (ETDEWEB)

    Vendeuvre, C.

    2006-01-15

    The limitations of petroleum resources implying a better valorisation of crude oil through the optimisation of production, refinery and petrochemistry processes, as well as the environmental regulations have strengthened the necessity of more detailed characterisation of petroleum products. In order to take up this challenge, efficient analytical tools have to be developed. This work demonstrates that comprehensive two-dimensional gas chromatography (GCxGC) constitutes a major advance compared to GC owing to its improved resolution power and to the structured chromatograms indicating the polarity and the volatility of hydrocarbons. The principle of GCxGC is based on the analysis of a whole sample in two independent dimensions of separation achieved using two GC columns of different selectivities; between the two columns a modulator device samples, focuses and re-injects small portions of the effluent from the first column into the second one. Since its introduction in 1991, GCxGC has known a rapid growth and has received a wide acceptance by the analytical science community. The competitive situation has considerably evolved during this thesis with the introduction of commercial systems and the two first sessions of an international symposium dedicated to this technique (Volendam, 2003 and Atlanta, 2004). The key points of the thesis concern the development of a GCxGC prototype system using dual jets CO{sub 2} technology and a data processing program; the evaluation of a retention model allowing a rational choice of operating conditions; and the application of this technique to various and complex issues. Thus, effluents from petrochemistry, refinery or pollution areas have been analysed according to the chemical classes of hydrocarbons and to their number of carbon atoms; a new method for obtaining distillation curves for each chemical group was also presented. Furthermore, the hyphenation of GCxGC with a specific sulphur detector revealed a great interest for

  11. Mutational analysis in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD): Identification of five mutations in the PKD1 gene.

    Science.gov (United States)

    Abdelwahed, Mayssa; Hilbert, Pascale; Ahmed, Asma; Mahfoudh, Hichem; Bouomrani, Salem; Dey, Mouna; Hachicha, Jamil; Kamoun, Hassen; Keskes-Ammar, Leila; Belguith, Neïla

    2018-05-31

    Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most frequent genetic disorder of the kidneys, is characterized by a typical presenting symptoms include cysts development in different organs and a non-cysts manifestations. ADPKD is caused by mutations in PKD1 or PKD2 genes. In this study, we aimed to search for molecular causative defects among PKD1 and PKD2 genes. Eighteen patients were diagnosed based on renal ultrasonography and renal/extra-renal manifestations. Then, Sanger sequencing was performed for PKD1 and PKD2 genes. Multiplex Ligation dependent Probe Amplification method (MLPA) methods was performed for both PKD genes. Mutational analysis of the PKD2 gene revealed the absence of variants and no deletions or duplications of both PKD genes were detected. But three novels mutations i.e. p.S463C exon 7; c. c.11156+2T>C IVS38 and c.8161-1G>A IVS22 and two previously reported c.1522T>C exon 7 and c.412C>T exon 4 mutations in the PKD1 gene were detected. Bioinformatics tools predicted that the novel variants have a pathogenic effects on splicing machinery, pre-mRNA secondary structure and stability and protein stability. Our results highlighted molecular features of Tunisian patients with ADPKD and revealed novel variations that can be utilized in clinical diagnosis and in the evaluation of living kidney donor. To the best of our knowledge, this is the first report of Autosomal Polycystic Kidney Disease in Tunisia. Copyright © 2017. Published by Elsevier B.V.

  12. Intragenic haplotype analysis of common HFE mutations in the ...

    Indian Academy of Sciences (India)

    mutation <100 generations ago in the Celtic populations of mainland Europe, with a ... 0.9–5.8%, evidencing regional differences in distribution across the ..... and H63D alleles in the HFE gene among various Jewish ethnic groups in Israel: a ...

  13. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    Science.gov (United States)

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation AnalysisPhouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.<...

  14. A novel genetic tool for clonal analysis of fourth chromosome mutations

    OpenAIRE

    Sousa-Neves, Rui; Schinaman, Joseph M.

    2012-01-01

    The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FR...

  15. Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

    Directory of Open Access Journals (Sweden)

    Lorena Suarez-Artiles

    2018-01-01

    Full Text Available Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr and c.2581G>C; p.(Ala861Pro abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.

  16. Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis.

    Science.gov (United States)

    Hetzel, Terence; Loeker, Denise; Teutenberg, Thorsten; Schmidt, Torsten C

    2016-10-01

    The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2 μm fully porous as well as the 2.7 μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2 μm fully porous particle packed column for ultra-fast liquid chromatography. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Ligand exchange chromatography for analysis and preparative separation of tritium-labelled amino acids

    International Nuclear Information System (INIS)

    Zolotarev, Yu.A.; Zaitsev, D.A.; Penkina, V.I.; Dostavalov, I.N.; Myasoedov, N.F.

    1988-01-01

    Racemic tritium-labelled amino acids were separated into optical isomers by chromatography on a chiral polyacrylamide sorbent filled with copper ions. The polyacrylamide sorbent is synthesized by Mannich's reaction through the action of formaldehyde and L-phenylalanine upon polyacrylamide Biogel P-4 in an alkali phosphate buffer. Tritium-labelled amino acids are eluted by a weak alkali solution of ammonium carbonate. Data are presented on the ligand exchange chromatography of amino acids depending on the degree to which the sorbent is filled with copper ions and on the eluent concentration. Amino acids are isolated from the eluent on short columns filled with sulfonated cation exchanger in the H + form. HPLC on modified silica gel sorbents is also used for the analysis of tritium-labelled optically active amino acids. Amino acids are eluted by a weakly acidic water-methanol solution containing ammonium acetate. UV and scintillation flow type detectors are used. (author) 7 refs.; 8 figs

  18. Identifying Natural syNergist from Pongamia pinnata Using High-Speed Counter-Current Chromatography Combined with Isobolographic Analysis

    Directory of Open Access Journals (Sweden)

    Hao Yin

    2017-03-01

    Full Text Available For identifying the synergistic compounds from Pongamia pinnata, an approach based on high-speed counter-current chromatography (HSCCC combined with isobolographic analysis was designed to detect the synergistic effects in the complex mixture [...

  19. EXTRACTION AND QUANTITATIVE ANALYSIS OF ELEMENTAL SULFUR FROM SULFIDE MINERAL SURFACES BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY. (R826189)

    Science.gov (United States)

    A simple method for the quantitative determination of elemental sulfur on oxidized sulfide minerals is described. Extraction of elemental sulfur in perchloroethylene and subsequent analysis with high-performance liquid chromatography were used to ascertain the total elemental ...

  20. Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

    Science.gov (United States)

    Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum

    2017-07-25

    Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

  1. Illicit Drug Analysis Using Two-Dimension Liquid Chromatography/Tandem Mass Spectrometry.

    Science.gov (United States)

    Mallet, Claude; Botch-Jones, Sabra

    2016-10-01

    For the identification of illicit drugs in forensic toxicological casework, analysis can be delayed and potentially compromised due to lengthy sample preparation techniques. For a complete forensic identification, a robust methodology is required and the current trend in forensic laboratories is the use of liquid chromatography coupled to mass spectrometry (LC/MS or LC/MS-MS). However, to achieve satisfactory results, extensive and time-consuming sample preparation protocols are required to reach sub-ng/mL levels. The concept of sequential 2D extraction was designed to capture the retention behavior of a target analyte in response to various extraction parameters. Therefore, optimized conditions can be selected to excise a region of interest during extraction. The utilization of multi-dimensional chromatography combined with a micro-extraction technique was evaluated to decrease sample preparation time while enhancing the separation integrity observed with current single-dimensional chromatography techniques. A wide range of illicit drugs were spiked in human urine and extracted using three extraction protocols for performance evaluation. The extraction process was performed using a reversed-phase solid phase extraction (SPE) in 1D, 2D-optimized, 2D-sequential and cumulative elution modes. The chosen 2D chromatography conditions that were used in this application were identified using a 6 × 6 automated methods development protocol (144 methods total). The extraction of urine samples containing target analytes was completed in less than 20 min. The analysis was performed using 200 µL of the final organic solvent (MeOH) extracts. The limit of detection for all drugs was measured at 100 pg/mL (ppt) from a 1 mL sample volume. Several analytes showed excellent signal at 10 pg/mL (ppt). © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Mutation analysis of the STRA6 gene in isolated and non-isolated anophthalmia/microphthalmia.

    Science.gov (United States)

    Chassaing, N; Ragge, N; Kariminejad, A; Buffet, A; Ghaderi-Sohi, S; Martinovic, J; Calvas, P

    2013-03-01

    PDAC syndrome [Pulmonary hypoplasia/agenesis, Diaphragmatic hernia/eventration, Anophthalmia/microphthalmia (A/M) and Cardiac Defect] is a condition associated with recessive mutations in the STRA6 gene in some of these patients. Recently, cases with isolated anophthalmia have been associated with STRA6 mutations. To determine the minimal findings associated with STRA6 mutations, we performed mutation analysis of the STRA6 gene in 28 cases with anophthalmia. In 7 of the cases the anophthalmia was isolated, in 14 cases it was associated with one of the major features included in PDAC and 7 had other abnormalities. Mutations were identified in two individuals: one with bilateral anophthalmia and some features included in PDAC, who was a compound heterozygote for a missense mutation and a large intragenic deletion, and the second case with all the major features of PDAC and who had a homozygous splicing mutation. This study suggests that STRA6 mutations are more likely to be identified in individuals with A/M and other abnormalities included in the PDAC spectrum, rather than in isolated A/M cases. © 2012 John Wiley & Sons A/S.

  3. Whole exome analysis identifies frequent CNGA1 mutations in Japanese population with autosomal recessive retinitis pigmentosa.

    Directory of Open Access Journals (Sweden)

    Satoshi Katagiri

    Full Text Available OBJECTIVE: The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP in the Japanese population. METHODS: In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed. RESULTS: Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients, EYS (three patients and SAG (one patient in eight patients and potential disease-causing gene variants of USH2A (two patients, EYS (one patient, TULP1 (one patient and C2orf71 (one patient in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation. CONCLUSIONS: This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients. CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.

  4. [Gene mutation analysis and prenatal diagnosis of a family with Bartter syndrome].

    Science.gov (United States)

    Li, Long; Ma, Na; Li, Xiu-Rong; Gong, Fei; DU, Juan

    2016-08-01

    To investigate the mutation of related genes and prenatal diagnosis of a family with Bartter syndrome (BS). The high-throughput capture sequencing technique and PCR-Sanger sequencing were used to detect pathogenic genes in the proband of this family and analyze the whole family at the genomic level. After the genetic cause was clarified, the amniotic fluid was collected from the proband's mother who was pregnant for 5 months for prenatal diagnosis. The proband carried compound heterozygous mutations of c.88C>T(p.Arg30*) and c.968+2T>A in the CLCNKB gene; c.88C>T(p.Arg30*) had been reported as a pathogenic mutation, and c.968+2T>A was a new mutation. Pedigree analysis showed that the two mutations were inherited from the mother and father, respectively. Prenatal diagnosis showed that the fetus did not inherit the mutations from parents and had no mutations at the two loci. The follow-up visit confirmed that the infant was in a healthy state, which proved the accuracy of genetic diagnosis and prenatal diagnosis. The compound heterozygous mutations c.88C>T(p.Arg30*) and c.968+2T>A in the CLCNKB gene are the cause of BS in the proband, and prenatal diagnosis can prevent the risk of recurrence of BS in this family.

  5. CFTR mutation analysis and haplotype associations in CF patients☆

    OpenAIRE

    Cordovado, S.K.; Hendrix, M.; Greene, C.N.; Mochal, S.; Earley, M.C.; Farrell, P.M.; Kharrazi, M.; Hannon, W.H.; Mueller, P.W.

    2011-01-01

    Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention’s NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and por...

  6. Role of BRAFV600E Mutation Analysis for Thyroid Nodules Classified as Indeterminate on Ultrasonography

    International Nuclear Information System (INIS)

    Nam, Sang Yu; Shin, Jung Hee; Han, Boo Kyung; Ko, Eun Young; Kang, Seok Seon; Hahn, Soo Yeon; Hwang, Ji Young; Nam, Mee Young; Kim, Jong Won; Chung, Jae Hoon

    2010-01-01

    We aimed to evaluate a possible role for BRAFV600E mutation analysis of aspiration specimens in the work up of thyroid nodules classified as indeterminate on US. A total of 122 nodules from 122 patients were prospectively classified as indeterminate nodules based on US findings. US-guided fine needle aspiration (FNA) was done for all 122 nodules. The presence of a BRAFV600E mutation in FNA specimens was determined by allele-specific PCR. US-indeterminate nodules were confirmed as malignant in 20.5% (25/122) of cases and benign in 76.2% (93/122) after FNA or surgery. A few (3.3% (4/122), remained indeterminate. A BRAFV600E mutation was identified in 14.8% (18/122) of US indeterminate nodules. Of those 18 nodules, three were benign and 13 were malignant after the initial FNA. One (0.8%, 1/122) with an initially benign cytology and a BRAFV600E mutation was confirmed to be malignant after surgery. The remaining two benign nodules with a mutation were not followed-up. All 9 initial FNA-nondiagnostic nodules were mutation negative but 2 (11.8%) of 17 indeterminate nodules on initial FNAs were mutation positive. BRAFV600E mutation analysis prevents false negative cytology for only 0.8% of cases and reduces ambiguous diagnoses for 1.6% of all US-indeterminate thyroid nodules. Therefore, adding BRAFV600E mutation analysis to FNA for US-indeterminate nodules is of limited usefulness

  7. The TREAT-NMD DMD Global Database: Analysis of More than 7,000 Duchenne Muscular Dystrophy Mutations

    Science.gov (United States)

    Bladen, Catherine L; Salgado, David; Monges, Soledad; Foncuberta, Maria E; Kekou, Kyriaki; Kosma, Konstantina; Dawkins, Hugh; Lamont, Leanne; Roy, Anna J; Chamova, Teodora; Guergueltcheva, Velina; Chan, Sophelia; Korngut, Lawrence; Campbell, Craig; Dai, Yi; Wang, Jen; Barišić, Nina; Brabec, Petr; Lahdetie, Jaana; Walter, Maggie C; Schreiber-Katz, Olivia; Karcagi, Veronika; Garami, Marta; Viswanathan, Venkatarman; Bayat, Farhad; Buccella, Filippo; Kimura, En; Koeks, Zaïda; van den Bergen, Janneke C; Rodrigues, Miriam; Roxburgh, Richard; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Zimowski, Janusz; Santos, Rosário; Neagu, Elena; Artemieva, Svetlana; Rasic, Vedrana Milic; Vojinovic, Dina; Posada, Manuel; Bloetzer, Clemens; Jeannet, Pierre-Yves; Joncourt, Franziska; Díaz-Manera, Jordi; Gallardo, Eduard; Karaduman, A Ayşe; Topaloğlu, Haluk; El Sherif, Rasha; Stringer, Angela; Shatillo, Andriy V; Martin, Ann S; Peay, Holly L; Bellgard, Matthew I; Kirschner, Jan; Flanigan, Kevin M; Straub, Volker; Bushby, Kate; Verschuuren, Jan; Aartsma-Rus, Annemieke; Béroud, Christophe; Lochmüller, Hanns

    2015-01-01

    Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations). PMID:25604253

  8. Mutation analysis of 272 Spanish families affected by autosomal recessive retinitis pigmentosa using a genotyping microarray.

    Science.gov (United States)

    Ávila-Fernández, Almudena; Cantalapiedra, Diego; Aller, Elena; Vallespín, Elena; Aguirre-Lambán, Jana; Blanco-Kelly, Fiona; Corton, M; Riveiro-Álvarez, Rosa; Allikmets, Rando; Trujillo-Tiebas, María José; Millán, José M; Cremers, Frans P M; Ayuso, Carmen

    2010-12-03

    Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive loss of vision. The aim of this study was to identify the causative mutations in 272 Spanish families using a genotyping microarray. 272 unrelated Spanish families, 107 with autosomal recessive RP (arRP) and 165 with sporadic RP (sRP), were studied using the APEX genotyping microarray. The families were also classified by clinical criteria: 86 juveniles and 186 typical RP families. Haplotype and sequence analysis were performed to identify the second mutated allele. At least one-gene variant was found in 14% and 16% of the juvenile and typical RP groups respectively. Further study identified four new mutations, providing both causative changes in 11% of the families. Retinol Dehydrogenase 12 (RDH12) was the most frequently mutated gene in the juvenile RP group, and Usher Syndrome 2A (USH2A) and Ceramide Kinase-Like (CERKL) were the most frequently mutated genes in the typical RP group. The only variant found in CERKL was p.Arg257Stop, the most frequent mutation. The genotyping microarray combined with segregation and sequence analysis allowed us to identify the causative mutations in 11% of the families. Due to the low number of characterized families, this approach should be used in tandem with other techniques.

  9. Analysis of trace dicyandiamide in stream water using solid phase extraction and liquid chromatography UV spectrometry.

    Science.gov (United States)

    Qiu, Huidong; Sun, Dongdi; Gunatilake, Sameera R; She, Jinyan; Mlsna, Todd E

    2015-09-01

    An improved method for trace level quantification of dicyandiamide in stream water has been developed. This method includes sample pretreatment using solid phase extraction. The extraction procedure (including loading, washing, and eluting) used a flow rate of 1.0mL/min, and dicyandiamide was eluted with 20mL of a methanol/acetonitrile mixture (V/V=2:3), followed by pre-concentration using nitrogen evaporation and analysis with high performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV). Sample extraction was carried out using a Waters Sep-Pak AC-2 Cartridge (with activated carbon). Separation was achieved on a ZIC(®)-Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) (50mm×2.1mm, 3.5μm) chromatography column and quantification was accomplished based on UV absorbance. A reliable linear relationship was obtained for the calibration curve using standard solutions (R(2)>0.999). Recoveries for dicyandiamide ranged from 84.6% to 96.8%, and the relative standard deviations (RSDs, n=3) were below 6.1% with a detection limit of 5.0ng/mL for stream water samples. Copyright © 2015. Published by Elsevier B.V.

  10. Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.

    Science.gov (United States)

    Bergmann, Marianne L; Sadjadi, Seyed; Schmedes, Anne

    2017-07-01

    The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Development of a sensitive and rapid method for rifampicin impurity analysis using supercritical fluid chromatography.

    Science.gov (United States)

    Li, Wei; Wang, Jun; Yan, Zheng-Yu

    2015-10-10

    A novel simple, fast and efficient supercritical fluid chromatography (SFC) method was developed and compared with RPLC method for the separation and determination of impurities in rifampicin. The separation was performed using a packed diol column and a mobile phase B (modifier) consisting of methanol with 0.1% ammonium formate (w/v) and 2% water (v/v). Overall satisfactory resolutions and peak shapes for rifampicin quinone (RQ), rifampicin (RF), rifamycin SV (RSV), rifampicin N-oxide (RNO) and 3-formylrifamycinSV (3-FR) were obtained by optimization of the chromatography system. With gradient elution of mobile phase, all of the impurities and the active were separated within 4 min. Taking full advantage of features of SFC (such as particular selectivity, non-sloping baseline in gradient elution, and without injection solvent effects), the method was successfully used for determination of impurities in rifampicin, with more impurity peaks detected, better resolution achieved and much less analysis time needed compared with conventional reversed-phase liquid chromatography (RPLC) methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Analysis of chlorophyll mutations induced by γ-rays in barley (hordeum vulgare)

    International Nuclear Information System (INIS)

    Wang Cailian; Shen Mei; Xu Gang; Zhao Kongnan; Chen Qiufang

    1991-06-01

    Thirty varieties of dormant barley seeds were irradiated with 137 Cs γ-rays. Dose-effect relations of chlorophyll mutation frequency in M 2 seedling and differences resulting from cultured types or radiosensitive types were investigated. Experimental results show that the relations between chlorophyll mutation frequency and doses can be fitted by a linear regression equation Y = A + BX. According to analysis of covariance, there is no considerable difference in various cultured types, but the difference of five different radiosensitive types is remarkable. The sensitive and intermediate types need much lower doses than other types to induce maximum chlorophyll mutation

  13. Cumulative BRCA mutation analysis in the Greek population confirms that homogenous ethnic background facilitates genetic testing.

    Science.gov (United States)

    Tsigginou, Alexandra; Vlachopoulos, Fotios; Arzimanoglou, Iordanis; Zagouri, Flora; Dimitrakakis, Constantine

    2015-01-01

    Screening for BRCA 1 and BRCA 2 mutations has long moved from the research lab to the clinic as a routine clinical genetic testing. BRCA molecular alteration pattern varies among ethnic groups which makes it already a less straightforward process to select the appropriate mutations for routine genetic testing on the basis of known clinical significance. The present report comprises an in depth literature review of the so far reported BRCA 1 and BRCA 2 molecular alterations in Greek families. Our analysis of Greek cumulative BRCA 1 and 2 molecular data, produced by several independent groups, confirmed that six recurrent deleterious mutations account for almost 60 % and 70 % of all BRCA 1 and 2 and BRCA 1 mutations, respectively. As a result, it makes more sense to perform BRCA mutation analysis in the clinic in two sequential steps, first conventional analysis for the six most prevalent pathogenic mutations and if none identified, a second step of New Generation Sequencing-based whole genome or whole exome sequencing would follow. Our suggested approach would enable more clinically meaningful, considerably easier and less expensive BRCA analysis in the Greek population which is considered homogenous.

  14. Clinical applications of fast liquid chromatography: a review on the analysis of cardiovascular drugs and their metabolites.

    Science.gov (United States)

    Baranowska, Irena; Magiera, Sylwia; Baranowski, Jacek

    2013-05-15

    One of the major challenges facing the medicine today is developing new therapies that enhance human health. To help address these challenges the utilization of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. In the last decade various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an increasingly important tool for monitoring drugs and their metabolites. Furthermore, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages since its introduction. This paper provides an overview of current trends in fast chromatography for the analysis of cardiovascular drugs and their metabolites in clinical applications. Current trends in fast liquid chromatographic separations involve monolith technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive complementary method to traditional methodology used for routine applications. The practical aspects of, recent developments in and the present status of fast chromatography for the analysis of biological fluids for therapeutic drug and metabolite monitoring, pharmacokinetic studies and bioequivalence studies are presented. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1 by denaturing high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Férec Claude

    2001-11-01

    Full Text Available Abstract Background R122, the primary autolysis site of the human cationic trypsinogen (PRSS1, constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC single nucleotide substitution, a c.365~366GC>AT (CGC>CAT gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. Materials and methods DNA samples containing either the known c.365G>A or c.365~366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC assay. Results DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C. Conclusions A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.

  16. Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

    Science.gov (United States)

    De Brouwer, Sara; De Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; Van Maerken, Tom; De Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H; Schramm, Alexander; Van Den Broecke, Caroline; Vermeulen, Joëlle; Van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

    2010-09-01

    Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.

  17. Fast and Simple Forensic Red Pen Ink Analysis Using Ultra-Performance Liquid Chromatography (UPLC)

    International Nuclear Information System (INIS)

    Lee, L.C.; Ying, S.L.; Wan Nur Syazwani Wan Mohamad Fuad; Ab Aziz Ishak; Khairul Osman

    2016-01-01

    Ultra-performance liquid chromatography (UPLC) is more effective than high performance liquid chromatography in terms of analysis speed and sensitivity. This paper presents a feasibility study on forensic red pen inks analysis using UPLC. A total of 12 varieties of red ball point pen inks were purchased from selected stationary shop. For each variety, four different individual pens were sampled to provide intra-variability within a particular variety of pen. The proposed approach is very simple that it only involved limited analysis step and chemicals. A total of 144 chromatograms were obtained from red ink entries extracted with 1.5 mL 80 % (v/v) acetonitrile. Peaks originated from pen inks were determined by comparing the chromatograms of both blank paper and blank solvent against that of ink samples. Subsequently, one-way ANOVA was conducted to discriminate all 66 possible pairs for red pen inks. Results showed that the proposed approach giving discriminating power of 95.45 %. The outcome of the study indicates that UPLC could be a fast and simple approach to red ball point pen inks analysis. (author)

  18. Analysis of a new morphogenetic mutation in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Mglinets, V.A.

    1987-01-01

    Somatic mosaicism for mutations monster and yellow was induced by gamma-irradiation of Drosophila melanogaster y/y; Dp(1; 2)sc 19 M(2)z/mn d embryos and larvae. Frequencies of mosaicism increased with the age of treated larvae, especially in the end of the 2nd larval instar. Autonomous expression of mn was observed throughout the whole range of larval age studied, though neither for all y/y spots nor for all parts of the spots. Dissimilarities in dynamics of mosaic spots and duplication induction suggest that the latter are not due to mn expression in somatic clones

  19. Isotopic analysis of H2, HD, D2 mixtures and analysis of ortho-para-hydrogen mixtures by gas chromatography

    International Nuclear Information System (INIS)

    Botter, F.; Perriere, G. de la; Tistchenko, S.

    1961-01-01

    This communication describes the present situation concerning the possibilities of vapor phase chromatography for the separation and analysis of mixtures of H 2 , HD and D 2 and of ortho- and para-hydrogen mixtures. Separation factors for physical adsorption of the various varieties of hydrogen have been deduced from chromatograms and have also been measured directly with a static method - the agreements is good. (author) [fr

  20. Mutational Analysis of PTPN11 Gene in Taiwanese Children with Noonan Syndrome

    Directory of Open Access Journals (Sweden)

    Chia-Sui Hung

    2007-01-01

    Full Text Available Noonan syndrome (NS is an autosomal dominant disorder presenting with characteristic facies, short stature, skeletal anomalies, and congenital heart defects. Mutations in protein-tyrosine phosphatase, nonreceptor-type 11 (PTPN11, encoding SHP-2, account for 33-50% of NS. This study screened for mutations in the PTPN11 gene in 34 Taiwanese patients with NS. Mutation analysis of the 15 coding exons and exon/intron boundaries was performed by polymerase chain reaction and direct sequencing of the PTPN11 gene. We identified 10 different missense mutations in 13 (38% patients, including a novel missense mutation (855T > G, F285L. These mutations were clustered in exon 3 (n = 6 encoding the N-SH2 domain, exon 4 (n = 2 encoding the C-SH2 domain, and in exons 8 (n = 2 and 13 (n = 3 encoding the PTP domain. In conclusion, this study provides further support that PTPN11 mutations are responsible for Noonan syndrome in Taiwanese patients. [J Formos Med Assoc 2007;106(2:169-172

  1. Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia[S

    Science.gov (United States)

    Mendoza-Barberá, Elena; Julve, Josep; Nilsson, Stefan K.; Lookene, Aivar; Martín-Campos, Jesús M.; Roig, Rosa; Lechuga-Sancho, Alfonso M.; Sloan, John H.; Fuentes-Prior, Pablo; Blanco-Vaca, Francisco

    2013-01-01

    During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia. PMID:23307945

  2. Clinical profile and mutation analysis of xeroderma pigmentosum in Indian patients.

    Science.gov (United States)

    Tamhankar, Parag M; Iyer, Shruti V; Ravindran, Shyla; Gupta, Neerja; Kabra, Madhulika; Nayak, Chitra; Kura, Mahendra; Sanghavi, Swapnil; Joshi, Rajesh; Chennuri, Vasundhara Sridhar; Khopkar, Uday

    2015-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by cutaneous and ocular photosensitivity and an increased risk of developing cutaneous neoplasms. Progressive neurological abnormalities develop in a quarter of XP patients. To study the clinical profile and perform a mutation analysis in Indian patients with xeroderma pigmentosum. Ten families with 13 patients with XP were referred to our clinic over 2 years. The genes XPA, XPB and XPC were sequentially analyzed till a pathogenic mutation was identified. Homozygous mutations in the XPA gene were seen in patients with moderate to severe mental retardation (6/10 families) but not in those without neurological features. Two unrelated families with a common family name and belonging to the same community from Maharashtra were found to have an identical mutation in the XPA gene, namely c.335_338delTTATinsCATAAGAAA (p.F112SfsX2). Testing of the XPC gene in two families with four affected children led to the identification of the novel mutations c.1243C>T or p.R415X and c.1677C>A or p.Y559X. In two families, mutations could not be identified in XPA, XPB and XPC genes. The sample size is small. Indian patients who have neurological abnormalities associated with XP should be screened for mutations in the XPA gene.

  3. Pseudo-absolute quantitative analysis using gas chromatography – Vacuum ultraviolet spectroscopy – A tutorial

    International Nuclear Information System (INIS)

    Bai, Ling; Smuts, Jonathan; Walsh, Phillip; Qiu, Changling; McNair, Harold M.; Schug, Kevin A.

    2017-01-01

    The vacuum ultraviolet detector (VUV) is a new non-destructive mass sensitive detector for gas chromatography that continuously and rapidly collects full wavelength range absorption between 120 and 240 nm. In addition to conventional methods of quantification (internal and external standard), gas chromatography - vacuum ultraviolet spectroscopy has the potential for pseudo-absolute quantification of analytes based on pre-recorded cross sections (well-defined absorptivity across the 120–240 nm wavelength range recorded by the detector) without the need for traditional calibration. The pseudo-absolute method was used in this research to experimentally evaluate the sources of sample loss and gain associated with sample introduction into a typical gas chromatograph. Standard samples of benzene and natural gas were used to assess precision and accuracy for the analysis of liquid and gaseous samples, respectively, based on the amount of analyte loaded on-column. Results indicate that injection volume, split ratio, and sampling times for splitless analysis can all contribute to inaccurate, yet precise sample introduction. For instance, an autosampler can very reproducibly inject a designated volume, but there are significant systematic errors (here, a consistently larger volume than that designated) in the actual volume introduced. The pseudo-absolute quantification capability of the vacuum ultraviolet detector provides a new means for carrying out system performance checks and potentially for solving challenging quantitative analytical problems. For practical purposes, an internal standardized approach to normalize systematic errors can be used to perform quantitative analysis with the pseudo-absolute method. - Highlights: • Gas chromatography diagnostics and quantification using VUV detector. • Absorption cross-sections for molecules enable pseudo-absolute quantitation. • Injection diagnostics reveal systematic errors in hardware settings. • Internal

  4. Pseudo-absolute quantitative analysis using gas chromatography – Vacuum ultraviolet spectroscopy – A tutorial

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Ling [Department of Chemistry & Biochemistry, The University of Texas at Arlington, Arlington, TX (United States); Smuts, Jonathan; Walsh, Phillip [VUV Analytics, Inc., Cedar Park, TX (United States); Qiu, Changling [Department of Chemistry & Biochemistry, The University of Texas at Arlington, Arlington, TX (United States); McNair, Harold M. [Department of Chemistry, Virginia Tech, Blacksburg, VA (United States); Schug, Kevin A., E-mail: kschug@uta.edu [Department of Chemistry & Biochemistry, The University of Texas at Arlington, Arlington, TX (United States)

    2017-02-08

    The vacuum ultraviolet detector (VUV) is a new non-destructive mass sensitive detector for gas chromatography that continuously and rapidly collects full wavelength range absorption between 120 and 240 nm. In addition to conventional methods of quantification (internal and external standard), gas chromatography - vacuum ultraviolet spectroscopy has the potential for pseudo-absolute quantification of analytes based on pre-recorded cross sections (well-defined absorptivity across the 120–240 nm wavelength range recorded by the detector) without the need for traditional calibration. The pseudo-absolute method was used in this research to experimentally evaluate the sources of sample loss and gain associated with sample introduction into a typical gas chromatograph. Standard samples of benzene and natural gas were used to assess precision and accuracy for the analysis of liquid and gaseous samples, respectively, based on the amount of analyte loaded on-column. Results indicate that injection volume, split ratio, and sampling times for splitless analysis can all contribute to inaccurate, yet precise sample introduction. For instance, an autosampler can very reproducibly inject a designated volume, but there are significant systematic errors (here, a consistently larger volume than that designated) in the actual volume introduced. The pseudo-absolute quantification capability of the vacuum ultraviolet detector provides a new means for carrying out system performance checks and potentially for solving challenging quantitative analytical problems. For practical purposes, an internal standardized approach to normalize systematic errors can be used to perform quantitative analysis with the pseudo-absolute method. - Highlights: • Gas chromatography diagnostics and quantification using VUV detector. • Absorption cross-sections for molecules enable pseudo-absolute quantitation. • Injection diagnostics reveal systematic errors in hardware settings. • Internal

  5. Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.

    Science.gov (United States)

    Revollo, Javier R; Crabtree, Nathaniel M; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Dobrovolsky, Vasily N

    2016-03-01

    Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. © 2015 Wiley Periodicals, Inc.

  6. The first USH2A mutation analysis of Japanese autosomal recessive retinitis pigmentosa patients: a totally different mutation profile with the lack of frequent mutations found in Caucasian patients.

    Science.gov (United States)

    Zhao, Yang; Hosono, Katsuhiro; Suto, Kimiko; Ishigami, Chie; Arai, Yuuki; Hikoya, Akiko; Hirami, Yasuhiko; Ohtsubo, Masafumi; Ueno, Shinji; Terasaki, Hiroko; Sato, Miho; Nakanishi, Hiroshi; Endo, Shiori; Mizuta, Kunihiro; Mineta, Hiroyuki; Kondo, Mineo; Takahashi, Masayo; Minoshima, Shinsei; Hotta, Yoshihiro

    2014-09-01

    Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease. The USH2A gene, which accounts for approximately 74-90% of Usher syndrome type 2 (USH2) cases, is also one of the major autosomal recessive RP (arRP) causative genes among Caucasian populations. To identify disease-causing USH2A gene mutations in Japanese RP patients, all 73 exons were screened for mutations by direct sequencing. In total, 100 unrelated Japanese RP patients with no systemic manifestations were identified, excluding families with obvious autosomal dominant inheritance. Of these 100 patients, 82 were included in this present study after 18 RP patients with very likely pathogenic EYS (eyes shut homolog) mutations were excluded. The mutation analysis of the USH2A revealed five very likely pathogenic mutations in four patients. A patient had only one very likely pathogenic mutation and the others had two of them. Caucasian frequent mutations p.C759F in arRP and p.E767fs in USH2 were not found. All the four patients exhibited typical clinical features of RP. The observed prevalence of USH2A gene mutations was approximately 4% among Japanese arRP patients, and the profile of the USH2A gene mutations differed largely between Japanese patients and previously reported Caucasian populations.

  7. Simple, specific analysis of organophosphorus and carbamate pesticides in sediments using column extraction and gas chromatography

    Science.gov (United States)

    Belisle, A.A.; Swineford, D.M.

    1988-01-01

    A simple, specific procedure was developed for the analysis of organophosphorus and carbamate pesticides in sediment. The wet soil was mixed with anhydrous sodium sulfate to bind water and the residues were column extracted in acetone:methylene chloride (1:l,v/v). Coextracted water was removed by additional sodium sulfate packed below the sample mixture. The eluate was concentrated and analyzed directly by capillary gas chromatography using phosphorus and nitrogen specific detectors. Recoveries averaged 93 % for sediments extracted shortly after spiking, but decreased significantly as the samples aged.

  8. Development of conjugate methods with gas chromatography for inorganic compounds analysis

    International Nuclear Information System (INIS)

    Baccan, N.

    1975-01-01

    The application of gas chromatography combined with mass spectrometry or with nuclear methods for the analysis of inorganic compounds is studied. The advantages of the use of a gas chromatograph coupled with a quadrupole mass spectrometer or with a high resolution radiation detector, are discussed. We also studied the formation and solvent extraction of metal chelates; an aliquot of the organic phase was directly injected into the gas chromatograph and the eluted compounds were detected by mass spectrometry or, when radioactive, by nuclear methods. (author)

  9. New insights into liquid chromatography for more eco-friendly analysis of pharmaceuticals.

    Science.gov (United States)

    Shaaban, Heba

    2016-10-01

    Greening the analytical methods used for analysis of pharmaceuticals has been receiving great interest aimed at eliminating or minimizing the amount of organic solvents consumed daily worldwide without loss in chromatographic performance. Traditional analytical LC techniques employed in pharmaceutical analysis consume tremendous amounts of hazardous solvents and consequently generate large amounts of waste. The monetary and ecological impact of using large amounts of solvents and waste disposal motivated the analytical community to search for alternatives to replace polluting analytical methodologies with clean ones. In this context, implementing the principles of green analytical chemistry (GAC) in analytical laboratories is highly desired. This review gives a comprehensive overview on different green LC pathways for implementing GAC principles in analytical laboratories and focuses on evaluating the greenness of LC analytical procedures. This review presents green LC approaches for eco-friendly analysis of pharmaceuticals in industrial, biological, and environmental matrices. Graphical Abstract Green pathways of liquid chromatography for more eco-friendly analysis of pharmaceuticals.

  10. Discrimination of honeys using colorimetric sensor arrays, sensory analysis and gas chromatography techniques.

    Science.gov (United States)

    Tahir, Haroon Elrasheid; Xiaobo, Zou; Xiaowei, Huang; Jiyong, Shi; Mariod, Abdalbasit Adam

    2016-09-01

    Aroma profiles of six honey varieties of different botanical origins were investigated using colorimetric sensor array, gas chromatography-mass spectrometry (GC-MS) and descriptive sensory analysis. Fifty-eight aroma compounds were identified, including 2 norisoprenoids, 5 hydrocarbons, 4 terpenes, 6 phenols, 7 ketones, 9 acids, 12 aldehydes and 13 alcohols. Twenty abundant or active compounds were chosen as key compounds to characterize honey aroma. Discrimination of the honeys was subsequently implemented using multivariate analysis, including hierarchical clustering analysis (HCA) and principal component analysis (PCA). Honeys of the same botanical origin were grouped together in the PCA score plot and HCA dendrogram. SPME-GC/MS and colorimetric sensor array were able to discriminate the honeys effectively with the advantages of being rapid, simple and low-cost. Moreover, partial least squares regression (PLSR) was applied to indicate the relationship between sensory descriptors and aroma compounds. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities.

    Science.gov (United States)

    Hicks, Michael B; Regalado, Erik L; Tan, Feng; Gong, Xiaoyi; Welch, Christopher J

    2016-01-05

    Supercritical fluid chromatography (SFC) has long been a preferred method for enantiopurity analysis in support of pharmaceutical discovery and development, but implementation of the technique in regulated GMP laboratories has been somewhat slow, owing to limitations in instrument sensitivity, reproducibility, accuracy and robustness. In recent years, commercialization of next generation analytical SFC instrumentation has addressed previous shortcomings, making the technique better suited for GMP analysis. In this study we investigate the use of modern SFC for enantiopurity analysis of several pharmaceutical intermediates and compare the results with the conventional HPLC approaches historically used for analysis in a GMP setting. The findings clearly illustrate that modern SFC now exhibits improved precision, reproducibility, accuracy and robustness; also providing superior resolution and peak capacity compared to HPLC. Based on these findings, the use of modern chiral SFC is recommended for GMP studies of stereochemistry in pharmaceutical development and manufacturing. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Liu, W.; Qian, C.; Comeau, K.; Francke, U. [Stanford Univ. Medical Center, Stanford, CA (United States)

    1994-09-01

    Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by variable manifestations in skeletal, cardiovascular and ocular systems. Mutations in the fibrillin gene on chromosome 15 (FBN1) have been shown to cause MFS. To examine the relationship between FBN1 gene mutations, fibrillin protein function and MFS phenotypes, we screened for alternations in the fibrillin coding sequence in fibroblast derived cDNA from MFS patients. To date, abnormally migrating bands in more than 20 unrelated MFS patients have been identified by using non-radioactive single-strand conformation analysis and silver staining. Five altered bands have been directly sequenced. Two missense mutations and three splice site mutations have been identified. Both missense mutations substitute another amino acid for a cysteine residue (C1402W and C1672R) in EGF-like motifs of the fibrillin polypeptide chain. The two splice site mutations are at nucleotide positions 6994+1 (G{yields}A), and 7205-2 (A{yields}G) and result in in-frame skipping of exon 56 and 58, respectively. Skipping of exon 56 occurs in 50% of mutant transcripts. Use of a cryptic splice site 51 bp upstream of the normal donor site results in half of the mutant transcripts containing part of exon 56. Both products contain in-frame deletions. Another splice site mutation, identified by exon screening from patient genomic DNA using intron primers, is at nucleotide position 2293+2 (T{yields}A), but the predicted exon skipping has not been detected at the RT-PCR level. This may be due to instability of the mutant transcript. Including the mutations reported here, a total of 8 out of 36 published FBN1 gene mutations involve exon skipping. It may be inferred that FBN1 exon skipping plays an important pathogenic role in MFS.

  13. Greening Reversed-Phase Liquid Chromatography Methods Using Alternative Solvents for Pharmaceutical Analysis

    Directory of Open Access Journals (Sweden)

    Moussa Yabré

    2018-05-01

    Full Text Available The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.

  14. Sequence analysis of tyrosinase gene in ocular and oculocutaneous albinism patients: introducing three novel mutations.

    Science.gov (United States)

    Khordadpoor-Deilamani, Faravareh; Akbari, Mohammad Taghi; Karimipoor, Morteza; Javadi, Gholamreza

    2015-01-01

    Albinism is a heterogeneous genetic disorder of melanin synthesis that results in hypopigmented eyes (in patients with ocular albinism) or hair, skin, and eyes (in individuals with oculocutaneous albinism). It is associated with decreased visual acuity, nystagmus, strabismus, and photophobia. The tyrosinase gene is known to be involved in both oculocutaneous albinism and autosomal recessive ocular albinism. In this study, we aimed to screen the mutations in the TYR gene in the nonsyndromic OCA and autosomal recessive ocular albinism patients from Iran. The tyrosinase gene was examined in 23 unrelated patients with autosomal recessive ocular albinism or nonsyndromic OCA using DNA sequencing and bioinformatics analysis. TYR gene mutations were identified in 14 (app. 60%) albinism patients. We found 10 mutations, 3 of which were novel. No mutation was found in our ocular albinism patients, but one of them was heterozygous for the p.R402Q polymorphism.

  15. Mutation analysis and evaluation of the cardiac localization of TMEM43 in arrhythmogenic right ventricular cardiomyopathy

    DEFF Research Database (Denmark)

    Christensen, A H; Andersen, C B; Tybjærg-Hansen, A

    2011-01-01

    Christensen AH, Andersen CB, Tybjærg-Hansen A, Haunso S, Svendsen JH. Mutation analysis and evaluation of the cardiac localization of TMEM43 in arrhythmogenic right ventricular cardiomyopathy. A single report has associated mutations in TMEM43 (LUMA) with a distinctive form of arrhythmogenic right...... with anti-TMEM43, anti-plakoglobin, anti-plakophilin-2, anti-connexin-43, and anti-emerin antibodies was performed on myocardium from TMEM43-positive patients (n = 3) and healthy controls (n = 3). The genetic screening identified heterozygous variants in two families: one reported mutation (c.1073C> T......; in two related patients) and one novel variant (c.705+ 7G> A; in one patient) of unknown significance. All three patients fulfilled Task Force criteria and did not carry mutations in any other ARVC-related gene. Immunostaining with TMEM43 antibody showed intense staining of the sarcolemma. The signal...

  16. [The mutation analysis of PAH gene and prenatal diagnosis in classical phenylketonuria family].

    Science.gov (United States)

    Yan, Yousheng; Hao, Shengju; Yao, Fengxia; Sun, Qingmei; Zheng, Lei; Zhang, Qinghua; Zhang, Chuan; Yang, Tao; Huang, Shangzhi

    2014-12-01

    To characterize the mutation spectrum of phenylalanine hydroxylase (PAH) gene and perform prenatal diagnosis for families with classical phenylketonuria. By stratified sequencing, mutations were detected in the exons and flaking introns of PAH gene of 44 families with classical phenylketonuria. 47 fetuses were diagnosed by combined sequencing with linkage analysis of three common short tandem repeats (STR) (PAH-STR, PAH-26 and PAH-32) in the PAH gene. Thirty-one types of mutations were identified. A total of 84 mutations were identified in 88 alleles (95.45%), in which the most common mutation have been R243Q (21.59%), EX6-96A>G (6.82%), IVS4-1G>A (5.86%) and IVS7+2T>A (5.86%). Most mutations were found in exons 3, 5, 6, 7, 11 and 12. The polymorphism information content (PIC) of these three STR markers was 0.71 (PAH-STR), 0.48 (PAH-26) and 0.40 (PAH-32), respectively. Prenatal diagnosis was performed successfully with the combined method in 47 fetuses of 44 classical phenylketonuria families. Among them, 11 (23.4%) were diagnosed as affected, 24 (51.1%) as carriers, and 12 (25.5%) as unaffected. Prenatal diagnosis can be achieved efficiently and accurately by stratified sequencing of PAH gene and linkage analysis of STR for classical phenylketonuria families.

  17. NMD Microarray Analysis for Rapid Genome-Wide Screen of Mutated Genes in Cancer

    Directory of Open Access Journals (Sweden)

    Maija Wolf

    2005-01-01

    Full Text Available Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD inhibition and microarray analysis (NMD microarrays in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization in order to identify inactivation of tumor suppressor genes in cancer. Such a “mutatomics” screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

  18. Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit

    DEFF Research Database (Denmark)

    Nagirnaja, Liina; Venclovas, Česlovas; Rull, Kristiina

    2012-01-01

    Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by appl...... of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGβ genes CGB5 and CGB8.......Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of h......CG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls...

  19. Comprehensive two-dimensional gas chromatography applied to illicit drug analysis.

    Science.gov (United States)

    Mitrevski, Blagoj; Wynne, Paul; Marriott, Philip J

    2011-11-01

    Multidimensional gas chromatography (MDGC), and especially its latest incarnation--comprehensive two-dimensional gas chromatography (GC × GC)--have proved advantageous over and above classic one-dimensional gas chromatography (1D GC) in many areas of analysis by offering improved peak capacity, often enhanced sensitivity and, especially in the case of GC × GC, the unique feature of 'structured' chromatograms. This article reviews recent advances in MDGC and GC × GC in drug analysis with special focus on ecstasy, heroin and cocaine profiling. Although 1D GC is still the method of choice for drug profiling in most laboratories because of its simplicity and instrument availability, GC × GC is a tempting proposition for this purpose because of its ability to generate a higher net information content. Effluent refocusing due to the modulation (compression) process, combined with the separation on two 'orthogonal' columns, results in more components being well resolved and therefore being analytically and statistically useful to the profile. The spread of the components in the two-dimensional plots is strongly dependent on the extent of retention 'orthogonality' (i.e. the extent to which the two phases possess different or independent retention mechanisms towards sample constituents) between the two columns. The benefits of 'information-driven' drug profiling, where more points of reference are usually required for sample differentiation, are discussed. In addition, several limitations in application of MDGC in drug profiling, including data acquisition rate, column temperature limit, column phase orthogonality and chiral separation, are considered and discussed. Although the review focuses on the articles published in the last decade, a brief chronological preview of the profiling methods used throughout the last three decades is given.

  20. VLCAD deficiency: Pitfalls in newborn screening and confirmation of diagnosis by mutation analysis

    DEFF Research Database (Denmark)

    Boneh, A; Andresen, Brage Storstein; Gregersen, Niels

    2006-01-01

    samples taken at age 48-72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false......-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype-phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients....

  1. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Paolo Cossu-Rocca

    Full Text Available Triple Negative Breast Cancer (TNBC accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

  2. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

    Science.gov (United States)

    Cossu-Rocca, Paolo; Orrù, Sandra; Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

    2015-01-01

    Triple Negative Breast Cancer (TNBC) accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

  3. Elemental analysis of oum er rbia moroccan river-water Using icp-aes and chromatography

    International Nuclear Information System (INIS)

    Zahry, F.; Bounakhla, M.; Labraimi, M.; Moutte, J.; Bilal, E.

    2001-01-01

    The analysis of water and suspended particulate matter (SPM) from Oum er Rbia Moroccan river for the content of 22 elements has been performed utilizing ICP-AES and chromatography methods. MPS samples were dissolved in nitric acid. The element analyzed are : Na, Mg, Al, Si, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Br, Ba and Pb. The detection limits are sufficient for most of the elements to be determined. Results were assessed for the accuracy by the analysis of a multi-elemental standard. The results for most of the elements obtained were accurate to about 2-15%. The objective of the analysis were to determine the geochemical areas on this river, to explain the mechanisms of sediment transport and nevertheless, to assess the pollution of the river by human activities. 48 samples were collected along the river, between its source in meddle atlas and the Atlantic ocean at Azemour city

  4. Analysis of antiepileptic drugs in biological fluids by means of electrokinetic chromatography.

    Science.gov (United States)

    Pucci, Vincenzo; Raggi, Maria Augusta

    2005-02-01

    An overview of the electrokinetic chromatographic methods for the analysis of antiepileptic drug levels in biological samples is presented. In particular, micellar electrokinetic capillary chromatography is a very suitable method for the determination of these drugs, because it allows a rapid, selective, and accurate analysis. In addition to the electrokinetic chromatographic studies on the determination of antiepileptic drugs, some information regarding sample pretreatment will also be reported: this is a critical step when the analysis of biological fluids is concerned. The electrokinetic chromatographic methods for the determination of recent antiepileptic drugs (e.g., lamotrigine, levetiracetam) and classical anticonvulsants (e.g., carbamazepine, phenytoin, ethosuximide, valproic acid) will be discussed in depth, and their pharmacological profiles will be briefly described as well.

  5. Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines

    DEFF Research Database (Denmark)

    Liu, Ying; Bodmer, Walter F

    2006-01-01

    A comprehensive analysis of the TP53 gene and its protein status was carried out on a panel of 56 colorectal cancer cell lines. This analysis was based on a combination of denaturing HPLC mutation screening of all exons of the p53 gene, sequencing the cDNA, and assessing the function of the p53 p...

  6. Gas chromatography-vacuum ultraviolet spectroscopy for analysis of fatty acid methyl esters.

    Science.gov (United States)

    Fan, Hui; Smuts, Jonathan; Bai, Ling; Walsh, Phillip; Armstrong, Daniel W; Schug, Kevin A

    2016-03-01

    A new vacuum ultraviolet (VUV) detector for gas chromatography was recently developed and applied to fatty acid methyl ester (FAME) analysis. VUV detection features full spectral acquisition in a wavelength range of 115-240nm, where virtually all chemical species absorb. VUV absorption spectra of 37 FAMEs, including saturated, monounsaturated, and polyunsaturated types were recorded. Unsaturated FAMEs show significantly different gas phase absorption profiles than saturated ones, and these classes can be easily distinguished with the VUV detector. Another advantage includes differentiating cis/trans-isomeric FAMEs (e.g. oleic acid methyl ester and linoleic acid methyl ester isomers) and the ability to use VUV data analysis software for deconvolution of co-eluting signals. As a universal detector, VUV also provides high specificity, sensitivity, and a fast data acquisition rate, making it a powerful tool for fatty acid screening when combined with gas chromatography. The fatty acid profile of several food oil samples (olive, canola, vegetable, corn, sunflower and peanut oils) were analyzed in this study to demonstrate applicability to real world samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Neutral monosaccharide composition analysis of plant-derived oligo- and polysaccharides by high performance liquid chromatography.

    Science.gov (United States)

    Yan, Jun; Shi, Songshan; Wang, Hongwei; Liu, Ruimin; Li, Ning; Chen, Yonglin; Wang, Shunchun

    2016-01-20

    A novel analytical method for neutral monosaccharide composition analysis of plant-derived oligo- and polysaccharides was developed using hydrophilic interaction liquid chromatography coupled to a charged aerosol detector. The effects of column type, additives, pH and column temperature on retention and separation were evaluated. Additionally, the method could distinguish potential impurities in samples, including chloride, sulfate and sodium, from sugars. The results of validation demonstrated that this method had good linearity (R(2) ≥ 0.9981), high precision (relative standard deviation ≤ 4.43%), and adequate accuracy (94.02-103.37% recovery) and sensitivity (detection limit: 15-40 ng). Finally, the monosaccharide compositions of the polysaccharide from Eclipta prostrasta L. and stachyose were successfully profiled through this method. This report represents the first time that all of these common monosaccharides could be well-separated and determined simultaneously by high performance liquid chromatography without additional derivatization. This newly developed method is convenient, efficient and reliable for monosaccharide analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Comparison of an Electronic Nose Based on Ultrafast Gas Chromatography, Comprehensive Two-Dimensional Gas Chromatography, and Sensory Evaluation for an Analysis of Type of Whisky

    Directory of Open Access Journals (Sweden)

    Paulina Wiśniewska

    2017-01-01

    Full Text Available Whisky is one of the most popular alcoholic beverages. There are many types of whisky, for example, Scotch, Irish, and American whisky (called bourbon. The whisky market is highly diversified, and, because of this, it is important to have a method which would enable rapid quality evaluation and authentication of the type of whisky. The aim of this work was to compare 3 methods: an electronic nose based on the technology of ultrafast gas chromatography (Fast-GC, comprehensive two-dimensional gas chromatography (GC × GC, and sensory evaluation. The selected whisky brands included 6 blended whiskies from Scotland, 4 blended whiskies from Ireland, and 4 bourbons produced in the USA. For data analysis, peak heights of chromatograms were used. The panelists who took part in sensory evaluations included 4 women and 4 men. The obtained data were analyzed by 2 chemometric methods: partial least squares discriminant analysis (PLS-DA and discrimination function analysis (DFA. E-nose and GC × GC allowed for differentiation between whiskies by type. Sensory analysis did not allow for differentiation between whiskies by type, but it allowed giving consumer preferences.

  9. Analysis of mutations in the entire coding sequence of the factor VIII gene

    Energy Technology Data Exchange (ETDEWEB)

    Bidichadani, S.I.; Lanyon, W.G.; Connor, J.M. [Glascow Univ. (United Kingdom)] [and others

    1994-09-01

    Hemophilia A is a common X-linked recessive disorder of bleeding caused by deleterious mutations in the gene for clotting factor VIII. The large size of the factor VIII gene, the high frequency of de novo mutations and its tissue-specific expression complicate the detection of mutations. We have used a combination of RT-PCR of ectopic factor VIII transcripts and genomic DNA-PCRs to amplify the entire essential sequence of the factor VIII gene. This is followed by chemical mismatch cleavage analysis and direct sequencing in order to facilitate a comprehensive search for mutations. We describe the characterization of nine potentially pathogenic mutations, six of which are novel. In each case, a correlation of the genotype with the observed phenotype is presented. In order to evaluate the pathogenicity of the five missense mutations detected, we have analyzed them for evolutionary sequence conservation and for their involvement of sequence motifs catalogued in the PROSITE database of protein sites and patterns.

  10. Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes.

    Science.gov (United States)

    Vaughn, Cecily P; Robles, Jorge; Swensen, Jeffrey J; Miller, Christine E; Lyon, Elaine; Mao, Rong; Bayrak-Toydemir, Pinar; Samowitz, Wade S

    2010-05-01

    Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long-range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long-range product spanning exons 11-15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty-seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long-range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2. (c) 2010 Wiley-Liss, Inc.

  11. Somatic mutation analysis of MYH11 in breast and prostate cancer

    International Nuclear Information System (INIS)

    Alhopuro, Pia; Karhu, Auli; Winqvist, Robert; Waltering, Kati; Visakorpi, Tapio; Aaltonen, Lauri A

    2008-01-01

    MYH11 (also known as SMMHC) encodes the smooth-muscle myosin heavy chain, which has a key role in smooth muscle contraction. Inversion at the MYH11 locus is one of the most frequent chromosomal aberrations found in acute myeloid leukemia. We have previously shown that MYH11 mutations occur in human colorectal cancer, and may also be associated with Peutz-Jeghers syndrome. The mutations found in human intestinal neoplasia result in unregulated proteins with constitutive motor activity, similar to the mutant myh11 underlying the zebrafish meltdown phenotype characterized by disrupted intestinal architecture. Recently, MYH1 and MYH9 have been identified as candidate breast cancer genes in a systematic analysis of the breast cancer genome. The aim of this study was to investigate the role of somatic MYH11 mutations in two common tumor types; breast and prostate cancers. A total of 155 breast cancer and 71 prostate cancer samples were analyzed for those regions in MYH11 (altogether 8 exons out of 42 coding exons) that harboured mutations in colorectal cancer in our previous study. In breast cancer samples only germline alterations were observed. One prostate cancer sample harbored a frameshift mutation c.5798delC, which we have previously shown to result in a protein with unregulated motor activity. Little evidence for a role of somatic MYH11 mutations in the formation of breast or prostate cancers was obtained in this study

  12. Mutation analysis of GJB2 gene and prenatal diagnosis in a non-syndromic deafness family

    Directory of Open Access Journals (Sweden)

    Xiao-hua CHEN

    2014-08-01

    Full Text Available Objective To identify the pathogenic gene in a non-syndromic deafness family, provide an accurate genetic consultation and early intervention for deaf family to reduce the incidence of congenital deafness. Methods Mutation analysis was carried out by polymerase chain reaction followed by DNA sequencing of coding region of GJB2 gene. The fetal DNA was extracted from the amniotic fluid cells by amniocentesis at 20 weeks during pregnancy. The genotype of the fetus was characterized for predicting the status of hearing. Results Complex heterozygous mutations 235delC and 176-191del16bp were detected in the proband of the family, heterozygous mutation 176-191del16bp was detected in the father, and 235delC was detected in the mother. Fetus carried 235delC heterozygous mutation inherited from his mother. Conclusions The proband's hearing loss is resulted from the complex heterozygous mutations 235delC and 176-191del16bp in GJB2 gene. Fetus is a heterozygous mutation 235delC carrier. Prenatal diagnosis for deafness assisted by genetic test can provide efficient guidance about offspring's hearing condition, and prevent another deaf-mute member from birth. DOI: 10.11855/j.issn.0577-7402.2014.07.09

  13. Mutation and Methylation Analysis of the Chromodomain-Helicase-DNA Binding 5 Gene in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kylie L. Gorringe

    2008-11-01

    Full Text Available Chromodomain, helicase, DNA binding 5 (CHD5 is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04. The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.

  14. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  15. [Clinical features and ACADVL gene mutation spectrum analysis of 11 Chinese patients with very long chain acyl-CoA dehydrogenase deficiency].

    Science.gov (United States)

    Jinjun, Cao; Wenjuan, Qiu; Ruinan, Zhang; Jun, Ye; Lianshu, Han; Huiwen, Zhang; Qigang, Zhang; Xuefan, Gu

    2015-04-01

    To investigate the clinical and laboratory features of very long chain acyl-CoA dehydrogenase deficiency ( VLCADD ) and the correlations between its genotype and phenotype. Eleven patients diagnosed as VLCADD of Shanghai Jiaotong University School of Medicine seen from September 2006 to May 2014 were included. There were 9 boys and 2 girls, whose age was 2 d-17 years. Analysis was performed on clinical features, routine laboratory examination, and tandem mass spectrometry (MS-MS) , gas chromatography mass spectrometry (GC-MS) and genetic analysis were conducted. All cases had elevated levels of blood tetradecanoylcarnitine (C14:1) recognized as the characteristic biomarker for VLCADD. The eleven patients were classified into three groups: six cases in neonatal onset group, three in infancy onset group form patients and two in late onset group. Neonatal onset patients were characterized by hypoactivity, hypoglycemia shortly after birth. Infancy onset patients presented hepatomegaly and hypoglycemia in infancy. The two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Six of the eleven patients died at the age of 2-8 months, including four neonatal onset and two infant onset patients, with one or two null mutations. The other two neonatal onset patients were diagnosed since early birth through neonatal screening and their clinical manifestation are almost normal after treatments. Among 11 patients, seventeen different mutations in the ACADVL gene were identified, with a total mutation detection rate of 95.45% (21/22 alleles), including eleven reported mutations ( p. S22X, p. G43D, p. R511Q, p. W427X, p. A213T, p. C215R, p. G222R, p. R450H, p. R456H, c. 296-297delCA, c. 1605 + 1G > T) and six novel mutations (p. S72F, p. Q100X, p. M437T, p. D466Y, c. 1315delG insAC, IVS7 + 4 A > G). The p. R450H was the most frequent mutation identified in three alleles (13.63%, 3/22 alleles), followed by p. S22X and p. D466Y mutations which

  16. Preimplantation genetic diagnosis for mitochondrial DNA mutations: analysis of one blastomere suffices.

    Science.gov (United States)

    Sallevelt, Suzanne C E H; Dreesen, Joseph C F M; Coonen, Edith; Paulussen, Aimee D C; Hellebrekers, Debby M E I; de Die-Smulders, Christine E M; Smeets, Hubert J M; Lindsey, Patrick

    2017-10-01

    Preimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset. For the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers. Only one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected. Our findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  17. Survival analysis of cancer risk reduction strategies for BRCA1/2 mutation carriers.

    Science.gov (United States)

    Kurian, Allison W; Sigal, Bronislava M; Plevritis, Sylvia K

    2010-01-10

    Women with BRCA1/2 mutations inherit high risks of breast and ovarian cancer; options to reduce cancer mortality include prophylactic surgery or breast screening, but their efficacy has never been empirically compared. We used decision analysis to simulate risk-reducing strategies in BRCA1/2 mutation carriers and to compare resulting survival probability and causes of death. We developed a Monte Carlo model of breast screening with annual mammography plus magnetic resonance imaging (MRI) from ages 25 to 69 years, prophylactic mastectomy (PM) at various ages, and/or prophylactic oophorectomy (PO) at ages 40 or 50 years in 25-year-old BRCA1/2 mutation carriers. With no intervention, survival probability by age 70 is 53% for BRCA1 and 71% for BRCA2 mutation carriers. The most effective single intervention for BRCA1 mutation carriers is PO at age 40, yielding a 15% absolute survival gain; for BRCA2 mutation carriers, the most effective single intervention is PM, yielding a 7% survival gain if performed at age 40 years. The combination of PM and PO at age 40 improves survival more than any single intervention, yielding 24% survival gain for BRCA1 and 11% for BRCA2 mutation carriers. PM at age 25 instead of age 40 offers minimal incremental benefit (1% to 2%); substituting screening for PM yields a similarly minimal decrement in survival (2% to 3%). Although PM at age 25 plus PO at age 40 years maximizes survival probability, substituting mammography plus MRI screening for PM seems to offer comparable survival. These results may guide women with BRCA1/2 mutations in their choices between prophylactic surgery and breast screening.

  18. Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations

    DEFF Research Database (Denmark)

    Wibrand, Flemming; Jeppesen, Tina Dysgaard; Frederiksen, Anja L

    2010-01-01

    We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large......, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM....

  19. Diagnostic and therapeutic implications of genetic heterogeneity in myeloid neoplasms uncovered by comprehensive mutational analysis

    Directory of Open Access Journals (Sweden)

    Sarah M. Choi

    2017-01-01

    Full Text Available While growing use of comprehensive mutational analysis has led to the discovery of innumerable genetic alterations associated with various myeloid neoplasms, the under-recognized phenomenon of genetic heterogeneity within such neoplasms creates a potential for diagnostic confusion. Here, we describe two cases where expanded mutational testing led to amendment of an initial diagnosis of chronic myelogenous leukemia with subsequent altered treatment of each patient. We demonstrate the power of comprehensive testing in ensuring appropriate classification of genetically heterogeneous neoplasms, and emphasize thoughtful analysis of molecular and genetic data as an essential component of diagnosis and management.

  20. Multiresidue analysis of pesticides in olive oil by gel permeation chromatography followed by gas chromatography-tandem mass-spectrometric determination

    International Nuclear Information System (INIS)

    Sanchez, Andres Garcia; Martos, Natividad Ramos; Ballesteros, Evaristo

    2006-01-01

    A method for the multiresidue analysis of olive oil samples for 26 pesticides is proposed. Residual pesticides are extracted from oil using an n-hexane/acetonitrile mixture, extracts being cleaned-up by gel permeation chromatography (GPC) for analysis by gas chromatography-tandem mass spectrometry (GC-MS/MS). Electron ionization and chemical ionization are employed in a single analysis for the determination of pesticides. Pesticide recoveries from virgin and refined olive oil spiked with 10, 100 and 250 μg/kg concentrations of the pesticides ranged from 83.8 to 110.3%. The proposed method features good sensitivity: its limits of quantification are low enough to allow pesticide residues to be determined at concentrations below the maximum residue levels legally accepted. The precision, expressed as relative standard deviation, ranges from 4.93 to 8.11%. Applicability was tested on 40 olive oil samples. Several pesticides were detected in most of the virgin olive oil samples. By contrast, refined olive samples contained few pesticides, and only endosulfan sulphate was detected in all

  1. Analysis of small carbohydrates in several bioactive botanicals by gas chromatography with mass spectrometry and liquid chromatography with tandem mass spectrometry.

    Science.gov (United States)

    Moldoveanu, Serban; Scott, Wayne; Zhu, Jeff

    2015-11-01

    Bioactive botanicals contain natural compounds with specific biological activity, such as antibacterial, antioxidant, immune stimulating, and taste improving. A full characterization of the chemical composition of these botanicals is frequently necessary. A study of small carbohydrates from the plant materials of 18 bioactive botanicals is further described. The study presents the identification of the carbohydrate using a gas chromatographic-mass spectrometric analysis that allows detection of molecules as large as maltotetraose, after changing them into trimethylsilyl derivatives. A number of carbohydrates in the plant (fructose, glucose, mannose, sucrose, maltose, xylose, sorbitol, and myo-, chiro-, and scyllo-inositols) were quantitated using a novel liquid chromatography with tandem mass spectrometric technique. Both techniques involved new method developments. The gas chromatography with mass spectrometric analysis involved derivatization and separation on a Rxi(®)-5Sil MS column with H2 as a carrier gas. The liquid chromatographic separation was obtained using a hydrophilic interaction type column, YMC-PAC Polyamine II. The tandem mass spectrometer used an electrospray ionization source in multiple reaction monitoring positive ion mode with the detection of the adducts of the carbohydrates with Cs(+) ions. The validated quantitative procedure showed excellent precision and accuracy allowing the analysis in a wide range of concentrations of the analytes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Phytochemical analysis of Hibiscus caesius using high performance liquid chromatography coupled with mass spectrometry.

    Science.gov (United States)

    Ain, Quratul; Naveed, Muhammad Na; Mumtaz, Abdul Samad; Farman, Muhammad; Ahmed, Iftikhar; Khalid, Nauman

    2015-09-01

    Various species in genus Hibiscus are traditionally known for their therapeutic attributes. The present study focused on the phytochemical analysis of a rather unexplored species Hibiscus caesius (H. caesius), using high-pressure liquid chromatography coupled with mass spectrometry (HPLC-MS). The analysis revealed five major compounds in the aqueous extract, viz. vanillic acid, protocatechoic acid, quercetin, quercetin glucoside and apigenin, being reported for the first time in H. caesius. Literature suggests that these compounds have important pharmacological traits such as anti-cancer, anti-inflammatory, anti-bacterial and hepatoprotective etc. however, this requires further pharmacological investigations at in vitro and in vivo scale. The above study concluded the medicinal potential of H. caesius.

  3. Core-Shell Columns in High-Performance Liquid Chromatography: Food Analysis Applications

    Science.gov (United States)

    Preti, Raffaella

    2016-01-01

    The increased separation efficiency provided by the new technology of column packed with core-shell particles in high-performance liquid chromatography (HPLC) has resulted in their widespread diffusion in several analytical fields: from pharmaceutical, biological, environmental, and toxicological. The present paper presents their most recent applications in food analysis. Their use has proved to be particularly advantageous for the determination of compounds at trace levels or when a large amount of samples must be analyzed fast using reliable and solvent-saving apparatus. The literature hereby described shows how the outstanding performances provided by core-shell particles column on a traditional HPLC instruments are comparable to those obtained with a costly UHPLC instrumentation, making this novel column a promising key tool in food analysis. PMID:27143972

  4. High performance thin layer chromatography fingerprint analysis of guava (Psidium guajava) leaves

    Science.gov (United States)

    Astuti, M.; Darusman, L. K.; Rafi, M.

    2017-05-01

    High-performance thin layer chromatography (HPTLC) fingerprint analysis is commonly used for quality control of medicinal plants in term of identification and authentication. In this study, we have been developed HPTLC fingerprint analysis for identification of guava (Psidium guajava) leaves raw material. A mixture of chloroform, acetone, and formic acid in the ratio 10:2:1 was used as the optimum mobile phase in HPTLC silica plate and with 13 bands were detected. As reference marker we chose gallic acid (Rf = 0.21) and catechin (Rf = 0.11). The two compound were detected as pale black bands at 366 nm after derivatization with sulfuric acid 10% v/v (in methanol) reagent. Validation of the method was met within validation criteria, so the developed method could be used for quality control of guava leaves.

  5. Improvement in Limit of Detection of Enzymatic Biogas Sensor Utilizing Chromatography Paper for Breath Analysis.

    Science.gov (United States)

    Motooka, Masanobu; Uno, Shigeyasu

    2018-02-02

    Breath analysis is considered to be an effective method for point-of-care diagnosis due to its noninvasiveness, quickness and simplicity. Gas sensors for breath analysis require detection of low-concentration substances. In this paper, we propose that reduction of the background current improves the limit of detection of enzymatic biogas sensors utilizing chromatography paper. After clarifying the cause of the background current, we reduced the background current by improving the fabrication process of the sensors utilizing paper. Finally, we evaluated the limit of detection of the sensor with the sample vapor of ethanol gas. The experiment showed about a 50% reduction of the limit of detection compared to previously-reported sensor. This result presents the possibility of the sensor being applied in diagnosis, such as for diabetes, by further lowering the limit of detection.

  6. Development of high performance liquid chromatography method for miconazole analysis in powder sample

    Science.gov (United States)

    Hermawan, D.; Suwandri; Sulaeman, U.; Istiqomah, A.; Aboul-Enein, H. Y.

    2017-02-01

    A simple high performance liquid chromatography (HPLC) method has been developed in this study for the analysis of miconazole, an antifungal drug, in powder sample. The optimized HPLC system using C8 column was achieved using mobile phase composition containing methanol:water (85:15, v/v), a flow rate of 0.8 mL/min, and UV detection at 220 nm. The calibration graph was linear in the range from 10 to 50 mg/L with r 2 of 0.9983. The limit of detection (LOD) and limit of quantitation (LOQ) obtained were 2.24 mg/L and 7.47 mg/L, respectively. The present HPLC method is applicable for the determination of miconazole in the powder sample with a recovery of 101.28 % (RSD = 0.96%, n = 3). The developed HPLC method provides short analysis time, high reproducibility and high sensitivity.

  7. Structural analysis of thermostabilizing mutations of cocaine esterase

    Energy Technology Data Exchange (ETDEWEB)

    Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan; Ko, Mei-Chuan; Macdonald, Joanne; Tamburi, Patricia; Yoon, Dan; Landry, Donald M.; Woods, James H.; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K. (Michigan); (Columbia); (Kentucky)

    2010-09-03

    Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures ({tau}{sub 1/2} {approx} 13 min at 37 C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.

  8. Analysis of glyphosate residues in cereals using liquid chromatography-mass spectrometry (LC-MS/MS)

    DEFF Research Database (Denmark)

    Granby, Kit; Johannesen, S.; Gabrielsen, Martin Vahl

    2003-01-01

    A fast and specific method for the determination of glyphosate in cereals is described. The method is based on extraction with water by ultrasonication. The samples are cleaned up and separated by high-performance liquid chromatography on a polystyrene-based reverse-phase column (clean-up) in ser......A fast and specific method for the determination of glyphosate in cereals is described. The method is based on extraction with water by ultrasonication. The samples are cleaned up and separated by high-performance liquid chromatography on a polystyrene-based reverse-phase column (clean...... monitored m/z 168--> 150 (glyphosate) and 170-->152 (internal standard 2- 13 (CN)-N-15-glyphosate) for quantification. The mean recovery was 85% ( n =32) at spiking levels from 0.03 to 0.33 mg kg(-1) . From 1998 to 2001, from the analysis of about 50 samples per annum, a reduction in the glyphosate residues...... was observed owing to a Danish trade decision not to use grain with glyphosate residues for milling or bread production....

  9. Quantitative analysis of benzodiazepines in vitreous humor by high-performance liquid chromatography

    Science.gov (United States)

    Bazmi, Elham; Behnoush, Behnam; Akhgari, Maryam; Bahmanabadi, Leila

    2016-01-01

    Objective: Benzodiazepines are frequently screened drugs in emergency toxicology, drugs of abuse testing, and in forensic cases. As the variations of benzodiazepines concentrations in biological samples during bleeding, postmortem changes, and redistribution could be biasing forensic medicine examinations, hence selecting a suitable sample and a validated accurate method is essential for the quantitative analysis of these main drug categories. The aim of this study was to develop a valid method for the determination of four benzodiazepines (flurazepam, lorazepam, alprazolam, and diazepam) in vitreous humor using liquid–liquid extraction and high-performance liquid chromatography. Methods: Sample preparation was carried out using liquid–liquid extraction with n-hexane: ethyl acetate and subsequent detection by high-performance liquid chromatography method coupled to diode array detector. This method was applied to quantify benzodiazepines in 21 authentic vitreous humor samples. Linear curve for each drug was obtained within the range of 30–3000 ng/mL with coefficient of correlation higher than 0.99. Results: The limit of detection and quantitation were 30 and 100 ng/mL respectively for four drugs. The method showed an appropriate intra- and inter-day precision (coefficient of variation forensic toxicology laboratory. PMID:27635251

  10. [Analysis of pigments from Rhodotorula glutinis by Raman spectroscopy and thin layer chromatography].

    Science.gov (United States)

    Yuan, Yu-feng; Tao, Zhan-hua; Wang, Xue; Li, Yong-qing; Liu, Jun-xian

    2012-03-01

    The pigments from Rhodotorula glutinis were separated by using thin layer chromatography, and the result showed that Rhodotorula glutinis cells could synthesize at least three kinds of pigments, which were beta-carotene, torulene, and torularhodin. The Raman spectra based on the three pigments were acquired, and original spectra were preprocessed by background elimination, baseline correction, and three-point-smoothing, then the averaged spectra from different pigments were investigated, and the result indicated that Raman shift which represents C-C bond was different, and the wave number of beta-carotene demonstrated the largest deviation, finally torulene and torularhodin in Rhodotorula glutinis had more content than beta-carotene. Quantitative analysis of Raman peak height ratio revealed that peak height ratio of pigments showed little difference, which could be used as parameters for further research on living cells, providing reference content of pigments. The above results suggest that Raman spectroscopy combined with thin layer chromatography can be applied to analyze pigments from Rhodotorula glutinis, provides abundant information about pigments, and serves as an effective method to study pigments.

  11. Re-analysis of radiation-induced specific locus mutations in the mouse

    International Nuclear Information System (INIS)

    Abrahamson, S.; Wolff, S.

    1976-01-01

    It is stated that a re-analysis of published data on mouse mutation rates induced by x-and gamma rays suggests that the kinetics of induction can be analysed by fitting that data to a parabolic curve. This is interpreted to mean that a substantial proportion of the induced mutations results from gross chromosomal changes such as deletions, some of which are one-track and some of which are two-track. This analysis is based on the assumption that the shape of the dose curve, which in the female is concave upward, reflects the manner in which the mutations are induced rather than representing a one-track (linear) curve whose shape has been modified by differential repair. (author)

  12. Amino-Acid Network Clique Analysis of Protein Mutation Non-Additive Effects: A Case Study of Lysozme

    Directory of Open Access Journals (Sweden)

    Dengming Ming

    2018-05-01

    Full Text Available Optimizing amino-acid mutations in enzyme design has been a very challenging task in modern bio-industrial applications. It is well known that many successful designs often hinge on extensive correlations among mutations at different sites within the enzyme, however, the underpinning mechanism for these correlations is far from clear. Here, we present a topology-based model to quantitively characterize non-additive effects between mutations. The method is based on the molecular dynamic simulations and the amino-acid network clique analysis. It examines if the two mutation sites of a double-site mutation fall into to a 3-clique structure, and associates such topological property of mutational site spatial distribution with mutation additivity features. We analyzed 13 dual mutations of T4 phage lysozyme and found that the clique-based model successfully distinguishes highly correlated or non-additive double-site mutations from those additive ones whose component mutations have less correlation. We also applied the model to protein Eglin c whose structural topology is significantly different from that of T4 phage lysozyme, and found that the model can, to some extension, still identify non-additive mutations from additive ones. Our calculations showed that mutation non-additive effects may heavily depend on a structural topology relationship between mutation sites, which can be quantitatively determined using amino-acid network k-cliques. We also showed that double-site mutation correlations can be significantly altered by exerting a third mutation, indicating that more detailed physicochemical interactions should be considered along with the network clique-based model for better understanding of this elusive mutation-correlation principle.

  13. Timing of the uv mutagenesis in yeast: a pedigree analysis of induced recessive mutation

    International Nuclear Information System (INIS)

    James, A.P.; Kilbey, B.J.

    1977-01-01

    The mechanism of uv-induced mutation in eukaryotes was studied in individual yeast cells by a procedure that combined pedigree analysis and tetrad analysis. The technique involved the induction of recessive lethals and semilethals in G1 diploid cells. Induced frequencies were 25 and 61% at survival levels of 90 and 77%, respectively. No evidence of gross chromosome aberrations was detected. Recessive mutations that affect only one strand or that affect both strands of the DNA molecule are induced much at random among a population of cells, and both types can occur within the same cell. However, the data confirm that two-strand mutations are in the majority after a low level of irradiation. The simplest explanation involves a mechanism whereby most mutations are fixed in both strands prior to the first round of post-irradiation DNA replication. The recessive mutational consequences of irradiation are exhausted at the conclusion of the first post-irradiation cell division, although dominant-lethal sectoring continues at a high level through the second post-irradiation division. It is concluded that pyrimidine dimers that persist to the second round of DNA replication are rare or ineffective

  14. The timing of UV mutagenesis in yeast: a pedigree analysis of induced recessive mutation.

    Science.gov (United States)

    James, A P; Kilbey, B J

    1977-10-01

    The mechanism of UV-induced mutation in eukaryotes was studied in individual yeast cells by a procedure that combined pedigree analysis and tetrad analysis. The technique involved the induction of recessive lethals and semilethals in G1 diploid cells. Induced frequencies were 25 and 61 percent at survival levels of 90 and 77 percent, respectively. No evidence of gross chromosome aberrations was detected. Recessive mutations that affect only one strand or that affect both strands of the DNA molecule are induced much at random among a population of cells, and both types can occur within the same cell. However, the data confirm that two-strand mutations are in the majority after a low level of irradiation. The simplest explanation involves a mechanism whereby most mutations are fixed in both strands prior to the first round of post-irradiation DNA replication. The recessive mutational consequences of irradiation are exhausted at the conclusion of the first post-irradiation cell division, although dominant-lethal sectoring continues at a high level through the second post-irradiation division. It is concluded that pyrimidine dimers that persist to the second round of DNA replication are rare or ineffective.

  15. Analysis of gene mutations in children with cholestasis of undefined etiology.

    Science.gov (United States)

    Matte, Ursula; Mourya, Reena; Miethke, Alexander; Liu, Cong; Kauffmann, Gregory; Moyer, Katie; Zhang, Kejian; Bezerra, Jorge A

    2010-10-01

    The discovery of genetic mutations in children with inherited syndromes of intrahepatic cholestasis allows for diagnostic specificity despite similar clinical phenotypes. Here, we aimed to determine whether mutation screening of target genes could assign a molecular diagnosis in children with idiopathic cholestasis. DNA samples were obtained from 51 subjects with cholestasis of undefined etiology and surveyed for mutations in the genes SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 by a high-throughput gene chip. Then, the sequence readouts for all 5 genes were analyzed for mutations and correlated with clinical phenotypes. Healthy subjects served as controls. Sequence analysis of the genes identified 14 (or 27%) subjects with missense, nonsense, deletion, and splice site variants associated with disease phenotypes based on the type of mutation and/or biallelic involvement in the JAG1, ATP8B1, ABCB11, or ABCB4 genes. These patients had no syndromic features and could not be differentiated by biochemical markers or histopathology. Among the remaining subjects, 10 (or ∼20%) had sequence variants in ATP8B1 or ABCB11 that involved only 1 allele, 8 had variants not likely to be associated with disease phenotypes, and 19 had no variants that changed amino acid composition. Gene sequence analysis assigned a molecular diagnosis in 27% of subjects with idiopathic cholestasis based on the presence of variants likely to cause disease phenotypes.

  16. RFLP analysis of rice semi-dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi-dwarf mutants induced by high energy argon ion radiation, Ar-10, and Xiang-Ar-1, were examined with restriction fragment length polymorphism (RFLP) analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes. Among the markers screened, 9 detected polymorphism were between Bianpizhen and Ar-10, and 11 detected polymorphism were between Xiangzhan and Xiang-Ar-1. Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5.15% and 6.39% for Ar-10 and Xiang-Ar-1 respectively. These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation. Genetic analysis and gene tagging of semi-dwarf mutation in one of the mutant line, Ar-10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4

  17. RFLP Analysis of rice semi dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi dwarf mutants induced by high energy argon ion radiation, Ar 10, and Xiang Ar 1, were examined with restriction fragment length polymorphism(RFLP)analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes.Among the markers screened, 9 detected polymorphism were between Bianpizhan and Ar 10, and 11 detected polymorphism were between Xiangzhan and Xiang Ar 1.Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5 15% and 6 39% for Ar 10 and Xiang Ar 1 respectively.These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation.Genetic analysis and gene tagging of semi dwarf mutation in one of the mutant line, Ar 10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4. (author)

  18. Analysis of the BRAFV600E mutation in 19 cases of Langerhans cell histiocytosis in Japan.

    Science.gov (United States)

    Sasaki, Yuya; Guo, Ying; Arakawa, Fumiko; Miyoshi, Hiroaki; Yoshida, Noriaki; Koga, Yuhki; Nakashima, Kazutaka; Kurita, Daisuke; Niino, Daisuke; Seto, Masao; Ohshima, Koichi

    2017-09-01

    Langerhans cell histiocytosis (LCH) is a rare disease characterized by clonal proliferation of CD1a- and CD207 (langerin)-positive dendritic cells. Mutated BRAF (p.V600E) is observed in histiocyte-related diseases and dendritic cell-related diseases, including LCH. BRAFV600E is observed in some LCH cases and is thought to be involved in maintaining MAPK activation. We retrospectively analyzed BRAFV600E in 19 patients diagnosed with LCH. In our study, direct sequencing for exon 15, a mutation hotspot, demonstrated that 4 out of the 19 patients (21%) harbored a GTG > GAG (valine > glutamic acid) base substitution, which encodes BRAFV600E. The clinical impact of BRAFV600E in such diseases is unclear. The frequency of BRAFV600E in our LCH patients from Japan was lower than that reported in the United States and in Germany. However, reports from Asia tend to show a lower rate of the BRAFV600E mutation. These results imply the possibility of different genetic backgrounds in the pathogenesis of LCH across various ethnicities. We also performed an immunohistochemical analysis to detect BRAFV600E using the mutation-specific monoclonal antibody. However, immunohistochemical analysis failed to detect any mutated protein in any of the 4 BRAFV600E-positive cases. This implies that at present, BRAFV600E should be assessed by direct sequencing. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Mutation analysis of the WFS1 gene in seven Danish Wolfram syndrome families; four new mutations identified

    DEFF Research Database (Denmark)

    Hansen, Lars; Eiberg, Hans Rudolf Lytchoff; Barrett, Timothy

    2005-01-01

    loss (LFSNHL). WFS1 variants were identified in eight subjects from seven families with WS, leading to the identification of four novel mutations, Q194X (nonsense), H313Y (missense), L313fsX360 (duplication frame shift) and F883fsX951 (deletion frame shift), and four previously reported mutations, A133...

  20. On-line liquid chromatography-gas chromatography: A novel approach for the analysis of phytosterol oxidation products in enriched foods.

    Science.gov (United States)

    Scholz, Birgit; Wocheslander, Stefan; Lander, Vera; Engel, Karl-Heinz

    2015-05-29

    A novel methodology for the automated qualitative and quantitative determination of phytosterol oxidation products in enriched foods via on-line liquid chromatography-gas chromatography (LC-GC) was established. The approach is based on the LC pre-separation of acetylated phytosterols and their corresponding oxides using silica as stationary phase and a mixture of n-hexane/methyl tert-butyl ether/isopropanol as eluent. Two LC-fractions containing (i) 5,6-epoxy- and 7-hydroxyphytosterols, and (ii) 7-ketophytosterols are transferred on-line to the GC for the analysis of their individual compositions on a medium polar trifluoropropylmethyl polysiloxane capillary column. Thus, conventionally employed laborious off-line purification and enrichment steps can be avoided. Validation data, including recovery, repeatability, and reproducibility of the method, were elaborated using an enriched margarine as example. The margarine was subjected to a heating procedure in order to exemplarily monitor the formation of phytosterol oxidation products. Quantification was performed using on-line LC-GC-FID, identification of the analytes was based on on-line LC-GC-MS. The developed approach offers a new possibility for the reliable and fast analysis of phytosterol oxidation products in enriched foods. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Quantitative Analysis of Tetramethylenedisulfotetramine ("Tetramine") Spiked into Beverages by Liquid Chromatography Tandem Mass Spectrometry with Validation by Gas Chromatography Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Owens, J; Hok, S; Alcaraz, A; Koester, C

    2008-11-13

    Tetramethylenedisulfotetramine, commonly known as tetramine, is a highly neurotoxic rodenticide (human oral LD{sub 50} = 0.1 mg/kg) used in hundreds of deliberate food poisoning events in China. Here we describe a method for quantitation of tetramine spiked into beverages, including milk, juice, tea, cola, and water and cleaned up by C8 solid phase extraction and liquid-liquid extraction. Quantitation by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) was based upon fragmentation of m/z 347 to m/z 268. The method was validated by gas chromatography mass spectrometry (GC/MS) operated in SIM mode for ions m/z 212, 240, and 360. The limit of quantitation was 0.10 {micro}g/mL by LC/MS/MS versus 0.15 {micro}g/mL for GC/MS. Fortifications of the beverages at 2.5 {micro}g/mL and 0.25 {micro}g/mL were recovered ranging from 73-128% by liquid-liquid extraction for GC/MS analysis, 13-96% by SPE and 10-101% by liquid-liquid extraction for LC/MS/MS analysis.

  2. Quantitative Analysis of Tetramethylenedisulfotetramine ('Tetramine') Spiked into Beverages by Liquid Chromatography Tandem Mass Spectrometry with Validation by Gas Chromatography Mass Spectrometry

    International Nuclear Information System (INIS)

    Owens, J.; Hok, S.; Alcaraz, A.; Koester, C.

    2008-01-01

    Tetramethylenedisulfotetramine, commonly known as tetramine, is a highly neurotoxic rodenticide (human oral LD 50 = 0.1 mg/kg) used in hundreds of deliberate food poisoning events in China. Here we describe a method for quantitation of tetramine spiked into beverages, including milk, juice, tea, cola, and water and cleaned up by C8 solid phase extraction and liquid-liquid extraction. Quantitation by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) was based upon fragmentation of m/z 347 to m/z 268. The method was validated by gas chromatography mass spectrometry (GC/MS) operated in SIM mode for ions m/z 212, 240, and 360. The limit of quantitation was 0.10 (micro)g/mL by LC/MS/MS versus 0.15 (micro)g/mL for GC/MS. Fortifications of the beverages at 2.5 (micro)g/mL and 0.25 (micro)g/mL were recovered ranging from 73-128% by liquid-liquid extraction for GC/MS analysis, 13-96% by SPE and 10-101% by liquid-liquid extraction for LC/MS/MS analysis.

  3. Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

    Science.gov (United States)

    Tian, Lingyang; Sato, Takuichi; Niwa, Kousuke; Kawase, Mitsuo; Tanner, Anne C R; Takahashi, Nobuhiro

    2014-01-01

    A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

  4. Rapid and Sensitive PCR-Dipstick DNA Chromatography for Multiplex Analysis of the Oral Microbiota

    Directory of Open Access Journals (Sweden)

    Lingyang Tian

    2014-01-01

    Full Text Available A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

  5. Mutation analysis of pre-mRNA splicing genes in Chinese families with retinitis pigmentosa

    Science.gov (United States)

    Pan, Xinyuan; Chen, Xue; Liu, Xiaoxing; Gao, Xiang; Kang, Xiaoli; Xu, Qihua; Chen, Xuejuan; Zhao, Kanxing; Zhang, Xiumei; Chu, Qiaomei; Wang, Xiuying

    2014-01-01

    Purpose Seven genes involved in precursor mRNA (pre-mRNA) splicing have been implicated in autosomal dominant retinitis pigmentosa (adRP). We sought to detect mutations in all seven genes in Chinese families with RP, to characterize the relevant phenotypes, and to evaluate the prevalence of mutations in splicing genes in patients with adRP. Methods Six unrelated families from our adRP cohort (42 families) and two additional families with RP with uncertain inheritance mode were clinically characterized in the present study. Targeted sequence capture with next-generation massively parallel sequencing (NGS) was performed to screen mutations in 189 genes including all seven pre-mRNA splicing genes associated with adRP. Variants detected with NGS were filtered with bioinformatics analyses, validated with Sanger sequencing, and prioritized with pathogenicity analysis. Results Mutations in pre-mRNA splicing genes were identified in three individual families including one novel frameshift mutation in PRPF31 (p.Leu366fs*1) and two known mutations in SNRNP200 (p.Arg681His and p.Ser1087Leu). The patients carrying SNRNP200 p.R681H showed rapid disease progression, and the family carrying p.S1087L presented earlier onset ages and more severe phenotypes compared to another previously reported family with p.S1087L. In five other families, we identified mutations in other RP-related genes, including RP1 p. Ser781* (novel), RP2 p.Gln65* (novel) and p.Ile137del (novel), IMPDH1 p.Asp311Asn (recurrent), and RHO p.Pro347Leu (recurrent). Conclusions Mutations in splicing genes identified in the present and our previous study account for 9.5% in our adRP cohort, indicating the important role of pre-mRNA splicing deficiency in the etiology of adRP. Mutations in the same splicing gene, or even the same mutation, could correlate with different phenotypic severities, complicating the genotype–phenotype correlation and clinical prognosis. PMID:24940031

  6. Mutation analysis for DJ-1 in sporadic and familial parkinsonism: screening strategy in parkinsonism.

    Science.gov (United States)

    Tomiyama, Hiroyuki; Li, Yuanzhe; Yoshino, Hiroyo; Mizuno, Yoshikuni; Kubo, Shin-Ichiro; Toda, Tatsushi; Hattori, Nobutaka

    2009-05-22

    DJ-1 mutations cause autosomal recessive parkinsonism (ARP). Although some reports of DJ-1 mutations have been published, there is lack of information on the prevalence of these mutations in large-scale studies of both familial and sporadic parkinsonism. In this genetic screening study, we analyzed the distribution and frequency of DJ-1 mutations by direct nucleotide sequencing of coding exons and exon-intron boundaries of DJ-1, in 386 parkin-negative parkinsonism patients (371 index cases: 67 probands of autosomal recessive parkinsonism families, 90 probands of autosomal dominant parkinsonism families, 201 patients with sporadic parkinsonism, and 13 with unknown family histories) from 12 countries (Japan 283, China 27, Taiwan 22, Korea 22, Israel 16, Turkey 5, Philippines 2, Bulgaria 2, Greece 2, Tunisia 1, USA 2, Ukraine 1, unknown 1). None had causative mutation in DJ-1, suggesting DJ-1 mutation is very rare among patients with familial and sporadic parkinsonism from Asian countries and those with other ethnic background. This is in contrast to the higher frequencies and worldwide distribution of parkin- and PINK1-related parkinsonism in ARP and sporadic parkinsonism. Thus, after obtaining clinical information, screening for mutations in (1) parkin, (2) PINK1, (3) DJ-1, (4) ATP13A2 should be conducted in that order, in ARP and sporadic parkinsonism, based on their reported frequencies. In addition, haplotype analysis should be employed to check for homozygosity of 1p36, which harbors a cluster of causative genes for ARP such as DJ-1, PINK1 and ATP13A2 in ARP and sporadic parkinsonism, especially in parkinsonism with consanguinity.

  7. Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

    Science.gov (United States)

    Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

    2017-01-01

    Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

  8. Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

    Science.gov (United States)

    Beltrán-Valero de Bernabé, D; Granadino, B; Chiarelli, I; Porfirio, B; Mayatepek, E; Aquaron, R; Moore, M M; Festen, J J; Sanmartí, R; Peñalva, M A; de Córdoba, S R

    1998-01-01

    Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles. PMID:9529363

  9. Mutational analysis of the cell cycle inhibitor Kip1/p27 in childhood leukemia.

    Science.gov (United States)

    Markaki, E-A; Stiakaki, E; Zafiropoulos, A; Arvanitis, D A; Katzilakis, N; Dimitriou, H; Spandidos, D A; Kalmanti, M

    2006-07-01

    Cyclin-dependent kinases (CDKs) and cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. Kip1/p27 is the main cyclin-dependent kinase inhibitor, which arrests cell division inhibiting G1-S transition. Kip1/p27 seems to play a critical role in the pathogenesis of several human malignancies and its lower expression has been shown to correlate with a poor prognosis in adult solid tumors. Bone marrow blasts from 49 children with leukemia, 37 acute lymphoblastic leukemia (ALL), and 12 acute myeloid leukemia (AML) were studied. Exon 3 of Kip1/p27 was amplified using the polymerase chain reaction technique (PCR). Single strand conformational polymorphism and heterodouplex analysis were performed to detect DNA sequence with altered conformations and were subsequently sequenced to document mutations. Mutations in Kip1/p27 gene were detected in 2 out of 3 T-ALL, 6 out of 12 AML patients, and only 1 out of 34 B lineage ALL cases. Although the patient groups are small, a highly significant relation of the mutation status with the type of leukemia (P = 0.0037) and the risk group according to treatment protocols (P = 0.00021) was estimated. A statistically significant difference in the white blood count was observed (P = 0.019) between the mutated and non-mutated patient groups although no statistically significant association of the mutation status with the hemoglobin and platelets values, karyotype, age, sex, disease progression, and outcome was determined. Based upon these results, the Kip1/p27 mutations should be considered for further prospective testing as an additional parameter for risk stratification and treatment of childhood leukemia. Copyright 2006 Wiley-Liss, Inc.

  10. Mutation analysis of the negative regulator cyclin G2 in gastric cancer

    African Journals Online (AJOL)

    Cyclin G2 is an unconventional cyclin which might have a potential negative role in carcinogenesis. In this study, the effect of cyclin G2 overexpression on gastric cell proliferation and expression levels of cyclin G2 in normal gastric cells and gastric cancer cells were investigated. Moreover, mutation analysis was performed ...

  11. USH2A mutation analysis in 70 Dutch families with Usher syndrome type II.

    NARCIS (Netherlands)

    Pennings, R.J.E.; Brinke, H. te; Weston, M.D.; Claassen, A.M.W.; Orten, D.J.; Weekamp, H.; Aarem, A. van; Huygen, P.L.M.; Deutman, A.F.; Hoefsloot, L.H.; Cremers, F.P.M.; Cremers, C.W.R.J.; Kimberling, W.J.; Kremer, J.M.J.

    2004-01-01

    Usher syndrome type II (USH2) is characterised by moderate to severe high-frequency hearing impairment, progressive visual loss due to retinitis pigmentosa and intact vestibular responses. Three loci are known for USH2, however, only the gene for USH2a (USH2A) has been identified. Mutation analysis

  12. Mutational analysis of the genome-linked protein of cowpea mosaic virus

    NARCIS (Netherlands)

    Carette, J.E.; Kujawa, A.; Gühl, K.; Verver, J.; Wellink, J.; Kammen, van A.

    2001-01-01

    In this study we have performed a mutational analysis of the cowpea mosaic comovirus (CPMV) genome-linked protein VPg to discern the structural requirements necessary for proper functioning of VPg. Either changing the serine residue linking VPg to RNA at a tyrosine or a threonine or changing the

  13. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    Science.gov (United States)

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASEStephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

  14. Linkage studies and mutation analysis of the PDEB gene in 23 families with Leber congenital amaurosis

    DEFF Research Database (Denmark)

    Riess, O; Weber, B; Nørremølle, Anne

    1992-01-01

    as to whether mutations in the human PDEB gene might cause LCA. We have previously cloned and characterized the human homologue of the mouse Pdeb gene and have mapped it to chromosome 4p16.3. In this study, a total of 23 LCA families of various ethnic backgrounds have been investigated. Linkage analysis using...

  15. Separation of Caffeine from Beverages and Analysis Using Thin-Layer Chromatography and Gas Chromatography-Mass Spectrometry

    Science.gov (United States)

    Torres y Torres, Janelle L.; Hiley, Shauna L.; Lorimor, Steven P.; Rhoad, Jonathan S.; Caldwell, Benjamin D.; Zweerink, Gerald L.; Ducey, Michael

    2015-01-01

    The Characterization and Analysis of a Product (CAP) project is used to introduce first-semester general chemistry students to chemical instrumentation through the analysis of caffeine-containing beverage products. Some examples of these products have included coffee, tea, and energy drinks. Students perform at least three instrumental experiments…

  16. Coupling of In Vitro Bioassays with Planar Chromatography in Effect-Directed Analysis.

    Science.gov (United States)

    Weiss, Stefan C; Egetenmeyer, Nicole; Schulz, Wolfgang

    Modern analytical test methods increasingly detect anthropogenic organic substances and their transformation products in water samples and in the environment. The presence of these compounds might pose a risk to the aquatic environment. To determine a possible (eco)toxicological risk, aquatic samples are tested using various bioassays, including sub-organismic assays such as the luminescent bacteria inhibition test, the acetylcholinesterase inhibition test, and the umu-test. The effect-directed analysis (EDA) combines physicochemical separation methods with biological (in vitro) tests. High-performance thin-layer chromatography (HPTLC) has proved to be particularly well suited for the separation of organic compounds and the subsequent analysis of effects by the application of the biotests directly on the surface of the HPTLC plate. The advantage of using HPTLC in comparison to high-performance liquid chromatography (HPLC) for EDA is that the solvent which is used as a mobile phase during chromatography is completely evaporated after the separation and therefore can no longer influence the applied bioassays.A prioritization during the complex identification process can be achieved when observed effects are associated with the separated zones in HPTLC. This increases the probability of identifying the substance responsible for an adverse effect from the multitude of organic trace substances in environmental samples. Furthermore, by comparing the pattern of biological effects of a separated sample, it is possible to track and assess changes in biological activity over time, over space, or in the course of a process, even without identifying the substance. HPTLC has already been coupled with various bioassays.Because HPTLC is a very flexible system, various detection techniques can be used and combined. In addition to the UV/Vis absorption and fluorescence measurements, TLC can also be coupled with a mass spectrometer (MS) for compound identification. In addition

  17. New insights into thyroglobulin gene: molecular analysis of seven novel mutations associated with goiter and hypothyroidism.

    Science.gov (United States)

    Citterio, Cintia E; Machiavelli, Gloria A; Miras, Mirta B; Gruñeiro-Papendieck, Laura; Lachlan, Katherine; Sobrero, Gabriela; Chiesa, Ana; Walker, Joanna; Muñoz, Liliana; Testa, Graciela; Belforte, Fiorella S; González-Sarmiento, Rogelio; Rivolta, Carina M; Targovnik, Héctor M

    2013-01-30

    The thyroglobulin (TG) gene is organized in 48 exons, spanning over 270 kb on human chromosome 8q24. Up to now, 62 inactivating mutations in the TG gene have been identified in patients with congenital goiter and endemic or non-endemic simple goiter. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report 13 patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and imaging evaluation. Single-strand conformation polymorphism (SSCP) analysis, endonuclease restriction analysis, sequencing of DNA, genotyping, population screening, and bioinformatics studies were performed. Molecular analyses revealed seven novel inactivating TG mutations: c.378C>A [p.Y107X], c.2359C>T [p.R768X], c.2736delG [p.R893fsX946], c.3842G>A [p.C1262Y], c.5466delA [p.K1803fsX1833], c.6000C>G [p.C1981W] and c.6605C>G [p.P2183R] and three previously reported mutations: c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.7006C>T [p.R2317X]. Six patients from two families were homozygous for p.R277X mutation, four were compound heterozygous mutations (p.Y107X/p.C1262Y, p.R893fsX946/p.A2215D, p.K1803fsX1832/p.R2317X), one carried three identified mutations (p.R277X/p.C1981W-p.P2183R) together with a hypothetical micro deletion and the remaining two siblings from another family with typical phenotype had a single p.R768X mutated allele. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of altered TG folding as a consequency of truncated TG proteins and missense mutations located in ACHE-like domain or that replace cysteine. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. Mutation Analysis of Consanguineous Moroccan Patients with Parkinson’s Disease Combining Microarray and Gene Panel

    Directory of Open Access Journals (Sweden)

    Ahmed Bouhouche

    2017-10-01

    Full Text Available During the last two decades, 15 different genes have been reported to be responsible for the monogenic form of Parkinson’s disease (PD, representing a worldwide frequency of 5–10%. Among them, 10 genes have been associated with autosomal recessive PD, with PRKN and PINK1 being the most frequent. In a cohort of 145 unrelated Moroccan PD patients enrolled since 2013, 19 patients were born from a consanguineous marriage, of which 15 were isolated cases and 4 familial. One patient was homozygous for the common LRRK2 G2019S mutation and the 18 others who did not carry this mutation were screened for exon rearrangements in the PRKN gene using Affymetrix Cytoscan HD microarray. Two patients were determined homozygous for PRKN exon-deletions, while another patient presented with compound heterozygous inheritance (3/18, 17%. Two other patients showed a region of homozygosity covering the 1p36.12 locus and were sequenced for the candidate PINK1 gene, which revealed two homozygous point mutations: the known Q456X mutation in exon 7 and a novel L539F variation in exon 8. The 13 remaining patients were subjected to next-generation sequencing (NGS that targeted a panel of 22 PD-causing genes and overlapping phenotypes. NGS data showed that two unrelated consanguineous patients with juvenile-onset PD (12 and 13 years carried the same homozygous stop mutation W258X in the ATP13A2 gene, possibly resulting from a founder effect; and one patient with late onset (76 years carried a novel heterozygous frameshift mutation in SYNJ1. Clinical analysis showed that patients with the ATP13A2 mutation developed juvenile-onset PD with a severe phenotype, whereas patients having either PRKN or PINK1 mutations displayed early-onset PD with a relatively mild phenotype. By identifying pathogenic mutations in 45% (8/18 of our consanguineous Moroccan PD series, we demonstrate that the combination of chromosomal microarray analysis and NGS is a powerful approach to

  19. Diagnosis of propionic acidemia by gas chromatography coupled to mass spectrometry in a case analysis

    International Nuclear Information System (INIS)

    Camayd Viera, Ivette; Robaina Jimenez, Zoe; Contreras Roura, Jiovanna

    2011-01-01

    Propionic acidemia is an inherited metabolic disease caused by a deficiency in the propionyl-CoA carboxilase, a biotin-dependent mitochondrial enzyme. The disorder is a clinically heterogeneous disease and one of the most frequently occurring organic acidurias. We report the first Cuban case with a severe form of propionic acidemia followed by acidosis and death. The diagnosis was carried out by gas chromatography coupled to mass spectrometry. Our aim is to highlight the importance of organic acids urine analysis as part of the first laboratory tests in undiagnosed seriously ill children. The definitive diagnosis is important as it serves as a clear guideline to establish a suitable treatment and allows geneticists to provide patients with a proper genetic counseling

  20. Integrating Paper Chromatography with Electrochemical Detection for the Trace Analysis of TNT in Soil

    Directory of Open Access Journals (Sweden)

    Patrick Ryan

    2015-07-01

    Full Text Available We report on the development of an electrochemical probe for the trace analysis of 2,4,6-trinitrotoluene (TNT in soil samples. The probe is a combination of graphite electrodes, filter paper, with ethylene glycol and choline chloride as the solvent/electrolyte. Square wave chromatovoltammograms show the probes have a sensitivity for TNT of 0.75 nA/ng and a limit of detection of 100 ng. In addition, by taking advantage of the inherent paper chromatography step, TNT can be separated in both time and cathodic peak potential from 4-amino-dinitrotolene co-spotted on the probe or in soil samples with the presence of methyl parathion as a possible interferent.

  1. [Analysis of phthalates in plastic food-packaging bags by thin layer chromatography].

    Science.gov (United States)

    Chen, Hui; Wang, Yuan; Zhu, Ruohua

    2006-01-01

    The method for simultaneous determination of four phthalates, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) in plastic food-packaging bags by thin layer chromatography (TLC) was developed. The plastic food-packaging bags were extracted with ethanol by ultrasonication, then the mixture was filtrated through membrane (0.45 microm). The mixture of ethyl acetate-anhydrous ether-isooctane (1 : 4 : 15, v/v) was used as developing agent on the TLC silica gel plate for development. The filtered liquid was spotted on the TLC plate dealt by acetone, and detected with scanning wavelength of 275 nm and reference wavelength of 340 nm. The qualitative analysis of the phthalates was performed using the R(f) values of the chromatogram. The quantitative analysis was performed with external standard method. Good linearities were obtained for DMP, DEP, DBP and DEHP. The detection limits were 2.1 ng for DMP, 2.4 ng for DEP, 3.4 ng for DBP and 4.0 ng for DEHP. The relative standard deviations (RSDs) of the four phthalates were 2.8% - 3.5%. The recoveries of the four phthalate standards in real sample were 78.58% - 111.04%. The method presented has the advantages of high precision, high sensitivity, small sample size, and simple pretreatment . The method was used to detect the four phthalates in the food-packaging bags. The contents in real samples were close to the results by gas chromatography.

  2. Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli

    International Nuclear Information System (INIS)

    Crosby, R.M.; Richardson, K.K.; Craft, T.R.; Benforado, K.B.; Liber, H.L.; Skopek, T.R.

    1988-01-01

    The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT - human lymphoblast colonies induced by eight repetitive 150 μM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli., DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism

  3. [Analysis of H63D mutation in hemochromatosis (HFE) gene in populations of central Eurasia].

    Science.gov (United States)

    Khusainova, R I; Khusnutdinova, N N; Litvinov, S S; Khusnutdinova, E K

    2013-02-01

    An analysis of the frequency of H63D (c. 187C>G) mutations in the HFEgene in 19 populations from Central Eurasia demonstrated that the distribution of the mutation in the region of interest was not uniform and that there were the areas of H63D accumulation. The investigation of three polymorphic variants, c.340+4T>C (rs2071303, IVS2(+4)T>C), c.893-44T>C (rs1800708, IVS4(-44)T>C), and c.1007-47G>A (rs1572982, IVS5(-47)A>G), in the HFE gene in individuals homozygous for H63D mutations in the HFE gene revealed the linkage of H63D with three haplotypes, *CTA, *TG, and *TTA. These findings indicated the partial spread of the mutation in Central Eurasia from Western Europe, as well as the possible repeated appearance of the mutation on the territory on interest.

  4. Screening of mutations affecting protein stability and dynamics of FGFR1—A simulation analysis

    Directory of Open Access Journals (Sweden)

    C. George Priya Doss

    2012-12-01

    Full Text Available Single amino acid substitutions in Fibroblast Growth Factor Receptor 1 (FGFR1 destabilize protein and have been implicated in several genetic disorders like various forms of cancer, Kallamann syndrome, Pfeiffer syndrome, Jackson Weiss syndrome, etc. In order to gain functional insight into mutation caused by amino acid substitution to protein function and expression, special emphasis was laid on molecular dynamics simulation techniques in combination with in silico tools such as SIFT, PolyPhen 2.0, I-Mutant 3.0 and SNAP. It has been estimated that 68% nsSNPs were predicted to be deleterious by I-Mutant, slightly higher than SIFT (37%, PolyPhen 2.0 (61% and SNAP (58%. From the observed results, P722S mutation was found to be most deleterious by comparing results of all in silico tools. By molecular dynamics approach, we have shown that P722S mutation leads to increase in flexibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. In addition, biophysical analysis revealed a clear insight of stability loss due to P722S mutation in FGFR1 protein. Majority of mutations predicted by these in silico tools were in good concordance with the experimental results.

  5. Prognostic Value of RUNX1 Mutations in AML: A Meta-Analysis

    Science.gov (United States)

    Jalili, Mahdi; Yaghmaie, Marjan; Ahmadvand, Mohammad; Alimoghaddam, Kamran; Mousavi, Seyed Asadollah; Vaezi, Mohammad; Ghavamzadeh, Ardeshir

    2018-02-26

    The RUNX1 (AML1) gene is a relatively infrequent mutational target in cases of acute myeloid leukemia (AML). Previous work indicated that RUNX1 mutations can have pathological and prognostic implications. To evaluate prognostic value, we conducted a meta-analysis of 4 previous published works with data for survival according to RUNX1 mutation status. Pooled hazard ratios for overall survival and disease-free survival were 1.55 (95% confidence interval (CI) = 1.11–2.15; p-value = 0.01) and 1.76 (95% CI = 1.24–2.52; p-value = 0.002), respectively, for cases positive for RUNX1 mutations. This evidence supports clinical implications of RUNX1 mutations in the development and progression of AML cases and points to the possibility of a distinct category within the newer WHO classification. Though it must be kept in mind that the present work was based on data extracted from observational studies, the findings suggest that the RUNX1 status can contribute to risk-stratification and decision-making in management of AML. Creative Commons Attribution License

  6. Systematic Analysis Reveals that Cancer Mutations Converge on Deregulated Metabolism of Arachidonate and Xenobiotics

    Directory of Open Access Journals (Sweden)

    Francesco Gatto

    2016-07-01

    Full Text Available Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network.

  7. Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

    Directory of Open Access Journals (Sweden)

    Zhi Xu

    Full Text Available Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7% in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

  8. [Analysis of USH2A gene mutation in a Chinese family affected with Usher syndrome].

    Science.gov (United States)

    Li, Pengcheng; Liu, Fei; Zhang, Mingchang; Wang, Qiufen; Liu, Mugen

    2015-08-01

    To investigate the disease-causing mutation in a Chinese family affected with Usher syndrome type II. All of the 11 members from the family underwent comprehensive ophthalmologic examination and hearing test, and their genomic DNA were isolated from venous leukocytes. PCR and direct sequencing of USH2A gene were performed for the proband. Wild type and mutant type minigene vectors containing exon 42, intron 42 and exon 43 of the USH2A gene were constructed and transfected into Hela cells by lipofectamine reagent. Reverse transcription (RT)-PCR was carried out to verify the splicing of the minigenes. Pedigree analysis and clinical diagnosis indicated that the patients have suffered from autosomal recessive Usher syndrome type II. DNA sequencing has detected a homozygous c.8559-2A>G mutation of the USH2A gene in the proband, which has co-segregated with the disease in the family. The mutation has affected a conserved splice site in intron 42, which has led to inactivation of the splice site. Minigene experiment has confirmed the retaining of intron 42 in mature mRNA. The c.8559-2A>G mutation in the USH2A gene probably underlies the Usher syndrome type II in this family. The splice site mutation has resulted in abnormal splicing of USH2A pre-mRNA.

  9. Identification of Missense Mutation (I12T in the BSND Gene and Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Hina Iqbal

    2011-01-01

    Full Text Available Nonsyndromic hearing loss is a paradigm of genetic heterogeneity with 85 loci and 39 nuclear disease genes reported so far. Mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness and nonsyndromic nearing loss. We studied a Pakistani consanguineous family. Clinical examinations of affected individuals did not reveal the presence of any associated signs, which are hallmarks of the Bartter syndrome type IV. Linkage analysis identified an area of 18.36 Mb shared by all affected individuals between markers D1S2706 and D1S1596. A maximum two-point LOD score of 2.55 with markers D1S2700 and multipoint LOD score of 3.42 with marker D1S1661 were obtained. BSND mutation, that is, p.I12T, cosegregated in all extant members of our pedigree. BSND mutations can cause nonsyndromic hearing loss, and it is a second report for this mutation. The respected protein, that is, BSND, was first modeled, and then, the identified mutation was further analyzed by using different bioinformatics tools; finally, this protein and its mutant was docked with CLCNKB and REN, interactions of BSND, respectively.

  10. Molecular genetic mutation analysis in Menkes-disease with prenatal diagnosis

    DEFF Research Database (Denmark)

    László, Aranka; Endreffy, Emoke; Tümer, Zeynep

    2010-01-01

    Menkes disease (MD) is an X-linked recessive multisystemic lethal, heredodegenerative disorder. Progressive neurodegeneration and connective tissue disturbances with microscopically kinky hair are the main symptoms. Molecular genetic mutation analysis was made at a Hungarian male infant suffering...... from MD and prenatal diagnosis was done in this MD loaded family. METHOD: The 12th exon of ATP7A gene has been analyzed by dideoxy-finger printing (DDF), polymerase chain reaction (PCR), direct sequencing of exon 12. The specific mutation was screened from chorionic villi of the maternal aunt at the 14......th gestational week. RESULTS: In the exon 12th a basic pair substitution with Arg 844 His change was detected leading to very severe fatal missense mutation....

  11. BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

    LENUS (Irish Health Repository)

    Stordal, Britta

    2013-06-01

    Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

  12. Hyphenated techniques in multidimensional gas chromatography for analysis of wine aroma and related odorants

    OpenAIRE

    Chin, Sung Tong

    2017-01-01

    The advent of analytical multidimensional gas chromatography (MDGC) techniques, with recent development in comprehensive two-dimensional gas chromatography (GC×GC) as well as microfluidic technology in conventional heart-cut MDGC, has achieved excellent separation efficiency for advanced characterization of complex volatile and semi-volatile samples, which is unlikely to be accomplished by single dimensional chromatography. This dissertation work has highlighted the potential opportunities fo...

  13. Simultaneous analysis of fourteen tertiary amine stimulants in human urine for doping control purposes by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

    International Nuclear Information System (INIS)

    Lu Jianghai; Wang San; Dong Ying; Wang Xiaobing; Yang Shuming; Zhang Jianli; Deng Jing; Qin Yang; Xu Youxuan; Wu Moutian; Ouyang Gangfeng

    2010-01-01

    A method for the simultaneous screening and confirmation of the presence of fourteen tertiary amine stimulants in human urine by gas chromatography-mass spectrometry (GC-MS) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Solid phase extraction (SPE) and liquid-liquid extraction (LLE) approaches were utilized for the pre-treatment of the urine samples. The study indicated that the capillary temperature played a significant role in the signal abundances of the protonated molecules of cropropamide and crotethamide under positive ion electrospray ionization (ESI) conditions. In addition, comparison studies of two different pre-treatment approaches as well as the two ionization modes were conducted. The LODs of the developed method for all the analytes were lower than the minimum required performance limit (MRPL) as set forth in the World Anti-Doping Agency (WADA) technical document for laboratories. The human urine sample obtained after oral administration of prolintane.HCl was successfully analyzed by the developed method, which demonstrated the applicability and reliability of the method for routine doping control analysis.

  14. Gas chromatography x gas chromatography-time-of-flight mass spectrometry analysis and antibacterial activity of essential oil from Amomum xanthophlebium

    International Nuclear Information System (INIS)

    Masila, A.; Aminah, I.; Yaakob, W.A.; Nazlina, I.

    2011-01-01

    Essential oils of fresh leaves, stem, rhizomes and whole aromatic plants of Amomum xanthophlebium (Zingiberaceae) were obtained by hydro distillation. Percentage yields of the leaf, stem and whole plant oils were 0.0032, 0.0074 and 0.0021 % whereas the rhizome oil obtained was very little. Chemical components of each oil and their percentages were determined by Gas Chromatography x Gas Chromatography-Time-of-Flight Mass Spectrometry (GCxGC-TOFMS). Analysis of A. xanthophlebium oils showed that they were dominated by terpenes. Main components in the leaves were allo-aromadendrene (3.41 %), (±)-globulol (2.58 %) and rosifoliol (2.55 %); stem, α-terpineol (4.25 %), rosifoliol (2.41 %) and bingpian (2.27 %); rhizomes, viridiflorol (5.72 %), (±)-globulol (5.23 %) and α-cadinol (4.81 %); whole plants, eucalyptol (4.11 %), l-α-terpineol (2.88 %) and rosifoliol (2.82 %). The stem oil of A. xanthophlebium showed antibacterial activity against Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) at the minimum inhibitory concentration of 80 mg/ ml. (author)

  15. Mutation analysis of the NSD1 gene in patients with autism spectrum disorders and macrocephaly

    Directory of Open Access Journals (Sweden)

    Delorme Richard

    2007-11-01

    Full Text Available Abstract Background Sotos syndrome is an overgrowth syndrome characterized by macrocephaly, advanced bone age, characteristic facial features, and learning disabilities, caused by mutations or deletions of the NSD1 gene, located at 5q35. Sotos syndrome has been described in a number of patients with autism spectrum disorders, suggesting that NSD1 could be involved in other cases of autism and macrocephaly. Methods We screened the NSD1 gene for mutations and deletions in 88 patients with autism spectrum disorders and macrocephaly (head circumference 2 standard deviations or more above the mean. Mutation analysis was performed by direct sequencing of all exons and flanking regions. Dosage analysis of NSD1 was carried out using multiplex ligation-dependent probe amplification. Results We identified three missense variants (R604L, S822C and E1499G in one patient each, but none is within a functional domain. In addition, segregation analysis showed that all variants were inherited from healthy parents and in two cases were also present in unaffected siblings, indicating that they are probably nonpathogenic. No partial or whole gene deletions/duplications were observed. Conclusion Our findings suggest that Sotos syndrome is a rare cause of autism spectrum disorders and that screening for NSD1 mutations and deletions in patients with autism and macrocephaly is not warranted in the absence of other features of Sotos syndrome.

  16. Knowledge-based analysis of functional impacts of mutations in ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... MicroRNAs are a class of important post-transcriptional regulators. ... In the analysis of a SNP in the seed region of hsa-miR-96 ... pathway' and 'Epidermal growth factor receptor signaling pathway' were significantly and ...

  17. Novel C16orf57 mutations in patients with Poikiloderma with Neutropenia: bioinformatic analysis of the protein and predicted effects of all reported mutations

    Directory of Open Access Journals (Sweden)

    Colombo Elisa A

    2012-01-01

    Full Text Available Abstract Background Poikiloderma with Neutropenia (PN is a rare autosomal recessive genodermatosis caused by C16orf57 mutations. To date 17 mutations have been identified in 31 PN patients. Results We characterize six PN patients expanding the clinical phenotype of the syndrome and the mutational repertoire of the gene. We detect the two novel C16orf57 mutations, c.232C>T and c.265+2T>G, as well as the already reported c.179delC, c.531delA and c.693+1G>T mutations. cDNA analysis evidences the presence of aberrant transcripts, and bioinformatic prediction of C16orf57 protein structure gauges the mutations effects on the folded protein chain. Computational analysis of the C16orf57 protein shows two conserved H-X-S/T-X tetrapeptide motifs marking the active site of a two-fold pseudosymmetric structure recalling the 2H phosphoesterase superfamily. Based on this model C16orf57 is likely a 2H-active site enzyme functioning in RNA processing, as a presumptive RNA ligase. According to bioinformatic prediction, all known C16orf57 mutations, including the novel mutations herein described, impair the protein structure by either removing one or both tetrapeptide motifs or by destroying the symmetry of the native folding. Finally, we analyse the geographical distribution of the recurrent mutations that depicts clusters featuring a founder effect. Conclusions In cohorts of patients clinically affected by genodermatoses with overlapping symptoms, the molecular screening of C16orf57 gene seems the proper way to address the correct diagnosis of PN, enabling the syndrome-specific oncosurveillance. The bioinformatic prediction of the C16orf57 protein structure denotes a very basic enzymatic function consistent with a housekeeping function. Detection of aberrant transcripts, also in cells from PN patients carrying early truncated mutations, suggests they might be translatable. Tissue-specific sensitivity to the lack of functionally correct protein accounts for the

  18. Characteristics and mutation analysis of Ph-positive leukemia patients with T315I mutation receiving tyrosine kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Xu PP

    2017-09-01

    Full Text Available Peipei Xu,1 Dan Guo,2 Xiaoyan Shao,1 Miaoxin Peng,1 Bing Chen2 1Department of Hematology, Drum Tower Hospital, School of Medicine, Nanjing University, 2Department of Hematology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, People’s Republic of China Background: TKIs are the first-line treatment for patients with Ph-positive (Ph+ leukemia. However, drug resistance is frequently observed, mainly due to mutations within the breakpoint cluster region-Abelson leukemia virus (BCR-ABL kinase domain. The T315I substitution confers complete resistance to TKIs. The aim of this study was to analyze the clinical characteristics of 17 patients with T315I mutation after TKI treatment and provide a basis for prognosis.Patients and methods: The clinical data of 17 TKI-resistant Ph+ leukemia patients who were found to have a ABL kinase domain mutation from September 2008 to January 2017 were collected. Karyotypes and BCR-ABL fusion gene were analyzed by R-banding and fluorescence in situ hybridization, respectively. Total RNA was extracted by TRIzol reagent, and the ABL kinase domain mutation was detected by direct sequencing.Results: A total of 17 patients reached effective remission including major molecular response and complete cytogenetic response. However, all the patients subsequently developed a T315I mutation after treatment with TKIs. The rate of the BCR-ABL fusion gene in most of the patients who developed the T315I mutation was significantly higher than that before the mutation. At initial diagnosis, patients average platelet count was 149.7×109/L, whereas the average platelet count was only 53.88×109/L after the T315I mutation (P<0.01. The results also showed that the survival time of patients with a high proportion of blast cells or a high number of white blood cells was obviously shortened.Conclusion: Patients platelet count decreased when detected with the T315I mutation compared with the initial

  19. Extending the range of compounds amenable for gas chromatography-mass spectrometric analysis.

    Science.gov (United States)

    Fialkov, Alexander B; Gordin, Alexander; Amirav, Aviv

    2003-04-04

    Gas chromatography-mass spectrometry (GC-MS) suffers from a major limitation in that an expanding number of thermally labile or low volatility compounds of interest are not amenable for analysis. We found that the elution temperatures of compounds from GC can be significantly lowered by reducing the column length, increasing the carrier gas flow rate, reducing the capillary column film thickness and lowering the temperature programming rate. Pyrene is eluted at 287 degrees C in standard GC-MS with a 30 m x 0.25 mm I.D. column with 1-microm DB5ms film and 1-ml/min He column flow rate. In contrast, pyrene is eluted at 79 degrees C in our "Supersonic GC-MS" system using a 1 m x 0.25 mm I.D. column with 0.1-microm DB5ms film and 100-ml/min He column flow rate. A simple model has been invoked to explain the significantly (up to 208 degrees C) lower elution temperatures observed. According to this model, every halving of the temperature programming rate, or number of separation plates (either through increased flow rate or due to reduced column length), results in approximately 20 degrees C lower elution temperature. These considerably lower elution temperatures enable the analysis of an extended range of thermally labile and low volatility compounds, that otherwise could not be analyzed by standard GC-MS. We demonstrate the analysis of large polycyclic aromatic hydrocarbons (PAHs) such as decacyclene with ten fused rings, well above the current GC limit of PAHs with six rings. Even a metalloporphirin such as magnesiumoctaethylporphin was easily analyzed with elution temperatures below 300 degrees C. Furthermore, a range of thermally labile compounds were analyzed including carbamates such as methomyl, aldicarb, aldicarbsulfone and oxamyl, explosives such as pentaerythritol tetranitrate, Tetryl and HMX, and drugs such as reserpine (608 a.m.u.). Supersonic GC-MS was used, based on the coupling of a supersonic molecular beam (SMB) inlet and ion sources with a bench

  20. Analysis of naphthenic acid mixtures as pentafluorobenzyl derivatives by gas chromatography-electron impact mass spectrometry.

    Science.gov (United States)

    Gutierrez-Villagomez, Juan Manuel; Vázquez-Martínez, Juan; Ramírez-Chávez, Enrique; Molina-Torres, Jorge; Trudeau, Vance L

    2017-01-01

    In this study, we report for the first time the efficiency of pentafluorobenzyl bromide (PFBBr) for naphthenic acid (NA) mixtures derivatization, and the comparison in the optimal conditions to the most common NAs derivatization reagents, BF 3 /MeOH and N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). Naphthenic acids are carboxylic acid mixtures of petrochemical origin. These compounds are important for the oil industry because of their corrosive properties, which can damage oil distillation infrastructure. Moreover, NAs are commercially used in a wide range of products such as paint and ink driers, wood and fabric preservatives, fuel additives, emulsifiers, and surfactants. Naphthenic acids have also been found in sediments after major oils spills in the United States and South Korea. Furthermore, the toxicity of the oil sands process-affected water (OSPW), product of the oil sands extraction activities in Canada's oil sands, has largely been attributed to NAs. One of the main challenges for the chromatographic analysis of these mixtures is the resolution of the components. The derivatization optimization was achieved using surface response analysis with molar ratio and time as factors for derivatization signal yield. After gas chromatography-electron impact mass spectrometry (GC/EIMS) analysis of a mixture of NA standards, it was found that the signal produced by PFB-derivatives was 2.3 and 1.4 times higher than the signal produced by methylated and MTBS-derivatives, respectively. The pentafluorobenzyl derivatives have a characteristic fragment ion at 181m/z that is diagnostic for the differentiation of carboxylic and non-carboxylic acid components within mixtures. In the analysis of a Sigma and a Merichem derivatized oil extract NA mixtures, it was found that some peaks lack the characteristic fragment ion; therefore they are not carboxylic acids. Open column chromatography was used to obtain a hexane and a methanol fraction of the Sigma and

  1. Analysis of particulate polycyclic aromatic hydrocarbons by on-line coupled supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry

    Science.gov (United States)

    Shimmo, Masahiko; Adler, Heidi; Hyötyläinen, Tuulia; Hartonen, Kari; Kulmala, Markku; Riekkola, Marja-Liisa

    An on-line supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry (SFE-LC-GC-MS) method was developed for the analysis of the particulate polycyclic aromatic hydrocarbons (PAHs). The limits of detection of the system for the quantification standards were in the range of 0.25-0.57 ng, while the limits of determinations for filter samples varied from 0.02 to 0.04 ng m -3 (24 h sampling). The linearity was excellent from 5 to 300 ng ( R2>0.967). The analysis could be carried out in a closed system without tedious manual sample pretreatment and with no risk of errors by contamination or loss of the analytes. The results of the SFE-LC-GC-MS method were comparable with those for Soxhlet and shake-flask extractions with GC-MS. The new method was applied to the analysis of PAHs collected by high-volume filter in the Helsinki area to study the seasonal trend of the concentrations. The individual PAH concentrations varied from 0.015 to more than 1 ng m -3, while total PAH concentrations varied from 0.81 to 5.68 ng m -3. The concentrations were generally higher in winter than in summer. The mass percentage of the total PAHs in total suspended particulates ranged from 2.85×10 -3% in July to 15.0×10 -3% in December. Increased emissions in winter, meteorological conditions, and more serious artefacts during the sampling in summer season may explain the concentration profiles.

  2. Evaluation and identification of factors related to KRAS and BRAF gene mutations in colorectal cancer: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Li Lin

    2016-01-01

    Conclusion: The meta-analysis reveals that KRAS has a slightly higher mutation rate in MSI-L/MSS tumors. Moreover, BRAF mutations have higher detection rates in right-sided colorectal cancer, which suggests that BRAF mutations are likely in CIMP-H tumors. Therefore, based on these findings, the molecular diagnostic tests to be conducted in colorectal cancer patients can be determined according to the location/clinical features of the tumor.

  3. Fast derivatization of fatty acids in different meat samples for gas chromatography analysis.

    Science.gov (United States)

    Figueiredo, Ingrid Lima; Claus, Thiago; Oliveira Santos Júnior, Oscar; Almeida, Vitor Cinque; Magon, Thiago; Visentainer, Jesuí Vergilio

    2016-07-22

    In order to analyze the composition of fatty acids employing gas chromatography as the separation method, a derivatization of lipids using esterification and transesterification reactions is needed. The methodologies currently available are time consuming and use large amounts of sample and reagents. Thus, this work proposes a new procedure to carry out the derivatization of fatty acids without the need for prior extraction of lipids. The use of small amounts of sample (100mg) allows the analysis to be performed in specific parts of animals, in most cases without having them slaughtered. Another benefit is the use of small amounts of reagents (only 2mL of NaOH/Methanol and H2SO4/Methanol). The use of an experimental design procedure (Design Expert software) allows the optimization of the alkaline and acid reaction times. The procedure was validated for five minutes in both steps. The method was validated for bovine fat, beef, chicken, pork, fish and shrimp meats. The results for the merit figures of accuracy (from 101.07% to 109.18%), precision (RSDintra-day (from 0.65 to 3.93%), RSDinter-day (from 1.57 to 5.22%)), linearity (R(2)=0.9864) and robustness confirmed that the new method is satisfactory within the linear range of 2-30% of lipids in the sample. Besides the benefits of minimizing the amount of samples and reagents, the procedure enables gas chromatography sample preparation in a very short time compared with traditional procedures. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. [Analysis of gene mutation of early onset epileptic spasm with unknown reason].

    Science.gov (United States)

    Yang, X; Pan, G; Li, W H; Zhang, L M; Wu, B B; Wang, H J; Zhang, P; Zhou, S Z

    2017-11-02

    Objective: To summarize the gene mutation of early onset epileptic spasm with unknown reason. Method: In this prospective study, data of patients with early onset epileptic spasm with unknown reason were collected from neurological department of Children's Hospital of Fudan University between March 2016 and December 2016. Patients with known disorders such as infection, metabolic, structural, immunological problems and known genetic mutations were excluded. Patients with genetic disease that can be diagnosed by clinical manifestations and phenotypic characteristics were also excluded. Genetic research methods included nervous system panel containing 1 427 epilepsy genes, whole exome sequencing (WES), analysis of copy number variation (CNV) and karyotype analysis of chromosome. The basic information, phenotypes, genetic results and the antiepileptic treatment of patients were analyzed. Result: Nine of the 17 cases with early onset epileptic spasm were boys and eight were girls. Patients' age at first seizure onset ranged from 1 day after birth to 8 months (median age of 3 months). The first hospital visit age ranged from 1 month to 2 years (median age of 4.5 months). The time of following-up ranged from 8 months to 3 years and 10 months. All the 17 patients had early onset epileptic spasm. Video electroencephalogram was used to monitor the spasm seizure. Five patients had Ohtahara syndrome, 10 had West syndrome, two had unclear classification. In 17 cases, 10 of them had detected pathogenic genes. Nine cases had point mutations, involving SCN2A, ARX, UNC80, KCNQ2, and GABRB3. Except one case of mutations in GABRB3 gene have been reported, all the other cases had new mutations. One patient had deletion mutation in CDKL5 gene. One CNV case had 6q 22.31 5.5MB repeats. Ten cases out of 17 were using 2-3 antiepileptic drugs (AEDs) and the drugs had no effect. Seven cases used adrenocorticotropic hormone (ACTH) and prednisone besides AEDs (a total course for 8 weeks

  5. Analysis of polycyclic aromatic hydrocarbons I. Determination by gas chromatography with glass and fused silica capillary columns

    International Nuclear Information System (INIS)

    Perez, M. M.; Gonzalez, D.

    1987-01-01

    A study of the analysis by gas chromatography of aromatic polycyclic hydrocarbons is presented. The separation has been carried out by glass and fused silica capillary column. The limitations and the advantages of the procedure are discussed in terms of separation efficiency, sensitivity and precision. (Author) 17 refs

  6. Comparative urine analysis by liquid chromatography-mass spectrometry and multivariate statistics : Method development, evaluation, and application to proteinuria

    NARCIS (Netherlands)

    Kemperman, Ramses F. J.; Horvatovich, Peter L.; Hoekman, Berend; Reijmers, Theo H.; Muskiet, Frits A. J.; Bischoff, Rainer

    2007-01-01

    We describe a platform for the comparative profiling of urine using reversed-phase liquid chromatography-mass spectrometry (LC-MS) and multivariate statistical data analysis. Urinary compounds were separated by gradient elution and subsequently detected by electrospray Ion-Trap MS. The lower limit

  7. Ionic liquids as silica deactivating agents in gas chromatography for direct analysis of primary amines in water

    NARCIS (Netherlands)

    Krzyzaniak, A.; Weggemans, W.M.A.; Schuur, B.; Haan, de A.B.

    2011-01-01

    Analysis of primary amines in aqueous samples remains a challenging analytical issue. The preferred approach by gas chromatography is hampered by interactions of free silanol groups with the highly reactive amine groups, resulting in inconsistent measurements. Here, we report a method for direct

  8. Ionic liquids as silica deactivating agents in gas chromatography for direct analysis or primary amines in water

    NARCIS (Netherlands)

    Krzyzaniak, A.; Weggemans, W.; Schuur, Boelo; de Haan, A.B.

    2011-01-01

    Analysis of primary amines in aqueous samples remains a challenging analytical issue. The preferred approach by gas chromatography is hampered by interactions of free silanol groups with the highly reactive amine groups, resulting in inconsistent measurements. Here, we report a method for direct

  9. Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry

    NARCIS (Netherlands)

    Gerssen, A.; McElhinney, M.; Mulder, P.P.J.; Bire, R.; Hess, P.; Boer, de J.

    2009-01-01

    The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC¿MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function

  10. Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry

    NARCIS (Netherlands)

    Gerssen, A.; McElhinney, A. M.; Mulder, P.P.J.; Bire, L.; Hess, P.; de Boer, J.

    2009-01-01

    The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function

  11. Analysis of polycyclic aromatic hydrocarbons. I. Determination by gas chromatography with glass and fused solica capillary columns

    International Nuclear Information System (INIS)

    Perez Garcia, M.; Gonzalez, D.

    1987-01-01

    A study of the analysis by gas chromatography of aromatic polycyclic hydrocarbons is presented. The separation has been carried out by glass and fused silice capillary column. The limitations and the advantages of the procedure are discussed in terms of separation efficiency, sensitivity and precision. (author). 3 figs., 17 refs

  12. Analysis of native human plasma proteins and haemoglobin for the presence of bityrosine by high-performance liquid chromatography

    DEFF Research Database (Denmark)

    Daneshvar, B; Frandsen, H; Dragsted, L O

    1997-01-01

    fluorescent substance, bityrosine. High-performance liquid chromatography (HPLC) analysis of acid hydrolyzed serum albumin after oxidation with peroxidase/H2O2 or with Cu++/H2O2 showed that bityrosine had been formed whereas oxidation of this protein with Fe(III)/ascorbate did not result in the formation...

  13. Glass bottle sampling solid phase microextraction gas chromatography mass spectrometry for breath analysis of drug metabolites.

    Science.gov (United States)

    Lu, Yan; Niu, Wenqi; Zou, Xue; Shen, Chengyin; Xia, Lei; Huang, Chaoqun; Wang, Hongzhi; Jiang, Haihe; Chu, Yannan

    2017-05-05

    Breath analysis is a non-invasive approach which may be applied to disease diagnosis and pharmacokinetic study. In the case of offline analysis, the exhaled gas needs to be collected and the sampling bag is often used as the storage vessel. However, the sampling bag usually releases some extra compounds, which may interfere with the result of the breath test. In this study, a novel breath sampling glass bottle was developed with a syringe needle sampling port for solid phase microextraction (SPME). Such a glass bottle scarcely liberates compounds and can be used to collect exhaled gas for ensuing analysis by gas chromatography-mass spectrometry (GC-MS). The glass bottle sampling SPME-GC-MS analysis was carried out to investigate the breath metabolites of myrtol, a multicompound drug normally used in the treatment of bronchitis and sinusitis. Four compounds, α-pinene, 2,3-dehydro-1,8-cineole, d-limonene and 1,8-cineole were found in the exhaled breath of all eight volunteers who had taken the myrtol. While for other ten subjects who had not used the myrtol, these compounds were undetectable. In the SPME-GC-MS analysis of the headspace of myrtol, three compounds were detected including α-pinene, d-limonene and 1,8-cineole. Comparing the results of breath and headspace analysis, it indicates that 2,3-dehydro-1,8-cineole in the breath is the metabolite of 1,8-cineole. It is the first time that this metabolite was identified in human breath. The study demonstrates that the glass bottle sampling SPME-GC-MS method is applicable to exhaled gas analysis including breath metabolites investigation of drugs like myrtol. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Occult HBV among Anti-HBc Alone: Mutation Analysis of an HBV Surface Gene and Pre-S Gene.

    Science.gov (United States)

    Kim, Myeong Hee; Kang, So Young; Lee, Woo In

    2017-05-01

    The aim of this study is to investigate the molecular characteristics of occult hepatitis B virus (HBV) infection in 'anti-HBc alone' subjects. Twenty-four patients with 'anti-HBc alone' and 20 control patients diagnosed with HBV were analyzed regarding S and pre-S gene mutations. All specimens were analyzed for HBs Ag, anti-HBc, and anti-HBs. For specimens with an anti-HBc alone, quantitative analysis of HBV DNA, as well as sequencing and mutation analysis of S and pre-S genes, were performed. A total 24 were analyzed for the S gene, and 14 were analyzed for the pre-S gene through sequencing. A total of 20 control patients were analyzed for S and pre-S gene simultaneously. Nineteen point mutations of the major hydrophilic region were found in six of 24 patients. Among them, three mutations, S114T, P127S/T, M133T, were detected in common. Only one mutation was found in five subjects of the control group; this mutation was not found in the occult HBV infection group, however. Pre-S mutations were detected in 10 patients, and mutations of site aa58-aa100 were detected in 9 patients. A mutation on D114E was simultaneously detected. Although five mutations from the control group were found at the same location (aa58-aa100), no mutations of occult HBV infection were detected. The prevalence of occult HBV infection is not low among 'anti-HBc alone' subjects. Variable mutations in the S gene and pre-S gene were associated with the occurrence of occult HBV infection. Further larger scale studies are required to determine the significance of newly detected mutations. © Copyright: Yonsei University College of Medicine 2017

  15. Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

    Science.gov (United States)

    Fuster, Oscar; Barragán, Eva; Bolufer, Pascual; Such, Esperanza; Valencia, Ana; Ibáñez, Mariam; Dolz, Sandra; de Juan, Inmaculada; Jiménez, Antonio; Gómez, Maria Teresa; Buño, Ismael; Martínez, Joaquín; Cervera, José; Montesinos, Pau; Moscardó, Federico; Sanz, Miguel Ángel

    2012-01-01

    During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

  16. Quantitative chromatography in the analysis of labelled compounds 1. Quantitative paper chromotography of amino acids by A spot comparison technique

    International Nuclear Information System (INIS)

    Barakat, M.F.; Farag, A.N.; El-Gharbawy, A.A.

    1974-01-01

    For the determination of the specific activity of labelled compounds separated by paper sheet chromatography, it was found essential to perfect the quantitative aspect of the paper chromatographic technique. Actually, so far paper chromatography has been used as a separation tool mainly and its use in quantification of the separated materials is by far less studied. In the present work, the quantitative analysis of amino acids by paper sheet chromatography has been carried out by methods, depending on the use of the relative spot area values for correcting the experimental data obtained. The results obtained were good and reproducible. The main advantage of the proposed technique is its extreme simplicity. No complicated equipment of procedures are necessary

  17. Analysis of 99mTc-labeled radiopharmaceuticals by high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Muto, Toshio

    1990-01-01

    High-performance liquid chromatography (HPLC) equiped with on line radiometric and optical detectors (i.e. radio-HPLC) have been applied to the radiochemical analysis of commonly-used 99m Tc-radio pharma ceuticals with a view point to check the radiochemical purities of the compounds. Chromatographic conditions were determined by examination of the types of column, mobile phase and pH. An aqueous size-exclusion (Shim-pack Diol-300) and reversed-phase column (Zorbax-ODS) were found to be suitable for 99m Tc-HSA and the other 99m Tc-agents, respectively. The analysis of low molecular weight 99m Tc-agents (e.g. 99m Tc-DTPA, 99m Tc-DMSA, 99m Tc-pyrophosphate, 99m Tc-phytic acid, 99m Tc-MDS, 99m Tc-HMDP) were done by reversed-phaseion pairing chromatography using a optimized mobile phase consisted on a mixture of 50 mM phosphate buffer (pH 7.0) and 2 mM TBA (tetra nbutyl) ammonium hydroxide) in 30 % methanol. The mobile phases for analysis of medium molecular weight 99m Tc-HSA were consisted of a mixture of 50 mM phosphate buffer (ph 7.0) in 30 % methanol, and a mixtures of 1 % SDS (sodium dodecyl sulfonate) in Tris buffer (pH. 7.0), respectively. It was apparent from the radio-chromatograms obtained from these chromatographic conditions, that impurity of 99m TcO 4 was observed in 99m Tc-pyrophosphate, 99m Tc-phytic acid, 99m Tc-MDP, 99m Tc-HMDP, and impurities of 99m Tc-labeled species and 99m TcO 4 , were observed in 99m Tc-HIDA, 99m Tc-HIDA, 99m Tc-HSA. The radiochemical impurities of the 99m Tc-radiopharmaceuticals were ranged between 90 and 100 %. From these results, radio-HPLC has been shown to be suitable method for analysis of 99m Tc-radiopharmaceuticals, with rapidity and excellent precision. (author)

  18. Analysis of mutations in the human HPRT gene induced by accelerated heavy-ion irradiation

    International Nuclear Information System (INIS)

    Kagawa, Yasuhiro; Yatagai, Fumio; Hanaoka, Fumio; Suzuki, Masao; Kase, Youko; Kobayashi, Akiko; Hirano, Masahiko; Kato, Takesi; Watanabe, Masami.

    1995-01-01

    Multiplex PCR analysis of HPRT(-) mutations in human embryo (HE) cells induced by 230 keV/μm carbon-ion irradiation showed no large deletion around the exon regions of the locus gene in contrast to the irradiations at different LETs. To identify these mutations, the sequence alterations in a cDNA of hprt gene were determined for 18 mutant clones in this study. Missing of exon 6 was the most frequent mutational event (10 clones), and missing of both exons 6 and 8 was next most frequent event (6 clones), then base substitutions (2 clones). These characteristics were not seen in a similar analysis of spontaneous mutations, which showed base substitution (5 clones), frameshift (2 clones), missing of both exons 2 and 3 (2 clones), and a single unidentified clone. Direct sequencing and restriction enzyme digestion of the genomic DNA of the mutants which showed missing of exons 6 and 8 in the cDNA, supports the possibility that they were induced by aberrant mRNA splicing. (author)

  19. High perfomance liquid chromatography fingerprint analysis for quality control of brotowali (Tinospora crispa)

    Science.gov (United States)

    Syarifah, V. B.; Rafi, M.; Wahyuni, W. T.

    2017-05-01

    Brotowali (Tinospora crispa) is widely used in Indonesia as ingredient of herbal medicine formulation. To ensure the quality, safety, and efficacy of herbal medicine products, its chemical constituents should be continuously evaluated. High performance liquid chromatography (HPLC) fingerprint is one of powerful technique for this quality control process. In this study, HPLC fingerprint analysis method was developed for quality control of brotowali. HPLC analysis was performed in C18 column and detection was performed using photodiode array detector. The optimum mobile phase for brotowali fingerprint was acetonitrile (ACN) and 0.1% formic acid in gradient elution mode at a flow rate of 1 mL/min. The number of peaks detected in HPLC fingerprint of brotowali was 32 peaks and 23 peaks for stems and leaves, respectively. Berberine as marker compound was detected at retention time of 20.525 minutes. Evaluation of analytical performance including precision, reproducibility, and stability prove that this HPLC fingerprint analysis was reliable and could be applied for quality control of brotowali.

  20. Headspace Analysis of Philippine Civet Coffee Beans Using Gas Chromatography-Mass Spectrometry and Electronic Nose

    Science.gov (United States)

    Ongo, E.; Sevilla, F.; Antonelli, A.; Sberveglieri, G.; Montevecchi, G.; Sberveglieri, V.; de Paola, E. L.; Concina, I.; Falasconi, M.

    2011-11-01

    Civet coffee, the most expensive and best coffee in the world, is an economically important export product of the Philippines. With a growing threat of food adulteration and counterfeiting, a need for quality authentication is essential to protect the integrity and strong market value of Philippine civet coffee. At present, there is no internationally accepted method of verifying whether a bean is an authentic civet coffee. This study presented a practical and promising approach to identify and establish the headspace qualitative profile of Philippine civet coffee using electronic nose (E-nose) and gas chromatography-mass spectrometry (GC-MS). E-nose analysis revealed that aroma characteristic is one of the most important quality indicators of civet coffee. The findings were supported by GC-MS analysis. Principal component analysis (PCA) exhibited a clearly separated civet coffees from their control beans. The chromatographic fingerprints indicated that civet coffees differed with their control beans in terms of composition and concentration of individual volatile constituents.

  1. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher

    2002-01-01

    Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  2. Mutational analysis of circulating tumor cells from colorectal cancer patients and correlation with primary tumor tissue.

    Directory of Open Access Journals (Sweden)

    Anna Lyberopoulou

    Full Text Available Circulating tumor cells (CTCs provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3 mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.

  3. Analysis of HFE gene mutations and HLA-A alleles in Brazilian patients with iron overload

    Directory of Open Access Journals (Sweden)

    Rodolfo Delfini Cançado

    Full Text Available CONTEXT AND OBJECTIVE: Hemochromatosis is a common inherited disorder of iron metabolism and one of the most important causes of iron overload. The objective was to analyze the presence of C282Y, H63D and S65C mutations in the HFE gene and HLA-A alleles for a group of Brazilian patients with iron overload, and to correlate genotype with clinical and laboratory variables. DESIGN AND SETTING: Prospective study, in Discipline of Hematology and Oncology, Faculdade de Ciências Médicas da Santa Casa de Misericórdia de São Paulo. METHODS: We studied 35 patients with iron overload seen at our outpatient unit between January 2001 and December 2003. Fasting levels of serum iron and ferritin, and total iron-binding capacity, were assayed using standard techniques. Determinations of C282Y, H63D and S65C mutations in the HFE gene and of HLA-A alleles were performed by polymerase chain reaction (PCR. RESULTS: Twenty-six out of 35 patients (74% presented at least one of the HFE gene mutations analyzed. Among these, five (14% were C282Y/C282Y, four (11% C282Y/H63D, one (3% H63D/H63D, six (17% C282Y/WT and ten (29% H63D/WT. No patients had the S65C mutation and nine (25% did not present any of the three HFE mutations. Four out of five patients with C282Y/C282Y genotype (80% and three out of four patients with C282Y/H63D genotype (75% were HLA A*03. CONCLUSION: Analysis of HFE gene mutations constitutes an important procedure in identifying patients with hereditary hemochromatosis, particularly for patients with iron overload.

  4. In silico analysis of a novel MKRN3 missense mutation in familial central precocious puberty.

    Science.gov (United States)

    Neocleous, Vassos; Shammas, Christos; Phelan, Marie M; Nicolaou, Stella; Phylactou, Leonidas A; Skordis, Nicos

    2016-01-01

    The onset of puberty is influenced by the interplay of stimulating and restraining factors, many of which have a genetic origin. Premature activation of the GnRH secretion in central precocious puberty (CPP) may arise either from gain-of-function mutations of the KISS1 and KISS1R genes or from loss-of-function manner mutations of the MKRN3 gene leading to MKRN3 deficiency. To explore the genetic causes responsible for CPP and the potential role of the RING finger protein 3 (MKRN3) gene. We investigated potential sequence variations in the intronless MKRN3 gene by Sanger sequencing of the entire 507 amino acid coding region of exon 1 in a family with two affected girls presented with CPP at the age of 6 and 5·7 years, respectively. A novel heterozygous g.Gly312Asp missense mutation in the MKRN3 gene was identified in these siblings. The imprinted MKRN3 missense mutation was also identified as expected in the unaffected father and followed as expected an imprinted mode of inheritance. In silico analysis of the altered missense variant using the computational algorithms Polyphen2, SIFT and Mutation Taster predicted a damage and pathogenic alteration causing CPP. The pathogenicity of the alteration at the protein level via an in silico structural model is also explored. A novel mutation in the MKRN3 gene in two sisters with CPP was identified, supporting the fundamental role of this gene in the suppression of the hypothalamic GnRH neurons. © 2015 John Wiley & Sons Ltd.

  5. Alagille syndrome in a Vietnamese cohort: mutation analysis and assessment of facial features.

    Science.gov (United States)

    Lin, Henry C; Le Hoang, Phuc; Hutchinson, Anne; Chao, Grace; Gerfen, Jennifer; Loomes, Kathleen M; Krantz, Ian; Kamath, Binita M; Spinner, Nancy B

    2012-05-01

    Alagille syndrome (ALGS, OMIM #118450) is an autosomal dominant disorder that affects multiple organ systems including the liver, heart, eyes, vertebrae, and face. ALGS is caused by mutations in one of two genes in the Notch Signaling Pathway, Jagged1 (JAG1) or NOTCH2. In this study, analysis of 21 Vietnamese ALGS individuals led to the identification of 19 different mutations (18 JAG1 and 1 NOTCH2), 17 of which are novel, including the third reported NOTCH2 mutation in Alagille Syndrome. The spectrum of JAG1 mutations in the Vietnamese patients is similar to that previously reported, including nine frameshift, three missense, two splice site, one nonsense, two whole gene, and one partial gene deletion. The missense mutations are all likely to be disease causing, as two are loss of cysteines (C22R and C78G) and the third creates a cryptic splice site in exon 9 (G386R). No correlation between genotype and phenotype was observed. Assessment of clinical phenotype revealed that skeletal manifestations occur with a higher frequency than in previously reported Alagille cohorts. Facial features were difficult to assess and a Vietnamese pediatric gastroenterologist was only able to identify the facial phenotype in 61% of the cohort. To assess the agreement among North American dysmorphologists at detecting the presence of ALGS facial features in the Vietnamese patients, 37 clinical dysmorphologists evaluated a photographic panel of 20 Vietnamese children with and without ALGS. The dysmorphologists were unable to identify the individuals with ALGS in the majority of cases, suggesting that evaluation of facial features should not be used in the diagnosis of ALGS in this population. This is the first report of mutations and phenotypic spectrum of ALGS in a Vietnamese population. Copyright © 2012 Wiley Periodicals, Inc.

  6. Recent advances in ultra-high performance liquid chromatography for the analysis of traditional chinese medicine

    Science.gov (United States)

    Traditional Chinese medicines (TCMs) have been widely used for the prevention and treatment of various diseases for thousands of years in China. Ultra Performance Liquid Chromatography (UHPLC) is a relatively new technique offering new possibilities in liquid chromatography. This paper reviews recen...

  7. Gas Chromatography-Mass Spectroscopic (GC-MS) Analysis of n ...

    African Journals Online (AJOL)

    tuber-regium (synonym Pleurotus tuber regium) using gas chromatography-mass spectroscopic (GC-. MS) techniques. Methods: The n-hexane extract of the sclerotia ... Soxhlet extraction and analysed using gas chromatography-mass spectroscopic (MS) techniques. ..... Phytochemical composition of Pleurotus tuber regium.

  8. Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates.

    Science.gov (United States)

    Moerdijk-Poortvliet, Tanja C W; Schierbeek, Henk; Houtekamer, Marco; van Engeland, Tom; Derrien, Delphine; Stal, Lucas J; Boschker, Henricus T S

    2015-07-15

    We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ(13)C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although LC/IRMS is expected to be more accurate and precise, no direct comparison has been reported. GC/IRMS with the aldonitrile penta-acetate (ANPA) derivatisation method was compared with LC/IRMS without derivatisation. A large number of glucose standards and a variety of natural samples were analysed for five neutral carbohydrates at natural abundance as well as at (13)C-enriched levels. Gas chromatography/chemical ionisation mass spectrometry (GC/CIMS) was applied to check for incomplete derivatisation of the carbohydrate, which would impair the accuracy of the GC/IRMS method. The LC/IRMS technique provided excellent precision (±0.08‰ and ±3.1‰ at natural abundance and enrichment levels, respectively) for the glucose standards and this technique proved to be superior to GC/IRMS (±0.62‰ and ±19.8‰ at natural abundance and enrichment levels, respectively). For GC/IRMS measurements the derivatisation correction and the conversion of carbohydrates into CO2 had a considerable effect on the measured δ(13)C values. However, we did not find any significant differences in the accuracy of the two techniques over the full range of natural δ(13)C abundances and (13)C-labelled glucose. The difference in the performance of GC/IRMS and LC/IRMS diminished when the δ(13)C values were measured in natural samples, because the chromatographic performance and background correction became critical factors, particularly for LC/IRMS. The derivatisation of carbohydrates for the GC/IRMS method was complete. Although both LC/IRMS and GC/IRMS are reliable techniques for compound-specific stable carbon isotope analysis of carbohydrates (provided that derivatisation is complete and the

  9. Rapid direct analysis to discriminate geographic origin of extra virgin olive oils by flash gas chromatography electronic nose and chemometrics.

    Science.gov (United States)

    Melucci, Dora; Bendini, Alessandra; Tesini, Federica; Barbieri, Sara; Zappi, Alessandro; Vichi, Stefania; Conte, Lanfranco; Gallina Toschi, Tullia

    2016-08-01

    At present, the geographical origin of extra virgin olive oils can be ensured by documented traceability, although chemical analysis may add information that is useful for possible confirmation. This preliminary study investigated the effectiveness of flash gas chromatography electronic nose and multivariate data analysis to perform rapid screening of commercial extra virgin olive oils characterized by a different geographical origin declared in the label. A comparison with solid phase micro extraction coupled to gas chromatography mass spectrometry was also performed. The new method is suitable to verify the geographic origin of extra virgin olive oils based on principal components analysis and discriminant analysis applied to the volatile profile of the headspace as a fingerprint. The selected variables were suitable in discriminating between "100% Italian" and "non-100% Italian" oils. Partial least squares discriminant analysis also allowed prediction of the degree of membership of unknown samples to the classes examined. Copyright © 2016. Published by Elsevier Ltd.

  10. The analysis of carbohydrates in milk powder by a new "heart-cutting" two-dimensional liquid chromatography method.

    Science.gov (United States)

    Ma, Jing; Hou, Xiaofang; Zhang, Bing; Wang, Yunan; He, Langchong

    2014-03-01

    In this study, a new"heart-cutting" two-dimensional liquid chromatography method for the simultaneous determination of carbohydrate contents in milk powder was presented. In this two dimensional liquid chromatography system, a Venusil XBP-C4 analysis column was used in the first dimension ((1)D) as a pre-separation column, a ZORBAX carbohydrates analysis column was used in the second dimension ((2)D) as a final-analysis column. The whole process was completed in less than 35min without a particular sample preparation procedure. The capability of the new two dimensional HPLC method was demonstrated in the determination of carbohydrates in various brands of milk powder samples. A conventional one dimensional chromatography method was also proposed. The two proposed methods were both validated in terms of linearity, limits of detection, accuracy and precision. The comparison between the results obtained with the two methods showed that the new and completely automated two dimensional liquid chromatography method is more suitable for milk powder sample because of its online cleanup effect involved. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  11. Analysis of perfluoroalkyl substances in cord blood by turbulent flow chromatography coupled to tandem mass spectrometry

    International Nuclear Information System (INIS)

    Llorca, Marta; Pérez, Francisca; Farré, Marinella; Agramunt, Sílvia; Kogevinas, Manolis; Barceló, Damià

    2012-01-01

    A fast on-line analytical method based on turbulent flow chromatography (TFC) in combination with tandem mass spectrometry has been applied for the first time for the analysis of eighteen perfluoroalkyl substances (PFASs), in cord blood. A simple and rapid sample pre-treatment was optimised consisting on protein precipitation of 100 μL of sample with acetonitrile (1:1) followed by centrifugation during 10 min. The method was adapted to be sensitive enough and robust with minimum sample injection volume requirements (20 μL). The optimised methodology presented method limits of detection (MLOD) between 0.031 and 0.76 μg/L, detection capabilities (CCα) in the range between 0.005 and 0.99 μg/L and decision limits (CCβ) ranging from 0.006 to 1.16 μg/L. The recoveries in blank blood were calculated by spiking experiments with a mixture of 18 PFASs and established between 70 and 126% for most of compounds. Isotopic dilution was carried out for quantification of selected analytes. In-house validation of this new approach was carried out according to the requirements in the 2002/657/EC Decision. Finally the good applicability of this new approach was proved by the analysis of 60 cord blood samples from two different Mediterranean cities, Barcelona (Spain) and Heraklion (Greece). Ions perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) were found at highest concentration and the more frequently compounds were PFHxS, PFOS and perfluorooctanoic acid (PFOA). The newly developed method proved to be suitable for large-scale epidemiologic studies, and to the data on PFASs exposure during pregnancy. -- Highlights: ► An on-line method has been developed for the analysis of 18 perfluoroalkyl substances. ► The method is based on turbulent flow chromatography tandem mass spectrometry. ► The method was applied in 60 cord blood samples from 2 Mediterranean cities. ► Acidic compounds were more frequently found and the method was proved to be suitable for

  12. Analysis of perfluoroalkyl substances in cord blood by turbulent flow chromatography coupled to tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Llorca, Marta; Perez, Francisca [Department of Environmental Chemistry, IDAEA-CSIC, Barcelona (Spain); Farre, Marinella, E-mail: mfuqam@cid.csic.es [Department of Environmental Chemistry, IDAEA-CSIC, Barcelona (Spain); Agramunt, Silvia [Centre for Research in Environmental Epidemiology (CREAL), Barcelona (Spain); IMIM (Hospital del Mar Research Institute), Barcelona (Spain); Kogevinas, Manolis [Centre for Research in Environmental Epidemiology (CREAL), Barcelona (Spain); IMIM (Hospital del Mar Research Institute), Barcelona (Spain); CIBER Epidemiologia y Salud Publica (CIBERESP), Barcelona (Spain); National School of Public Health, Athens (Greece); Barcelo, Damia [Department of Environmental Chemistry, IDAEA-CSIC, Barcelona (Spain); Catalan Institute for Water Research (ICRA), Girona (Spain); King Saud University, Riyadh (Saudi Arabia)

    2012-09-01

    A fast on-line analytical method based on turbulent flow chromatography (TFC) in combination with tandem mass spectrometry has been applied for the first time for the analysis of eighteen perfluoroalkyl substances (PFASs), in cord blood. A simple and rapid sample pre-treatment was optimised consisting on protein precipitation of 100 {mu}L of sample with acetonitrile (1:1) followed by centrifugation during 10 min. The method was adapted to be sensitive enough and robust with minimum sample injection volume requirements (20 {mu}L). The optimised methodology presented method limits of detection (MLOD) between 0.031 and 0.76 {mu}g/L, detection capabilities (CC{alpha}) in the range between 0.005 and 0.99 {mu}g/L and decision limits (CC{beta}) ranging from 0.006 to 1.16 {mu}g/L. The recoveries in blank blood were calculated by spiking experiments with a mixture of 18 PFASs and established between 70 and 126% for most of compounds. Isotopic dilution was carried out for quantification of selected analytes. In-house validation of this new approach was carried out according to the requirements in the 2002/657/EC Decision. Finally the good applicability of this new approach was proved by the analysis of 60 cord blood samples from two different Mediterranean cities, Barcelona (Spain) and Heraklion (Greece). Ions perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) were found at highest concentration and the more frequently compounds were PFHxS, PFOS and perfluorooctanoic acid (PFOA). The newly developed method proved to be suitable for large-scale epidemiologic studies, and to the data on PFASs exposure during pregnancy. -- Highlights: Black-Right-Pointing-Pointer An on-line method has been developed for the analysis of 18 perfluoroalkyl substances. Black-Right-Pointing-Pointer The method is based on turbulent flow chromatography tandem mass spectrometry. Black-Right-Pointing-Pointer The method was applied in 60 cord blood samples from 2 Mediterranean cities

  13. Comprehensive two-dimensional gas chromatography for biogas and biomethane analysis.

    Science.gov (United States)

    Hilaire, F; Basset, E; Bayard, R; Gallardo, M; Thiebaut, D; Vial, J

    2017-11-17

    The gas industry is going to be revolutionized by being able to generate bioenergy from biomass. The production of biomethane - a green substitute of natural gas - is growing in Europe and the United-States of America. Biomethane can be injected into the gas grid or used as fuel for vehicles after compression. Due to various biomass inputs (e.g. agricultural wastes, sludges from sewage treatment plants, etc.), production processes (e.g. anaerobic digestion, municipal solid waste (MSW) landfills), seasonal effects and purification processes (e.g. gas scrubbers, pressure swing adsorption, membranes for biogas upgrading), the composition and quality of biogas and biomethane produced is difficult to assess. All previous publications dealing with biogas analysis reported that hundreds of chemicals from ten chemical families do exist in trace amounts in biogas. However, to the best of our knowledge, no study reported a detailed analysis or the implementation of comprehensive two-dimensional gas chromatography (GC×GC) for biogas matrices. This is the reason why the benefit of implementing two-dimensional gas chromatography for the characterization of biogas and biomethane samples was evaluated. In a first step, a standard mixture of 89 compounds belonging to 10 chemical families, representative of those likely to be found, was used to optimize the analytical method. A set consisting of a non-polar and a polar columns, respectively in the first and the second dimension, was used with a modulation period of six seconds. Applied to ten samples of raw biogas, treated biogas and biomethane collected on 4 industrial sites (two MSW landfills, one anaerobic digester on a wastewater treatment plant and one agricultural biogas plant), this analytical method provided a "fingerprint" of the gases composition at the molecular level in all biogas and biomethane samples. Estimated limits of detection (far below the μgNm -3 ) coupled with the resolution of GC×GC allowed the comparison

  14. Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses.

    Directory of Open Access Journals (Sweden)

    Huma Saleem

    Full Text Available The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV and cotton leaf curl Kokhran virus (CLCuKoV, led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4 and CLCuKoV-CP gene (2.706X10-4, whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.

  15. Recent advancements and future trends in environmental analysis: Sample preparation, liquid chromatography and mass spectrometry.

    Science.gov (United States)

    Pérez-Fernández, Virginia; Mainero Rocca, Lucia; Tomai, Pierpaolo; Fanali, Salvatore; Gentili, Alessandra

    2017-08-29

    Among the thousands of chemicals having potential to enter the environment, the NORMAN network has identified at least 700 substances categorized into 20 classes in the European surface waters. Pesticides, pharmaceuticals, disinfection by-products, wood preservation and industrial chemicals are the prominent classes. Since the impact of these substances on aquatic life and human health might be dramatic, action is urgently required at multiple levels; one of them is just related to the development of more and more sensible and selective analytical methods. This review highlights the latest advancements and trends in liquid chromatography-mass spectrometry based environmental analysis. Specific sections are dedicated to novelties in sample preparation, chromatographic separation and mass spectrometry detection of emerging pollutants. The review also offers insights on last generation chromatographic and extraction materials, technological progresses and innovative methodological approaches for target and non-target analysis. As numerous papers have been published in this field, this overview covers the most representative and original works published in the 2011-2016 period. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Analysis of 62 synthetic cannabinoids by gas chromatography-mass spectrometry with photoionization.

    Science.gov (United States)

    Akutsu, Mamoru; Sugie, Ken-Ichi; Saito, Koichi

    2017-01-01

    Gas chromatography-mass spectrometry (GC-MS) in electron ionization (EI) mode is one of the most commonly used techniques for analysis of synthetic cannabinoids, because the GC-EI-MS spectra contain characteristic fragment ions for identification of a compound; however, the information on its molecular ions is frequently lacking. To obtain such molecular ion information, GC-MS in chemical ionization (CI) mode is frequently used. However, GC-CI-MS requires a relatively tedious process using reagent gas such as methane or isobutane. In this study, we show that GC-MS in photoionization (PI) mode provided molecular ions in all spectra of 62 synthetic cannabinoids, and 35 of the 62 compounds showed only the molecular radical cations. Except for the 35 compounds, the PI spectra showed very simple patterns with the molecular peak plus only a few fragment peak(s). An advantage is that the ion source for GC-PI-MS can easily be used for GC-EI-MS as well. Therefore, GC-EI/PI-MS will be a useful tool for the identification of synthetic cannabinoids contained in a dubious product. To the best of our knowledge, this is the first report to use GC-PI-MS for analysis of synthetic cannabinoids.

  17. Simultaneous analysis of multiple neurotransmitters by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry.

    Science.gov (United States)

    Tufi, Sara; Lamoree, Marja; de Boer, Jacob; Leonards, Pim

    2015-05-22

    Neurotransmitters are endogenous metabolites that allow the signal transmission across neuronal synapses. Their biological role is crucial for many physiological functions and their levels can be changed by several diseases. Because of their high polarity, hydrophilic interaction liquid chromatography (HILIC) is a promising tool for neurotransmitter analysis. Due to the large number of HILIC stationary phases available, an evaluation of the column performances and retention behaviors has been performed on five different commercial HILIC packing materials (silica, amino, amide and two zwitterionic stationary phases). Several parameters like the linear correlation between retention and the distribution coefficient (logD), the separation factor k and the column resolution Rs have been investigated and the column performances have been visualized with a heat map and hierarchical clustering analysis. An optimized and validated HILIC-MS/MS method based on the ZIC-cHILIC column is proposed for the simultaneous detection and quantification of twenty compounds consisting of neurotransmitters, precursors and metabolites: 3-methoxytyramine (3-MT), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxy-L-tripthophan, acetylcholine, choline, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, epinephrine, γ-aminobutyric acid (GABA), glutamate, glutamine, histamine, histidine, L-tryptophan, L-tyrosine, norepinephrine, normetanephrine, phenylalanine, serotonin and tyramine. The method was applied to neuronal metabolite profiling of the central nervous system of the freshwater snail Lymnaea stagnalis. This method is suitable to explore neuronal metabolism and its alteration in different biological matrices. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Proteomic analysis of Taenia ovis metacestodes by high performance liquid chromatography-coupled tandem mass spectrometry.

    Science.gov (United States)

    Zheng, Yadong

    2017-03-15

    Taenia ovis metacestodes reside in the muscle of sheep and goats, and may cause great economic loss due to condemnation of carcasses if not effectively controlled. Although advances have been made in the control of T. ovis infection, our knowledge of T. ovis biology is limited. Herein the protein profiling of T. ovis metacestodes was determined by liquid chromatography-linked tandem mass spectrometry. A total of 966 proteins were identified and 25.1% (188/748) were annotated to be associated with metabolic pathways. Consistently, GO analysis returned a metabolic process (16.27%) as one of two main biological process terms. Moreover, it was found that 24 proteins, including very low-density lipoprotein receptor, enolase, paramyosin and endophilin B1, were abundant in T. ovis metacestodes. These proteins may be associated with motility, metabolism, signaling, stress, drug resistance and immune responses. Furthermore, comparative analysis of 5 cestodes revealed the presence of Taenia-specific enolases. These data provide clues for better understanding of T. ovis biology, which is informative for effective control of infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Morphological, spectral and chromatography analysis and forensic comparison of PET fibers.

    Science.gov (United States)

    Farah, Shady; Tsach, Tsadok; Bentolila, Alfonso; Domb, Abraham J

    2014-06-01

    Poly(ethylene terephthalate) (PET) fiber analysis and comparison by spectral and polymer molecular weight determination was investigated. Plain fibers of PET, a common textile fiber and plastic material was chosen for this study. The fibers were analyzed for morphological (SEM and AFM), spectral (IR and NMR), thermal (DSC) and molecular weight (MS and GPC) differences. Molecular analysis of PET fibers by Gel Permeation Chromatography (GPC) allowed the comparison of fibers that could not be otherwise distinguished with high confidence. Plain PET fibers were dissolved in hexafluoroisopropanol (HFIP) and analyzed by GPC using hexafluoroisopropanol:chloroform 2:98 v/v as eluent. 14 PET fiber samples, collected from various commercial producers, were analyzed for polymer molecular weight by GPC. Distinct differences in the molecular weight of the different fiber samples were found which may have potential use in forensic fiber comparison. PET fibers with average molecular weights between about 20,000 and 70,000 g mol(-1) were determined using fiber concentrations in HFIP as low as 1 μg mL(-1). This GPC analytical method can be applied for exclusively distinguish between PET fibers using 1 μg of fiber. This method can be extended to forensic comparison of other synthetic fibers such as polyamides and acrylics. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Molecular Ionization-Desorption Analysis Source (MIDAS) for Mass Spectrometry: Thin-Layer Chromatography

    Science.gov (United States)

    Winter, Gregory T.; Wilhide, Joshua A.; LaCourse, William R.

    2016-02-01

    Molecular ionization-desorption analysis source (MIDAS), which is a desorption atmospheric pressure chemical ionization (DAPCI) type source, for mass spectrometry has been developed as a multi-functional platform for the direct sampling of surfaces. In this article, its utility for the analysis of thin-layer chromatography (TLC) plates is highlighted. Amino acids, which are difficult to visualize without staining reagents or charring, were detected and identified directly from a TLC plate. To demonstrate the full potential of MIDAS, all active ingredients from an analgesic tablet, separated on a TLC plate, were successfully detected using both positive and negative ion modes. The identity of each of the compounds was confirmed from their mass spectra and compared against standards. Post separation, the chemical signal (blue permanent marker) as reference marks placed at the origin and solvent front were used to calculate retention factor (Rf) values from the resulting ion chromatogram. The quantitative capabilities of the device were exhibited by scanning caffeine spots on a TLC plate of increasing sample amount. A linear curve based on peak are, R2 = 0.994, was generated for seven spots ranging from 50 to 1000 ng of caffeine per spot.

  1. Detection of non-milk fat in milk fat by gas chromatography and linear discriminant analysis.

    Science.gov (United States)

    Gutiérrez, R; Vega, S; Díaz, G; Sánchez, J; Coronado, M; Ramírez, A; Pérez, J; González, M; Schettino, B

    2009-05-01

    Gas chromatography was utilized to determine triacylglycerol profiles in milk and non-milk fat. The values of triacylglycerol were subjected to linear discriminant analysis to detect and quantify non-milk fat in milk fat. Two groups of milk fat were analyzed: A) raw milk fat from the central region of Mexico (n = 216) and B) ultrapasteurized milk fat from 3 industries (n = 36), as well as pork lard (n = 2), bovine tallow (n = 2), fish oil (n = 2), peanut (n = 2), corn (n = 2), olive (n = 2), and soy (n = 2). The samples of raw milk fat were adulterated with non-milk fats in proportions of 0, 5, 10, 15, and 20% to form 5 groups. The first function obtained from the linear discriminant analysis allowed the correct classification of 94.4% of the samples with levels <10% of adulteration. The triacylglycerol values of the ultrapasteurized milk fats were evaluated with the discriminant function, demonstrating that one industry added non-milk fat to its product in 80% of the samples analyzed.

  2. Possibilities of gas-phase radio-chromatography application to permanent-gas analysis

    International Nuclear Information System (INIS)

    Dupuis, M.C.; Charrier, G.; Alba, C.; Massimino, D.

    1970-01-01

    The gas-phase radio-chromatography technique has been applied to the rapid analysis of permanent gases (H 2 , O 2 , N 2 , A, Kr, Xe, CO, CH 4 ) labelled with radioactive indicators ( 3 H, 37 A, 85 Kr, 133 Xe). After calibration, the components of such a mixture can be identified and their concentrations measured in less than two hours, using a sample volume of from 0.1 to 10 cm 3 . The minimum detectable activity is of the order of 10 -4 μC for each radioactive isotope. The measurements are reproducible to about 2 to 3 per cent. This work has been mainly concerned with the influence of parameters affecting the response of the radioactivity detector (ionization chamber or gas flow proportional counter). The method has very numerous applications both theoretically, for the study of chromatographic phenomena under ideal conditions (infinitesimal concentrations made possible by the use of radioactive tracers), and practically, for rapid and accurate radiochemical analysis of radioactive gas mixtures. (authors) [fr

  3. Comparative analysis of peak-detection techniques for comprehensive two-dimensional chromatography.

    Science.gov (United States)

    Latha, Indu; Reichenbach, Stephen E; Tao, Qingping

    2011-09-23

    Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful technology for separating complex samples. The typical goal of GC×GC peak detection is to aggregate data points of analyte peaks based on their retention times and intensities. Two techniques commonly used for two-dimensional peak detection are the two-step algorithm and the watershed algorithm. A recent study [4] compared the performance of the two-step and watershed algorithms for GC×GC data with retention-time shifts in the second-column separations. In that analysis, the peak retention-time shifts were corrected while applying the two-step algorithm but the watershed algorithm was applied without shift correction. The results indicated that the watershed algorithm has a higher probability of erroneously splitting a single two-dimensional peak than the two-step approach. This paper reconsiders the analysis by comparing peak-detection performance for resolved peaks after correcting retention-time shifts for both the two-step and watershed algorithms. Simulations with wide-ranging conditions indicate that when shift correction is employed with both algorithms, the watershed algorithm detects resolved peaks with greater accuracy than the two-step method. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Clinical and mutation analysis of 51 probands with anophthalmia and/or severe microphthalmia from a single center

    Science.gov (United States)

    Gerth-Kahlert, Christina; Williamson, Kathleen; Ansari, Morad; Rainger, Jacqueline K; Hingst, Volker; Zimmermann, Theodor; Tech, Stefani; Guthoff, Rudolf F; van Heyningen, Veronica; FitzPatrick, David R

    2013-01-01

    Clinical evaluation and mutation analysis was performed in 51 consecutive probands with severe eye malformations – anophthalmia and/or severe microphthalmia – seen in a single specialist ophthalmology center. The mutation analysis consisted of bidirectional sequencing of the coding regions of SOX2, OTX2, PAX6 (paired domain), STRA6, BMP4, SMOC1, FOXE3, and RAX, and genome-wide array-based copy number assessment. Fifteen (29.4%) of the 51 probands had likely causative mutations affecting SOX2 (9/51), OTX2 (5/51), and STRA6 (1/51). Of the cases with bilateral anophthalmia, 9/12 (75%) were found to be mutation positive. Three of these mutations were large genomic deletions encompassing SOX2 (one case) or OTX2 (two cases). Familial inheritance of three intragenic, plausibly pathogenic, and heterozygous mutations was observed. An unaffected carrier parent of an affected child with an identified OTX2 mutation confirmed the previously reported nonpenetrance for this disorder. Two families with SOX2 mutations demonstrated a parent and child both with significant but highly variable eye malformations. Heterozygous loss-of-function mutations in SOX2 and OTX2 are the most common genetic pathology associated with severe eye malformations and bi-allelic loss-of-function in STRA6 is confirmed as an emerging cause of nonsyndromal eye malformations. PMID:24498598

  5. Identification of Mutation of Glucose-6-Phosphate Dehy-drogenase (G6PD) in Iran: Meta- analysis Study.

    Science.gov (United States)

    Moosazadeh, Mahmood; Nekoei-Moghadam, Mahmood; Aliram-Zany, Maryam; Amiresmaili, Mohammadreza

    2013-09-01

    Glucose-6-phosphate dehydrogenase is one of the most common genetic deficiencies, which approximately 400 million people in the world suffer from. According to authors' initial search, numerous studies have been carried out in Iran regarding molecular variants of this enzyme. Thus, this meta-analysis presented a reliable estimation about prevalence of different types of molecular mutations of G6PD Enzyme in Iran. Keywords "glucose 6 phosphate dehydrogenase or G6PD, Mediterranean or Chatham or Cosenza and mutation, Iran or Iranian and their Persian equivalents" were searched in different databases. Moreover, reference list of the published studies were examined to increase sensitivity and to select more studies. After studying titles and abstracts of retrieved articles, excluding the repeated and unrelated ones, and evaluating quality of articles, documents were selected. Data was analyzed using STATA. After performing systematic review, 22 papers were entered this meta-analysis and 1698 subjects were examined concerning G6PD molecular mutation. In this meta-analysis, prevalence of Mediterranean mutation, Chatham mutation and Cosenza mutation in Iran was estimated 78.2%, 9.1% and 0.5% respectively. This meta-analysis showed that in spite of prevalence of different types of G6PD molecular mutations in center, north, north-west and west of Iran, the most common molecular mutations in people with G6PD deficiency in Iran, like other Mediterranean countries and countries around Persian Gulf, were Mediterranean mutation, Chatham mutation and Cosenza mutation. It is also recommended that future studies may focus on races and regions which haven't been taken into consideration up to now.

  6. An Usher syndrome type 1 patient diagnosed before the appearance of visual symptoms by MYO7A mutation analysis.

    Science.gov (United States)

    Yoshimura, Hidekane; Iwasaki, Satoshi; Kanda, Yukihiko; Nakanishi, Hiroshi; Murata, Toshinori; Iwasa, Yoh-ichiro; Nishio, Shin-ya; Takumi, Yutaka; Usami, Shin-ichi

    2013-02-01

    Usher syndrome type 1 (USH1) appears to have only profound non-syndromic hearing loss in childhood and retinitis pigmentosa develops in later years. This study examined the frequency of USH1 before the appearance of visual symptoms in Japanese deaf children by MYO7A mutation analysis. We report the case of 6-year-old male with profound hearing loss, who did not have visual symptoms. The frequency of MYO7A mutations in profound hearing loss children is also discussed. We sequenced all exons of the MYO7A gene in 80 Japanese children with severe to profound non-syndromic HL not due to mutations of the GJB2 gene (ages 0-14 years). A total of nine DNA variants were found and six of them were presumed to be non-pathogenic variants. In addition, three variants of them were found in two patients (2.5%) with deafness and were classified as possible pathogenic variants. Among them, at least one nonsense mutation and one missense mutation from the patient were confirmed to be responsible for deafness. After MYO7A mutation analysis, the patient was diagnosed with RP, and therefore, also diagnosed with USH1. This is the first case report to show the advantage of MYO7A mutation analysis to diagnose USH1 before the appearance of visual symptoms. We believed that MYO7A mutation analysis is valid for the early diagnosis of USH1. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. PKD2 mutation in an Iranian autosomal dominant polycystic kidney disease family with misleading linkage analysis data

    Directory of Open Access Journals (Sweden)

    Mona Entezam

    2016-06-01

    Conclusion: Although analysis of additive informative polymorphic markers can overcome the misleading haplotype data, it is limited because of the lack of other highly polymorphic microsatellite markers closer to the gene. Direct mutation screening can identify the causative mutation in the apparently unlinked pedigree; moreover, it is the only approach to achieve the confirmed diagnosis in individuals with equivocal imaging results.

  8. Identification of novel BRCA founder mutations in Middle Eastern breast cancer patients using capture and Sanger sequencing analysis.

    Science.gov (United States)

    Bu, Rong; Siraj, Abdul K; Al-Obaisi, Khadija A S; Beg, Shaham; Al Hazmi, Mohsen; Ajarim, Dahish; Tulbah, Asma; Al-Dayel, Fouad; Al-Kuraya, Khawla S

    2016-09-01

    Ethnic differences of breast cancer genomics have prompted us to investigate the spectra of BRCA1 and BRCA2 mutations in different populations. The prevalence and effect of BRCA 1 and BRCA 2 mutations in Middle Eastern population is not fully explored. To characterize the prevalence of BRCA mutations in Middle Eastern breast cancer patients, BRCA mutation screening was performed in 818 unselected breast cancer patients using Capture and/or Sanger sequencing. 19 short tandem repeat (STR) markers were used for founder mutation analysis. In our study, nine different types of deleterious mutation were identified in 28 (3.4%) cases, 25 (89.3%) cases in BRCA 1 and 3 (10.7%) cases in BRCA 2. Seven recurrent mutations identified accounted for 92.9% (26/28) of all the mutant cases. Haplotype analysis was performed to confirm c.1140 dupG and c.4136_4137delCT mutations as novel putative founder mutation, accounting for 46.4% (13/28) of all BRCA mutant cases and 1.6% (13/818) of all the breast cancer cases, respectively. Moreover, BRCA 1 mutation was significantly associated with BRCA 1 protein expression loss (p = 0.0005). Our finding revealed that a substantial number of BRCA mutations were identified in clinically high risk breast cancer from Middle East region. Identification of the mutation spectrum, prevalence and founder effect in Middle Eastern population facilitates genetic counseling, risk assessment and development of cost-effective screening strategy. © 2016 UICC.

  9. F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

    Science.gov (United States)

    Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

    2014-05-01

    Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

  10. Pan-Cancer Mutational and Transcriptional Analysis of the Integrator Complex

    Directory of Open Access Journals (Sweden)

    Antonio Federico

    2017-04-01

    Full Text Available The integrator complex has been recently identified as a key regulator of RNA Polymerase II-mediated transcription, with many functions including the processing of small nuclear RNAs, the pause-release and elongation of polymerase during the transcription of protein coding genes, and the biogenesis of enhancer derived transcripts. Moreover, some of its components also play a role in genome maintenance. Thus, it is reasonable to hypothesize that their functional impairment or altered expression can contribute to malignancies. Indeed, several studies have described the mutations or transcriptional alteration of some Integrator genes in different cancers. Here, to draw a comprehensive pan-cancer picture of the genomic and transcriptomic alterations for the members of the complex, we reanalyzed public data from The Cancer Genome Atlas. Somatic mutations affecting Integrator subunit genes and their transcriptional profiles have been investigated in about 11,000 patients and 31 tumor types. A general heterogeneity in the mutation frequencies was observed, mostly depending on tumor type. Despite the fact that we could not establish them as cancer drivers, INTS7 and INTS8 genes were highly mutated in specific cancers. A transcriptome analysis of paired (normal and tumor samples revealed that the transcription of INTS7, INTS8, and INTS13 is significantly altered in several cancers. Experimental validation performed on primary tumors confirmed these findings.

  11. Genetic analysis of suppressors of the PF10 mutation in Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Dutcher, S.K.; Gibbons, W.; Inwood, W.B.

    1988-01-01

    A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another

  12. The importance of conventional radiography in the mutational analysis of skeletal dysplasias (the TRPV4 mutational family)

    Energy Technology Data Exchange (ETDEWEB)

    Nemec, Stefan F.; Cohn, Daniel H.; Krakow, Deborah; Funari, Vincent A.; Rimoin, David L.; Lachman, Ralph S. [Medical Genetics Institute, Cedars Sinai Medical Center, International Skeletal Dysplasia Registry, Los Angeles, CA (United States)

    2012-01-15

    The spondylo and spondylometaphyseal dysplasias (SMDs) are characterized by vertebral changes and metaphyseal abnormalities of the tubular bones, which produce a phenotypic spectrum of disorders from the mild autosomal-dominant brachyolmia to SMD Kozlowski to autosomal-dominant metatropic dysplasia. Investigations have recently drawn on the similar radiographic features of those conditions to define a new family of skeletal dysplasias caused by mutations in the transient receptor potential cation channel vanilloid 4 (TRPV4). This review demonstrates the significance of radiography in the discovery of a new bone dysplasia family due to mutations in a single gene. (orig.)

  13. Supercritical Fluid Extraction and Ultra Performance Liquid Chromatography of Respiratory Quinones for Microbial Community Analysis in Environmental and Biological Samples

    OpenAIRE

    Hanif, Muhammad; Atsuta, Yoichi; Fujie, Koichi; Daimon, Hiroyuki

    2012-01-01

    Microbial community structure plays a significant role in environmental assessment and animal health management. The development of a superior analytical strategy for the characterization of microbial community structure is an ongoing challenge. In this study, we developed an effective supercritical fluid extraction (SFE) and ultra performance liquid chromatography (UPLC) method for the analysis of bacterial respiratory quinones (RQ) in environmental and biological samples. RQ profile analysi...

  14. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    Science.gov (United States)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  15. Applications of liquid phase chromatographies for the analysis of streams arising at the back end of nuclear fuel cycle

    International Nuclear Information System (INIS)

    Deshingkar, D.S.; Yalmali, Vrunda S.; Wattal, P.K.

    2000-06-01

    India has opted for a closed fuel cycle comprising of reprocessing and recycling technology. The back end of such nuclear fuel cycle involves the reprocessing of spent nuclear fuels for recovery of plutonium and depleted uranium by Purex technology. Wastes arising from the reprocessing plant are classified as high, intermediate and low level wastes (HLW, ILW, LLW). HLW is mixture of over 50 elements present in different chemical forms. The accurate analyses of dissolver solution and HLW are the most challenging but essential tasks for reprocessing plant operations and also for further development of treatment methods. Inductively coupled plasma - atomic emission spectroscopy and atomic absorption spectroscopy techniques are suitable for analysis of metallic anions. Ion chromatography has proven capability to analyse number of cations or anions at ppm or even ppb level in single run. The report reviews the literature regarding the title subject. To assess the technical feasibility of ion chromatography for waste analysis, a simulated PHWR-HLW analogue was prepared. The PHWR-HLW analogue and ground water samples were analysed on DIONEX-DX 500 and Metrohm IC. Results obtained clearly demonstrated the usefulness of ion chromatography as vital analytical tool. HLW and other process or waste streams arising at the back end of nuclear fuel cycle can be analysed for alkali, alkaline earth, rare earth and transition metal cations and important anions. Use of fraction collector along with ion chromatography can enhance it's sensitivity to few Bq/ml for radioactive samples. (author)

  16. Qualitative and quantitative analysis of pyrolysis oil by gas chromatography with flame ionization detection and comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry.

    Science.gov (United States)

    Sfetsas, Themistoklis; Michailof, Chrysa; Lappas, Angelos; Li, Qiangyi; Kneale, Brian

    2011-05-27

    Pyrolysis oils have attracted a lot of interest, as they are liquid energy carriers and general sources of chemicals. In this work, gas chromatography with flame ionization detector (GC-FID) and two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) techniques were used to provide both qualitative and quantitative results of the analysis of three different pyrolysis oils. The chromatographic methods and parameters were optimized and solvent choice and separation restrictions are discussed. Pyrolysis oil samples were diluted in suitable organic solvent and were analyzed by GC×GC-TOFMS. An average of 300 compounds were detected and identified in all three samples using the ChromaToF (Leco) software. The deconvoluted spectra were compared with the NIST software library for correct matching. Group type classification was performed by use of the ChromaToF software. The quantification of 11 selected compounds was performed by means of a multiple-point external calibration curve. Afterwards, the pyrolysis oils were extracted with water, and the aqueous phase was analyzed both by GC-FID and, after proper change of solvent, by GC×GC-TOFMS. As previously, the selected compounds were quantified by both techniques, by means of multiple point external calibration curves. The parameters of the calibration curves were calculated by weighted linear regression analysis. The limit of detection, limit of quantitation and linearity range for each standard compound with each method are presented. The potency of GC×GC-TOFMS for an efficient mapping of the pyrolysis oil is undisputable, and the possibility of using it for quantification as well has been demonstrated. On the other hand, the GC-FID analysis provides reliable results that allow for a rapid screening of the pyrolysis oil. To the best of our knowledge, very few papers have been reported with quantification attempts on pyrolysis oil samples using GC×GC-TOFMS most of which make use of the

  17. Separation techniques: Chromatography

    Science.gov (United States)

    Coskun, Ozlem

    2016-01-01

    Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

  18. RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

    Science.gov (United States)

    Moss, Walter N

    2018-03-01

    With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. ENVIRONMENTAL ANALYSIS BY AB INITIO QUANTUM MECHANICAL COMPUTATION AND GAS CHROMATOGRAPHY/FOURIER TRANSFORM INFRARED SPECTROMETRY.

    Science.gov (United States)

    Computational chemistry, in conjunction with gas chromatography/mass spectrometry/Fourier transform infrared spectrometry (GC/MS/FT-IR), was used to tentatively identify seven tetrachlorobutadiene (TCBD) isomers detected in an environmental sample. Computation of the TCBD infrare...

  20. Development and validation of reverse phase high performance liquid chromatography for citral analysis from essential oils.

    Science.gov (United States)

    Gaonkar, Roopa; Yallappa, S; Dhananjaya, B L; Hegde, Gurumurthy

    2016-11-15

    Citral is a widely used monoterpene aldehyde in aromatherapy, food and pesticide industries. A new validated reverse phase high performance liquid chromatography (RP - HPLC) procedure for the detection and quantification of cis-trans isomers of citral was developed. The RP-HPLC analysis was carried out using Enable C - 18G column (250×4.6mm, 5μ), with acetonitrile and water (70: 30) mobile phase in isocratic mode at 1mL/min flow. A photodiode array (PDA) detector was set at 233nm for the detection of citral. The method showed linearity, selectivity and accuracy for citral in the range of 3-100μg/mL. In order to compare the new RP-HPLC method with the available methods, one of the commercially available essential oil from Cymbopogon flexuosus was analyzed using new RP-HPLC method and the same was analyzed using GC-MS for the comparison of the method for the detection of citral. The GC-MS analysis was done using mass selective detector (MSD) showed citral content to be of 72.76%; wherein the new method showed to contain that same at 74.98%. To prove the application of the new method, essential oils were extracted from lemongrass, lemon leaves and mosambi peels by steam distillation. The citral content present in the essential and also in the condensate was analyzed. The method was found to be suitable for the analysis of citral in essential oils and water based citral formulations with a very good resolution of its components geranial and neral. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Analysis of azaarenes in pan fried meat and its gravy by liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Błaszczyk, Urszula; Janoszka, Beata

    2008-07-01

    A method for analysis of six azaarenes (benzo[h]quinoline, benzo[a]acridine, benzo[c]acridine, dibenzo[a,c]acridine, dibenzo[a,j]acridine and dibenzo[a,h]acridine) in thermally treated high-protein food has been described. The clean-up procedure used based on alkaline hydrolysis, tandem solid phase extraction on columns filled with Extrelut - diatomaceous earth and cation exchanger (propyl sulfonic acid), enabled a selective isolation of carcinogenic compounds belonging to benzoacridines and dibenzoacridines from samples of cooked meat and its gravy. The isolated fractions of aza-PAHs were analysed by high-performance liquid chromatography with fluorescence detection. The detection limits for the azaarenes were between 0.0001ng and 0.005ng loaded on column. The recoveries for the four-ring and five-ring azaarenes were from 55% to 67%. Two types of dishes prepared from pork by pan-frying were investigated. Total contents of the benzoacridines and dibenzoacridines determined in cooked meat were 1.57 and 2.50ng/g in collar and chop samples, respectively; their gravies contained 0.34 and 0.59ng of these azaarenes per g of cooked meat. Copyright © 2007 Elsevier Ltd. All rights reserved.

  2. Isotope-dilution gas chromatography-mass spectrometry method for the analysis of hydroxyurea.

    Science.gov (United States)

    Garg, Uttam; Scott, David; Frazee, Clint; Kearns, Gregory; Neville, Kathleen

    2015-06-01

    Hydroxyurea is used in the treatment of various malignancies and sickle cell disease. There are limited studies on the pharmacokinetics of hydroxyurea, particularly in pediatric patients. An accurate, precise, and sensitive method is needed to support such studies and to monitor therapeutic adherence. We describe a novel gas chromatography-mass spectrometry (GC-MS) method for the determination of hydroxyurea concentration in plasma using stable labeled hydroxyurea C N2 as an internal standard. The method involved an organic extraction followed by the preparation of trimethylsilyl (TMS) derivatives of hydroxyurea for GC-MS selected ion-monitoring analysis. The following mass-to-charge (m/z) ratio ions for silated hydroxyurea and hydroxyurea C N2 were monitored: hydroxyurea-quantitative ion 277, qualifier ions 292 and 249; hydroxyurea C N2-quantitative ion 280, qualifier ion 295. This method was evaluated for reportable range, accuracy, within-run and between-run imprecisions, and limits of quantification. The reportable range for the method was 0.1-100 mcg/mL. All results were accurate within an allowable error of 15%. Within-run and between-run imprecisions were hydroxyurea described here is accurate, sensitive, precise, and robust. Its characteristics make the method suitable for supporting pharmacokinetic studies and/or clinical therapeutic monitoring.

  3. Determination of extractable organohalogens in apple by neutron activation analysis and gas chromatography

    International Nuclear Information System (INIS)

    Zhang, H.; Chai, Z.F.; Sun, H.B.; Zhang, J.L.; Sun, J.Q.

    2005-01-01

    In addition to compounds such as dichlorodiphenyl trichloroethanes (DDTs) and polychlorinated biphenyls (PCBs), which originate from by now well-known anthropogenic sources, there is increasing awareness of other halogenated compounds coming from a number of different processes, and probably also from natural source. To provide the reliable results and data for evaluation of hazard of extractable persistent organohalogen (EPOX) to food safety and to further characterize halogenated compounds in fruits, the concentrations of extractable organohalogen (EOX), EPOX, and identified organochlorine compounds including DDTs, hexachlorinated cyclohexanes (HCHs), chlordanes and PCBs in apple samples from Chinese homegrown and imports were determined by neutron activation analysis (NAA) and gas chromatography. The results indicated that the levels of EPOX in apples was in the order of EPOCl >> EPOBr > EPOI, which showed that EPOCl was the major fraction of the EPOX pollutions. Also, most chlorinated compounds in apple samples existed as inorganic species and water-soluble polar compounds, and about 1/10-4/5 of EOX was sulfuric acid-resistant EPOX contaminants. Meanwhile, about more than 94 % of EPOCl in detected specimens cannot be accounted for by DDTs, HCHs, chlordanes and PCBs. Furthermore, the T-test result indicated that the EPOCl levels in apple were significantly higher than those in peeled apple , which stated that people consuming peeled apple will get lesser intakes of EPOCl pollutants than they consume unpeeled apple.

  4. Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors.

    Science.gov (United States)

    Chen, Tao; Fan, Jun; Gao, Ruiqi; Wang, Tai; Yu, Ying; Zhang, Weiguang

    2016-10-07

    Chiral stationary phase-high performance liquid chromatography coupled with various detectors has been one of most commonly used methods for analysis and separation of chiral compounds over the past decades. Various detectors exhibit different characteristics in qualitative and quantitative studies under different chromatographic conditions. Herein, a comparative evaluation of HPLC coupled with ultraviolet, optical rotation, refractive index, and evaporative light scattering detectors has been conducted for qualitative and quantitative analyses of metalaxyl racemate. Effects of separation conditions on the peak area ratio between two enantiomers, including sample concentration, column temperature, mobile phase composition, as well as flow rate, have been investigated in detail. In addition, the limits of detection, the limits of quantitation, quantitative range and precision for these two enantiomers by using four detectors have been also studied. As indicated, the chromatographic separation conditions have been slight effects on ultraviolet and refractive index detections and the peak area ratio between two enantiomers remains almost unchanged, but the evaporative light scattering detection has been significantly affected by the above-mentioned chromatographic conditions and the corresponding peak area ratios varied greatly. Moreover, the limits of detection, the limits of quantitation, and the quantitative ranges of two enantiomers with UV detection were remarkably lower by 1-2 magnitudes than the others. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Analysis of oxidised heavy paraffininc products by high temperature comprehensive two-dimensional gas chromatography.

    Science.gov (United States)

    Potgieter, H; Bekker, R; Beigley, J; Rohwer, E

    2017-08-04

    Heavy petroleum fractions are produced during crude and synthetic crude oil refining processes and they need to be upgraded to useable products to increase their market value. Usually these fractions are upgraded to fuel products by hydrocracking, hydroisomerization and hydrogenation processes. These fractions are also upgraded to other high value commercial products like lubricant oils and waxes by distillation, hydrogenation, and oxidation and/or blending. Oxidation of hydrogenated heavy paraffinic fractions produces high value products that contain a variety of oxygenates and the characterization of these heavy oxygenates is very important for the control of oxidation processes. Traditionally titrimetric procedures are used to monitor oxygenate formation, however, these titrimetric procedures are tedious and lack selectivity toward specific oxygenate classes in complex matrices. Comprehensive two-dimensional gas chromatography (GC×GC) is a way of increasing peak capacity for the comprehensive analysis of complex samples. Other groups have used HT-GC×GC to extend the carbon number range attainable by GC×GC and have optimised HT-GC×GC parameters for the separation of aromatics, nitrogen-containing compounds as well as sulphur-containing compounds in heavy petroleum fractions. HT-GC×GC column combinations for the separation of oxygenates in oxidised heavy paraffinic fractions are optimised in this study. The advantages of the HT-GC×GC method in the monitoring of the oxidation reactions of heavy paraffinic fraction samples are illustrated. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Analysis of Fluconazole in Human Urine Sample by High Performance Liquid Chromatography Method

    International Nuclear Information System (INIS)

    Hermawan, D; Ali, N A Md; Ibrahim, W A Wan; Sanagi, M M

    2013-01-01

    A method for determination of fluconazole, antifungal drug in human urine by using reversed-phased high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detector was developed. Optimization HPLC conditions were carried out by changing the flow rate and composition of mobile phase. The optimum separation conditions at a flow rate 0.85 mL/min with a composition of mobile phase containing methanol:water (70:30, v/v) with UV detection at a wavelength 254 nm was able to analyze fluconazole within 3 min. The excellent linearity was obtained in the range of concentration 1 to 10 μg/mL with r 2 = 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.39 μg/mL and 1.28 μg/mL, respectively. Solid phase extraction (SPE) method using octadecylsilane (C18) as a sorbent was used to clean-up and pre-concentrated of the urine sample prior to HPLC analysis. The average recoveries of fluconazole in spiked urine sample was 72.4% with RSD of 3.21% (n=3).

  7. Multiclass analysis of antibiotic residues in honey by ultraperformance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Vidal, Jose Luis Martínez; Aguilera-Luiz, María Del Mar; Romero-González, Roberto; Frenich, Antonia Garrido

    2009-03-11

    A method has been developed and validated for the simultaneous analysis of different veterinary drug residues (macrolides, tetracyclines, quinolones, and sulfonamides) in honey. Honey samples were dissolved with Na(2)EDTA, and veterinary residues were extracted from the supernatant by solid-phase extraction (SPE), using OASIS HLB cartridges. The separation and determination was carried out by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), using an electrospay ionization source (ESI) in positive mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two ion transitions per compound to provide a high degree of sensitivity and specificity. The method was validated, and mean recoveries were evaluated at three concentration levels (10, 50, and 100 microg/kg), ranging from 70 to 120% except for doxycycline, erythromycin, and tylmicosin with recovery higher than 50% at the three levels assayed. Relative standard deviations (RSDs) of the recoveries were less than 20% within the intraday precision and less than 25% within the interday precision. The limits of quantification (LOQs) were always lower than 4 microg/kg. The developed procedure was applied to 16 honey samples, and erythromycin, sarafloxacin, and tylosin were found in a few samples.

  8. Comparison of derivatization/ionization techniques for liquid chromatography tandem mass spectrometry analysis of oxylipins.

    Science.gov (United States)

    Meckelmann, Sven W; Hellhake, Stefan; Steuck, Maryvonne; Krohn, Michael; Schebb, Nils Helge

    2017-05-01

    The performance of two derivatization and ionization techniques for the quantitative reversed phase liquid chromatography (LC)- mass spectrometry (MS) analysis of hydroxy fatty acids (OH-PUFA) in plasma was evaluated: One used AMPP (N-(4-aminomethylphenyl)pyridinium chloride) leading to a positive charged amid-derivate which can be detected by electrospray ionization (ESI)-MS. Second yielded penta fluorobenzyl bromide (PFB) ester derivates allowing detection in electron capture atmospheric pressure chemical ionization (ecAPCI)-MS. The sensitivity of detection of a comprehensive set of hydroxy fatty acids of n6- and n3- poly unsaturated fatty acids was investigated. On the SCIEX3200 MS the applied PFB derivatization led to poor limits of detection (LOD) of 10-100nM (0.1-1pmol/0.03-0.3ng on column). By contrast, AMPP derivatization led to a similar sensitivity compared to the standard ESI(-) of non derivatized analytes (LOD about 1nM (10fmol/3pg on column)). For several analytes, including 9-HETE, 11-HETE and 17-HDHA the AMPP derivatization improved sensitivity enabling their detection in human plasma. However, precision was reduced by AMPP derivatization and variation in IS recovery indicated a strong matrix influence on the MS-signal. In sum, with the instrumentation used, neither of these derivatization methods improves in our hands the LC-MS based quantification of oxylipins. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Liquid chromatography-tandem mass spectrometry for analysis of intestinal permeability of loperamide in physiological buffer.

    Directory of Open Access Journals (Sweden)

    Miriam S Rubelt

    Full Text Available Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS. To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC-MS/MS for the determination of loperamide in HEPES-buffered Ringer's solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d(3 were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer's solution as a matrix compound.

  10. Analysis of Dyes Extracted from Millimeter-Size Nylon Fibers by Micellar Electrokinetic Chromatography

    International Nuclear Information System (INIS)

    Lewis, L.A.

    2001-01-01

    The Learning Objective is to present to the forensic community a potential qualitative/quantitative method for trace-fiber color comparisons using micellar electrokinetic chromatography (MEKC). Developing a means of analyzing extracted dye constituents from millimeter-size nylon fiber samples was the objective of this research initiative. Aside from ascertaining fiber type, color evaluation and source comparison of trace-fiber evidence plays a critical role in forensic-fiber examinations. Literally thousands of dyes exist to date, including both natural and synthetic compounds. Typically a three-color-dye combination is employed to affect a given color on fiber material. The result of this practice leads to a significant number of potential dye combinations capable of producing a similar color and shade. Since a typical forensic fiber sample is 2 mm or less in length, an ideal forensic dye analysis would qualitatively and quantitatively identify the extracted dye constituents from a sample size of 1 mm or smaller. The goal of this research was to develop an analytical method for comparing individual dye constituents from trace-fiber evidence with dyes extracted from a suspected source, while preserving as much of the original evidence as possible

  11. Analysis of residual toluene in food packaging via headspace extraction method using gas chromatography

    International Nuclear Information System (INIS)

    Lim, Ying Chin; Mohd Marsin Sanagi

    2008-01-01

    Polymeric materials are used in many food contact applications as packaging material. The presence of residual toluene in this food packaging material can migrate into food and thus affect the quality of food. In this study, a manual headspace analysis was successfully designed and developed. The determination of residual toluene was carried out with standard addition method and multiple headspace extraction, MHE) method using gas chromatography-flame ionization detector, GC-FID). Identification of toluene was performed by comparison of its retention time with standard toluene and GC-MS. It was found that the suitable heating temperature was 180 degree Celsius with an optimum heating time of 10 minutes. The study also found that the concentration of residual toluene in multicolored sample was higher compared to mono colored sample whereas residual toluene in sample analyzed using standard addition method was higher compared to MHE method. However, comparison with the results obtained from De Paris laboratory, France found that MHE method gave higher accuracy for sample with low analyte concentration. On the other hand, lower accuracy was obtained for sample with high concentration of residual toluene due to systematic errors. Comparison between determination methods showed that MHE method is more precise compared to standard addition method. (author)

  12. Application of extraction paper chromatography in the analysis of Pu in aqueous samples containing 241Am

    International Nuclear Information System (INIS)

    Jagasia, Poonam; Dhami, P.S.; Tripathi, S.C.; Gandhi, P.M.

    2015-01-01

    Closing the fuel cycle is an inevitable option for sustained growth and is hallmark of Indian Atomic Energy Programme. Analysis of fissile component especially plutonium is very important for almost all the process streams at the back end of the fuel cycle. Among the various Isotopes of plutonium, 241 Pu is β emitter and decays to 241 Am (T 1/2 of ∼432 y) which is an α emitter and having γ energy of ∼60 keV. Its specific activity is about 56 times higher than that Pu. Therefore, Pu stream is always associated with 241 Am and its activity increases with time needing its separation prior to radiometric assay. Among the various separation techniques, solvent extraction method employing thenoytrifluoroacetone (TTA) is commonly employed for its selective separation under particular experimental conditions. In an alternate approach, 2-ethyl hexyl 2-ethyl hexyl phosphonic acid (KSM-17) based extraction paper chromatography (EPC) was attempted for separation of 241 Am from plutonium prior to radiometry. Pu 4+ forms strong complex with KSM-17 and is held at the point of application whereas 241 Am migrates towards the solvent front under the experimental conditions. During migration clear separation is observed in ∼15 minutes using 0.5 M HNO 3 as developing reagent. After separation is achieved, 1 cm 2 spot area containing Pu is desorbed in 1 mL xylene phase and used for radiometric counting

  13. Applicability of multisyringe chromatography coupled to cold-vapor atomic fluorescence spectrometry for mercury speciation analysis

    International Nuclear Information System (INIS)

    Guzmán-Mar, J.L.; Hinojosa-Reyes, L.; Serra, A.M.; Hernández-Ramírez, A.; Cerdà, V.

    2011-01-01

    Graphical abstract: An automatic system, based on the applicability of multisyringe chromatography (MSC) coupled to cold-vapor atomic fluorescence spectrometry (CV/AFS) detection is developed for mercury speciation. Highlights: ► The on-line coupling of MSC to CV/AFS was developed for mercury speciation analysis. ► The speciation of MeHg + , Hg 2+ and EtHg + was achieved on a RP C18 monolithic column. ► The hyphenated system provided higher sample throughput compared to HPLC–CV/AFS. ► The limits of detection for mercury species were comparable or better than those reported by HPLC–CV/AFS. ► The developed method also provided low instrumental and operational costs. - Abstract: In this paper, a novel automatic approach for the speciation of inorganic mercury (Hg 2+ ), methylmercury (MeHg + ) and ethylmercury (EtHg + ) using multisyringe chromatography (MSC) coupled to cold-vapor atomic fluorescence spectrometry (CV/AFS) was developed. For the first time, the separation of mercury species was accomplished on a RP C18 monolithic column using a multi-isocratic elution program. The elution protocol involved the use of 0.005% 2-mercapthoethanol in 240 mM ammonium acetate (pH 6)–acetonitrile (99:1, v/v), followed by 0.005% 2-mercapthoethanol in 240 mM ammonium acetate (pH 6)–acetonitrile (90:10, v/v). The eluted mercury species were then oxidized under post-column UV radiation and reduced using tin(II) chloride in an acidic medium. Subsequently, the generated mercury metal were separated from the reaction mixture and further atomized in the flame atomizer and detected by AFS. Under the optimized experimental conditions, the limits of detection (3σ) were found to be 0.03, 0.11 and 0.09 μg L −1 for MeHg + , Hg 2+ and EtHg + , respectively. The relative standard deviation (RSD, n = 6) of the peak height for 3, 6 and 3 μg L −1 of MeHg + , Hg 2+ and EtHg + (as Hg) ranged from 2.4 to 4.0%. Compared with the conventional HPLC–CV/AFS hyphenated systems

  14. Analysis of perfluorinated carboxylic acids in soils II: optimization of chromatography and extraction.

    Science.gov (United States)

    Washington, John W; Henderson, W Matthew; Ellington, J Jackson; Jenkins, Thomas M; Evans, John J

    2008-02-15

    With the objective of detecting and quantitating low concentrations of perfluorinated carboxylic acids (PFCAs), including perfluorooctanoic acid (PFOA), in soils, we compared the analytical suitability of liquid chromatography columns containing three different stationary phases, two different liquid chromatography-tandem mass spectrometry (LC/MS/MS) systems, and eight combinations of sample-extract pretreatments, extractions and cleanups on three test soils. For the columns and systems we tested, we achieved the greatest analytical sensitivity for PFCAs using a column with a C(18) stationary phase in a Waters LC/MS/MS. In this system we achieved an instrument detection limit for PFOA of 270 ag/microL, equating to about 14 fg of PFOA on-column. While an elementary acetonitrile/water extraction of soils recovers PFCAs effectively, natural soil organic matter also dissolved in the extracts commonly imparts significant noise that appears as broad, multi-nodal, asymmetric peaks that coelute with several PFCAs. The intensity and elution profile of this noise is highly variable among soils and it challenges detection of low concentrations of PFCAs by decreasing the signal-to-noise contrast. In an effort to decrease this background noise, we investigated several methods of pretreatment, extraction and cleanup, in a variety of combinations, that used alkaline and unbuffered water, acetonitrile, tetrabutylammonium hydrogen sulfate, methyl-tert-butyl ether, dispersed activated carbon and solid-phase extraction. For the combined objectives of complete recovery and minimization of background noise, we have chosen: (1) alkaline pretreatment; (2) extraction with acetonitrile/water; (3) evaporation to dryness; (4) reconstitution with tetrabutylammonium-hydrogen-sulfate ion-pairing solution; (5) ion-pair extraction to methyl-tert-butyl ether; (6) evaporation to dryness; (7) reconstitution with 60/40 acetonitrile/water (v/v); and (8) analysis by LC/MS/MS. Using this method, we

  15. Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients.

    Science.gov (United States)

    Zhytnik, Lidiia; Maasalu, Katre; Reimann, Ene; Prans, Ele; Kõks, Sulev; Märtson, Aare

    2017-08-15

    Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country. We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5'UTR and 3'UTR regions, to identify causative OI mutations. We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains. Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.

  16. The analysis of aqueous mixtures using liquid chromatography-electrospray mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Steven [Iowa State Univ., Ames, IA (United States)

    1999-02-12

    The focus of this dissertation is the use of chromatographic methods coupled with electrospray mass spectrometry (ES-MS) for the determination of both organic and inorganic compounds in aqueous solutions. The combination of liquid chromatography (LC) methods and ES-MS offers one of the foremost methods for determining compounds in complex aqueous solutions. In this work, LC-ES-MS methods are devised using ion exclusion chromatography, reversed phase chromatography, and ion exchange chromatography, as well as capillary electrophoresis (CE). For an aqueous sample, these LC-ES-MS and CE-ES-MS techniques require no sample preparation or analyte derivatization, which makes it possible to observe a wide variety of analytes as they exist in solution. The majority of this work focuses on the use of LC-ES-MS for the determination of unknown products and intermediates formed during electrochemical incineration (ECI), an experimental waste remediation process. This report contains a general introduction to the project and the general conclusions. Four chapters have been removed for separate processing. Titles are: Chapter 2: Determination of small carboxylic acids by ion exclusion chromatography with electrospray mass spectrometry; Chapter 3: Electrochemical incineration of benzoquinone in aqueous media using a quaternary metal oxide electrode in the absence of a soluble supporting electrolyte; Chapter 4: The determination of electrochemical incineration products of 4-chlorophenol by liquid chromatography-electrospray mass spectrometry; and Chapter 5: Determination of small carboxylic acids by capillary electrophoresis with electrospray mass spectrometry.

  17. Mutation analysis of the cathepsin C gene in Indian families with Papillon-Lefèvre syndrome

    Directory of Open Access Journals (Sweden)

    Srivastava Satish

    2003-07-01

    Full Text Available Abstract Background PLS is a rare autosomal recessive disorder characterized by early onset periodontopathia and palmar plantar keratosis. PLS is caused by mutations in the cathepsin C (CTSC gene. Dipeptidyl-peptidase I encoded by the CTSC gene removes dipeptides from the amino-terminus of protein substrates and mainly plays an immune and inflammatory role. Several mutations have been reported in this gene in patients from several ethnic groups. We report here mutation analysis of the CTSC gene in three Indian families with PLS. Methods Peripheral blood samples were obtained from individuals belonging to three Indian families with PLS for genomic DNA isolation. Exon-specific intronic primers were used to amplify DNA samples from individuals. PCR products were subsequently sequenced to detect mutations. PCR-SCCP and ASOH analyses were used to determine if mutations were present in normal control individuals. Results All patients from three families had a classic PLS phenotype, which included palmoplantar keratosis and early-onset severe periodontitis. Sequence analysis of the CTSC gene showed three novel nonsense mutations (viz., p.Q49X, p.Q69X and p.Y304X in homozygous state in affected individuals from these Indian families. Conclusions This study reported three novel nonsense mutations in three Indian families. These novel nonsense mutations are predicted to produce truncated dipeptidyl-peptidase I causing PLS phenotype in these families. A review of the literature along with three novel mutations reported here showed that the total number of mutations in the CTSC gene described to date is 41 with 17 mutations being located in exon 7.

  18. Comprehensive analysis of pharmaceutical products using simultaneous mixed-mode (ion-exchange/reversed-phase) and hydrophilic interaction liquid chromatography.

    Science.gov (United States)

    Kazarian, Artaches A; Nesterenko, Pavel N; Soisungnoen, Phimpha; Burakham, Rodjana; Srijaranai, Supalax; Paull, Brett

    2014-08-01

    Liquid chromatographic assays were developed using a mixed-mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve-mediated column switching and was based upon a single high-performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion-exchange, (ii) mixed-mode interactions under an applied dual gradient (reversed-phase/ion-exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C18 column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed-mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well-resolved unknown peaks. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Mutation analysis of β-thalassemia in East-Western Indian population: a recent molecular approach

    Directory of Open Access Journals (Sweden)

    Shah PS

    2017-05-01

    Full Text Available Parth S Shah,1 Nidhi D Shah,2 Hari Shankar P Ray,3 Nikunj B Khatri,3 Ketan K Vaghasia,3 Rutvik J Raval,4 Sandip C Shah,3 Mandava V Rao5 1Department of Medicine, Lahey Hospital and Medical Center, Boston, MA, 2Department of Pediatrics, Nassau University Medical Centre, New York, NY, USA; 3Supratech Micropath Laboratory and Research Institute, 4Department of Zoology, 5School of Sciences, Gujarat University, Ahmedabad, Gujarat, India Background: β-Thalassemia is the most prevalent genetic disorder in India. Its traits and coinheritance vary from mild to severe conditions, resulting in thalassemia minor, intermediate, and major, depending upon many factors.Purpose: The objective of this study was to identify the incidence of β-thalassemia traits, their coinheritance, and mutations, as well as to support the patients already diagnosed with β-thalassemia in East-Western Indian population for better management.Patients and methods: Seventy-five referral cases for β-thalassemia were analyzed for various β-thalassemia traits, heterozygosity, and homozygosity conditions. Blood phenotypic parameters using cell counter and capillary electrophoresis were investigated. Analyses of eight common mutations of thalassemia in India were carried out using polymerase chain reaction-amplification refractory mutation system, end point polymerase chain reaction, and DNA sequencing methods.Results: Of these (75 referral cases from East-Western Indian region, 68 were positive for β-thalassemia (90.67%. The majority of case types were of β-thalassemia minor (49, 65.33%, followed by HbE traits (6, 8.0% and β-thalassemia major, including heterozygous and homozygous (5, 6.66%; 4, 5.33% types and then HbE homozygous (2, 2.66%, as well as one each of the HbE/β-thalassemia and HbD/β-thalassemia (1, 1.34% combination. Mutation analysis also revealed that the highest frequency of mutation was c.92+5G>C (41, 60.29% followed by deletion 619bp (9, 13.23% and c.79G>A (8, 11

  20. Molecular analysis of the eighteen most frequent mutations in the BRCA1 gene in 63 Chilean breast cancer families

    Directory of Open Access Journals (Sweden)

    LILIAN JARA

    2004-01-01

    Full Text Available BRCA1 gene mutations account for nearly all families with multiple cases of both early onset breast and/or ovarian cancer and about 30% of hereditary breast cancer. Although to date more than 1,237 distinct mutations, polymorphisms, and variants have been described, several mutations have been found to be recurrent in this gene. We have analyzed 63 Chilean breast/ovarian cancer families for eighteen frequent BRCA1 mutations. The analysis of the five exons and two introns in which these mutations are located was made using mismatch PCR assay, ASO hybridization assay, restriction fragment analysis, allele specific PCR assay and direct sequentiation techniques. Two BRCA1 mutations (185delAG and C61G and one variant of unknown significance (E1250K were found in four of these families. Also, a new mutation (4185delCAAG and one previously described polymorphism (E1038G were found in two other families. The 185delAG was found in a 3.17 % of the families and the others were present only in one of the families of this cohort. Therefore these mutations are not prominent in the Chilean population. The variant of unknown significance and the polymorphism detected could represent a founder effect of Spanish origin

  1. PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

    Science.gov (United States)

    Wimmer, Katharina; Wernstedt, Annekatrin

    2014-01-01

    The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

  2. Optimization of heteroduplex analysis for the detection of BRCA mutations and SNPs

    Directory of Open Access Journals (Sweden)

    Lucian Negura

    2011-02-01

    Full Text Available BRCA1 and BRCA2 are tumour suppressor genes whose mutant phenotypes predispose to breast and ovarian cancer. Screening for mutations in these genes is now standard practice for hereditary breast and ovarian cancer (HBOC cases in Europe, and permits medical follow-up and genetic counselling adapted to the needs of individuals in such families. Currently, most laboratories performing diagnostic analysis of the BRCA genes use PCR of exons and intron-exon boundaries coupled to a pre-screening step to identify anomalous amplicons. The techniques employed for the detection of mutations and SNPs have evolved over time and vary in sensitivity, specificity and cost-effectiveness. As a variant for pre-screening techniques, we chose the recently developed Surveyor® heteroduplex cleavage method as a sensitive and specific technique to reveal anomalous amplicons of the BRCA genes, using only basic laboratory equipment and agarose gel electrophoresis. Here we present the detection of either mutations or SNPs within the BRCA1 exon 7, using heteroduplex analysis (HA by mismatch-specific endonuclease, confirmed by dideoxy sequencing.

  3. Analysis of esterase isozyme and SSR for mutagenic progenies induced by space mutation in mustard

    International Nuclear Information System (INIS)

    Shen Jinjuan; Liu Yihua; Zhang Zhaorong; Ran Guangkui; Zhao Shouzhong; Xiao Li

    2012-01-01

    Seeds of five mustard (Brassica juncea Coss) varieties were carried into outer space by 'Shijian No.8' satellite. After five years' consecutive planting and selection, ten relatively stable mutant lines were obtained, which had significant variation in agronomic and economic characters. The mutant lines and their original varieties without space mutation treatment as control were studied by esterase isozyme and SSR analyses. Electrophoresis analysis of esterase isozymes indicated that there were differences between mutant lines and their controls in enzyme types and enzyme activity. Different mustard varieties had different enzymographs, and so did the mutants induced by space mutation, which shows different sensitivity among different mustard varieties. The SSR analysis showed that large differences were found in the SSR loci between mutant lines and their original variety, the variation frequency was between 9.52% and 57.14% with an average frequency of 26.19% for all the mutant lines. Among the mutant SSR loci, about 56.36% showed changes in band number and 43.64% in molecular weight. These results indicated that the ten mutant lines had large genetic difference in phenotype, genomic sequence and gene expression, and the outer space mutation would be an effective method to develop new mustard germplasm and variety. (authors)

  4. Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs.

    Science.gov (United States)

    Christoforides, Alexis; Carpten, John D; Weiss, Glen J; Demeure, Michael J; Von Hoff, Daniel D; Craig, David W

    2013-05-04

    The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations--changes specific to a tumor and not within an individual's germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific. We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity. We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.

  5. Mutation analysis of SDHB and SDHC: novel germline mutations in sporadic head and neck paraganglioma and familial paraganglioma and/or pheochromocytoma

    Directory of Open Access Journals (Sweden)

    Wong Nora

    2006-01-01

    Full Text Available Abstract Background Germline mutations of the SDHD, SDHB and SDHC genes, encoding three of the four subunits of succinate dehydrogenase, are a major cause of hereditary paraganglioma and pheochromocytoma, and demonstrate that these genes are classic tumor suppressors. Succinate dehydrogenase is a heterotetrameric protein complex and a component of both the Krebs cycle and the mitochondrial respiratory chain (succinate:ubiquinone oxidoreductase or complex II. Methods Using conformation sensitive gel electrophoresis (CSGE and direct DNA sequencing to analyse genomic DNA from peripheral blood lymphocytes, here we describe the mutation analysis of the SDHB and SDHC genes in 37 patients with sporadic (i.e. no known family history head and neck paraganglioma and five pheochromocytoma and/or paraganglioma families. Results Two sporadic patients were found to have a SDHB splice site mutation in intron 4, c.423+1G>A, which produces a mis-spliced transcript with a 54 nucleotide deletion, resulting in an 18 amino acid in-frame deletion. A third patient was found to carry the c.214C>T (p.Arg72Cys missense mutation in exon 4 of SDHC, which is situated in a highly conserved protein motif that constitutes the quinone-binding site of the succinate: ubiquinone oxidoreductase (SQR complex in E. coli. Together with our previous results, we found 27 germline mutations of SDH genes in 95 cases (28% of sporadic head and neck paraganglioma. In addition all index patients of five families showing hereditary pheochromocytoma-paraganglioma were found to carry germline mutations of SDHB: four of which were novel, c.343C>T (p.Arg115X, c.141G>A (p.Trp47X, c.281G>A (p.Arg94Lys, and c.653G>C (p.Trp218Ser, and one reported previously, c.136C>T, p.Arg46X. Conclusion In conclusion, these data indicate that germline mutations of SDHB and SDHC play a minor role in sporadic head and neck paraganglioma and further underline the importance of germline SDHB mutations in cases of

  6. Analysis of morphology, DNA and isozyme of leaf mutation in Brassica napus L

    International Nuclear Information System (INIS)

    Luo Zhen; Hu Dongwei; Li Xiaobai

    2008-01-01

    This paper aims to study the rule of irradiating effects, provide the effective way of analyzing mutant, and discuss the production application of mutant. By irradiating the 040B of Brassica napus L with . 0Co γ- ray, an obvious leaf mutation (ML) with large leaf area was found. The ML which has been inherited stably after three generations was compared with wide-type (CK) on the morphologic, DNA and isozymic levels. Results showed that S 4 and S17 from RAPD were two molecular markers which can express good polymorphism and have close relationships with leaf mutation sites. And in the analysis of EST and POD between ML and CK, the polymorphisms also proved that many discrepancies exist between ML and CK on the protein level. In addition, the research results in question can be applied to the breeding and genetic research of Brassica napus L

  7. Perceptual characterization and analysis of aroma mixtures using gas chromatography recomposition-olfactometry.

    Directory of Open Access Journals (Sweden)

    Arielle J Johnson

    Full Text Available This paper describes the design of a new instrumental technique, Gas Chromatography Recomposition-Olfactometry (GC-R, that adapts the reconstitution technique used in flavor chemistry studies by extracting volatiles from a sample by headspace solid-phase microextraction (SPME, separating the extract on a capillary GC column, and recombining individual compounds selectively as they elute off of the column into a mixture for sensory analysis (Figure 1. Using the chromatogram of a mixture as a map, the GC-R instrument allows the operator to "cut apart" and recombine the components of the mixture at will, selecting compounds, peaks, or sections based on retention time to include or exclude in a reconstitution for sensory analysis. Selective recombination is accomplished with the installation of a Deans Switch directly in-line with the column, which directs compounds either to waste or to a cryotrap at the operator's discretion. This enables the creation of, for example, aroma reconstitutions incorporating all of the volatiles in a sample, including instrumentally undetectable compounds as well those present at concentrations below sensory thresholds, thus correcting for the "reconstitution discrepancy" sometimes noted in flavor chemistry studies. Using only flowering lavender (Lavandula angustifola 'Hidcote Blue' as a source for volatiles, we used the instrument to build mixtures of subsets of lavender volatiles in-instrument and characterized their aroma qualities with a sensory panel. We showed evidence of additive, masking, and synergistic effects in these mixtures and of "lavender' aroma character as an emergent property of specific mixtures. This was accomplished without the need for chemical standards, reductive aroma models, or calculation of Odor Activity Values, and is broadly applicable to any aroma or flavor.

  8. Perceptual Characterization and Analysis of Aroma Mixtures Using Gas Chromatography Recomposition-Olfactometry

    Science.gov (United States)

    Johnson, Arielle J.; Hirson, Gregory D.; Ebeler, Susan E.

    2012-01-01

    This paper describes the design of a new instrumental technique, Gas Chromatography Recomposition-Olfactometry (GC-R), that adapts the reconstitution technique used in flavor chemistry studies by extracting volatiles from a sample by headspace solid-phase microextraction (SPME), separating the extract on a capillary GC column, and recombining individual compounds selectively as they elute off of the column into a mixture for sensory analysis (Figure 1). Using the chromatogram of a mixture as a map, the GC-R instrument allows the operator to “cut apart" and recombine the components of the mixture at will, selecting compounds, peaks, or sections based on retention time to include or exclude in a reconstitution for sensory analysis. Selective recombination is accomplished with the installation of a Deans Switch directly in-line with the column, which directs compounds either to waste or to a cryotrap at the operator's discretion. This enables the creation of, for example, aroma reconstitutions incorporating all of the volatiles in a sample, including instrumentally undetectable compounds as well those present at concentrations below sensory thresholds, thus correcting for the “reconstitution discrepancy" sometimes noted in flavor chemistry studies. Using only flowering lavender (Lavandula angustifola ‘Hidcote Blue’) as a source for volatiles, we used the instrument to build mixtures of subsets of lavender volatiles in-instrument and characterized their aroma qualities with a sensory panel. We showed evidence of additive, masking, and synergistic effects in these mixtures and of “lavender' aroma character as an emergent property of specific mixtures. This was accomplished without the need for chemical standards, reductive aroma models, or calculation of Odor Activity Values, and is broadly applicable to any aroma or flavor. PMID:22912722

  9. High Performance Thin Layer Chromatography method for analysis of 3,4-methylenedioxymethamphetamine in seized tablets

    Directory of Open Access Journals (Sweden)

    Boris E. Duffau

    2015-12-01

    Full Text Available Context: Consumption of synthetic drugs had increased in recent years, used as a recreational drug by young people who presume that consumption of this drug is harmless for health; however clinical studies have shown that this stimulant and its metabolites are toxic. Due to these reasons, chemical analysis of this illicit drug is crucial from the points of view of occupational medicine, toxicology, and law enforcement with the aim of pursuit the traffic of illegal drug. Aims: Implement and fully validate a rapid and simple method for detection and quantitation of MDMA by High-Performance Thin Layer Chromatography in seized samples. Methods: With the implemented method was analyzed 12 positive samples seized by Chilean police, to found the concentration of MDMA in ecstasy tablets. Results: The method was fully validated, the linearity of the method was evaluated by the calibration curve between 51.0 – 510.0 µg/band (R2 0.9977; limit of detection was 12.1 µg per band, and limit of quantitation was 36.8 µg per band. The precision of the method (RSD was lower than 5.0%. Accuracy was evaluated by determination of the percentage of MDMA recovered by the assay (99.13%, and relative Uncertainty was 6.66%. With this method, it was analyzed real seized samples of MDMA, results showed that all samples contained MDMA and concentration was between 18.15 – 59.84 % w/w. Conclusions: The method is selective, sensitive, and specific, with possible application in forensic analysis. To the best of our knowledge, this is the first report about concentration of MDMA in ecstasy pills in Chile.

  10. Chemically modified glasses for analysis of hydrogen isotopes by gas-chromatography

    International Nuclear Information System (INIS)

    Stanciu, Vasile; Stefanescu, Doina

    1999-01-01

    Hydrogen isotope separation process by such methods as cryogenic distillation or thermal diffusion method is one of the key technologies of the tritium separation from heavy water of CANDU reactors and in the tritium fuel cycle for a thermonuclear fusion reactor. In each process, the analytical techniques for measuring contents of hydrogen isotope mixture are necessary. An extensive experimental research has been carried out in order to produce the most suitable absorbent and define the best operating conditions for selective separation and analysis of hydrogen isotope by gas-chromatography. This paper describes the preparation of adsorbent materials utilised as stationary phase in the gas-chromatographic column for hydrogen isotope separation and treatment (activation) of stationary phase. Modified thermo-resisting glass with Fe(NH 4 ) 2 (SO 4 ) 2 6H 2 O and Cr 2 O 3 , respectively, have been experimentally investigated at 77 K for H 2 , HD and D 2 separation and the results of chromatographic runs are also reported and discussed. The gas-chromatographic apparatus used is composed of a Hewlett-Packard 7620A gas-chromatograph equipped with a gas carrier flow rate controller and a thermal conductivity detector (TCD). The apparatus comprises also a Dewar vessel containing the separation column. The hydrogen isotopes H 2 , HD, D 2 and their mixture have been obtained in our laboratories. The best operating conditions of the adsorbent column Fe (III)/glass and Cr 2 O 3 /glass, i.e. granulometry, column length, pressure-drop along the column, carrier gas flow rate, sample volume have been studied by means of the analysis of the retention times, separation factors and HETP. (authors)

  11. Metal/glass composites for analysis of hydrogen isotopes by gas-chromatography

    International Nuclear Information System (INIS)

    Nicolae, Constantin Adrian; Sisu, Claudia; Stefanescu, Doina; Stanciu, Vasile

    1999-01-01

    The separation process of hydrogen isotopes by cryogenic distillation or thermal diffusion is a key technology for tritium separation from heavy water in CANDU reactor and for tritium fuel cycle in thermonuclear fusion reactor. In each process, analytical techniques for analyzing the hydrogen isotope mixture are required. An extensive experimental research has been carried out in order to produce the most suitable adsorbents and to establish the best operating conditions for selective separation and analysis of hydrogen isotopes by gas-chromatography. This paper describes the preparation of adsorbent materials used as stationary phases in the gas-chromatographic column for hydrogen isotope separation and the treatment (activation) of stationary phases. Modified thermoresisting glass with Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O and Cr 2 O 3 respectively have been experimentally investigated at 77 K for H 2 , HD and D 2 separation and the results of chromatographic runs are reported and discussed. The gas-chromatographic apparatus used in this study is composed of a Hewlett-Packard 7620A gas-chromatograph equipped with a gas carrier flow rate controller and a thermal conductivity detector. The apparatus comprises also a Dewar vessel containing the separation column. The hydrogen isotopes, H 2 , HD, D 2 , and their mixture have been obtained in our laboratories. The best operating conditions and parameters of the Fe 3+ /glass adsorbent column , i.e. granulometry, column length, pressure-drop along the column, carrier gas flow rate and sample volume have been studied by means of the analysis of the retention times, separation factors and HETP. (authors)

  12. Analysis of antithyroid drugs in surface water by using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Pérez-Fernández, Virginia; Marchese, Stefano; Gentili, Alessandra; García, María Ángeles; Curini, Roberta; Caretti, Fulvia; Perret, Daniela

    2014-11-07

    This paper describes development and validation of a new method for the simultaneous determination of six antithyroid drugs (ATDs) in surface waters by using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Target compounds include two ATD classes: thiouracil derivatives (thiouracil (TU), methyl-thiouracil (MTU), propyl-thiouracil (PTU), phenyl-thiouracil (PhTU)) and imidazole derivatives (tapazole (TAP), and mercaptobenzimidazole (MBI)). Sensitivity and selectivity of the LC-multiple reaction monitoring (MRM) analysis allowed applying a simple pre-concentration procedure and "shooting" the concentrated sample into the LC-MS/MS system without any other treatment. Recoveries were higher than 75% for all analytes. Intra-day precision and inter-day precision, calculated as relative standard deviation (RSD), were below 19 and 22%, respectively. Limits of detection (LODs) ranged from 0.05 to 0.25 μg/L; limits of quantitation (LOQs) varied between 0.15 and 0.75 μg/L. The validated method was successfully applied to the analysis of ATD residues in surface water samples collected from the Tiber River basin and three lakes of Lazio (central Italy). The analytes were quantified based on matrix-matched calibration curves with mercaptobenzimidazole-d4 (MBI-d4) as the internal standard (IS). The most widespread compound was TAP, one of the most common ATDs used in human medicine, but also TU and MBI were often detected in the analysed samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Radiochemical neutron activation analysis of high pure palladium and platinum by ion exchange chromatography

    International Nuclear Information System (INIS)

    Sadikov, I.I.; Zinov'ev, V.G.; Sadikova, Z.O.; Salimov, M.I.

    2006-01-01

    Full text: The palladium and platinum are widely used for jewel manufacture because of their beautiful white color. However the most part of these metals are widely adopted in the world as catalysts. Many works on analytical chemistry of platinum group elements published during last years are devoted to determination of platinum and palladium in other materials. There are no articles on analysis technique of the palladium and platinum purity published during last 20 years. Available publications are very old and are published till 70th of the last century, and implement chemical and spectral methods. At the same time, the palladium and platinum are very suitable for NAA. Therefore the purpose of our research was development of high-sensitivity and multielement techniques of radiochemical neutron activation analysis of a high pure palladium and platinum. Research of nuclear characteristics of palladium and platinum has shown that radioactive nuclides with different yields are formed under the reactor neutrons. 109 , 111 , 111m Pd, 109m , 111 Ag, 191 , 197 , 199 Pt, 199 Au are the most important among them. 109Pd separation factor is equal to 1*10 5 at palladium analysis, whereas 197 Pt and 199 Au separation factor is equal to 1*10 4 at the platinum analysis every other day after irradiation. Palladium and platinum can be separated by precipitation, extraction and ion exchange methods. For separation of radioactive nuclide of the matrix elements from the impurity elements we used ion exchange chromatography system Dowex-1x8 - 1 M HNO 3 for palladium and Dowex-1x8 - 0.1 M HNO 3 for platinum. At the HNO 3 acid concentrations variation from 0,1 M to 1 M more then 25 elements have distribution factors less than 1 and 10 elements have distribution factors 5 while matrix elements have distribution factors higher than 100. It allows an effective separation of these elements from palladium and platinum. Optimum sizes of the chromatographic column and the column effluent

  14. High-Throughput Analysis of Sucrose Fatty Acid Esters by Supercritical Fluid Chromatography/Tandem Mass Spectrometry

    Science.gov (United States)

    Hori, Katsuhito; Tsumura, Kazunobu; Fukusaki, Eiichiro; Bamba, Takeshi

    2014-01-01

    Supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry was applied to the profiling of sucrose fatty acid esters (SEs). The SFC conditions (column and modifier gradient) were optimized for the effective separation of SEs. In the column test, a silica gel reversed-phase column was selected. Then, the method was used for the detailed characterization of commercial SEs and the successful analysis of SEs containing different fatty acids. The present method allowed for fast and high-resolution separation of monoesters to tetra-esters within a shorter time (15 min) as compared to the conventional high-performance liquid chromatography. The applicability of our method for the analysis of SEs was thus demonstrated. PMID:26819875

  15. Sensitive method for the analysis of carbohydrates by gas chromatography of 3H-labeled alditol acetates

    International Nuclear Information System (INIS)

    Prehm, P.; Scheid, A.

    1978-01-01

    A highly sensitive method has been developed for the analysis of carbohydrates from glycoproteins or lipopolysaccharides. The method is based on labeling the carbohydrates with [ 3 H] sodium borohydride, acetylating the resulting alditols and separating them by gas chromatography. The gas effluent is fractionated by trapping on silicone-coated glass beads and the amount of radioactivity is determined. This permits the quantitation of as little as 0.2 nmoles monosaccharide with an accuracy of 10 to 15%. (Auth)

  16. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    Science.gov (United States)

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  17. Flow Injection Analysis and Liquid Chromatography for Multifunctional Chemical Analysis (MCA) Systems

    Science.gov (United States)

    Mayo, Ana V.; Loegel, Thomas N.; Bretz, Stacey Lowery; Danielson, Neil D.

    2013-01-01

    The large class sizes of first-year chemistry labs makes it challenging to provide students with hands-on access to instrumentation because the number of students typically far exceeds the number of research-grade instruments available to collect data. Multifunctional chemical analysis (MCA) systems provide a viable alternative for large-scale…

  18. Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

    Science.gov (United States)

    Gelhaus, Stacy L.; Mesaros, A. Clementina; Blair, Ian A.

    2011-01-01

    The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products. While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic

  19. Multiplex gas chromatography: an alternative concept for gas chromatographic analysis of planetary atmospheres

    Science.gov (United States)

    Valentin, J. R.

    1989-01-01

    -Cassini entry probe, which is being jointly planned by NASA and the European Space Agency (ESA), might be launched as early as 1994. As in the Pioneer mission, limited time--perhaps only 3-4 h--will be available for the completion of all analyses while the probe descends through the atmosphere. A conventional GC or GC-MS system would be able to analyze no more than two aerosol and two gas samples during the probe's descent. Conventional GC also is limited by the sensitivity of the detector and by the sample volume. For the Titan mission, the sensitivity problems will be worse because the atmospheric pressure at the time of instrument deployment is expected to be of such limitations, alternative GC analysis techniques have been investigated for future NASA missions. Multiplex gas chromatography has been investigated as a possible candidate for chemical analysis within a spacecraft or other restricted environment, and chemical modulators have been developed and used when needed with this technique to reduce the size and weight of the instrumentation. Also, several new multiplex techniques have been developed for use in specific applications.

  20. Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

    Science.gov (United States)

    Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

    2017-07-01

    Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

  1. Computational Analysis of Epidermal Growth Factor Receptor Mutations Predicts Differential Drug Sensitivity Profiles toward Kinase Inhibitors.

    Science.gov (United States)

    Akula, Sravani; Kamasani, Swapna; Sivan, Sree Kanth; Manga, Vijjulatha; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

    2018-05-01

    A significant proportion of patients with lung cancer carry mutations in the EGFR kinase domain. The presence of a deletion mutation in exon 19 or L858R point mutation in the EGFR kinase domain has been shown to cause enhanced efficacy of inhibitor treatment in patients with NSCLC. Several less frequent (uncommon) mutations in the EGFR kinase domain with potential implications in treatment response have also been reported. The role of a limited number of uncommon mutations in drug sensitivity was experimentally verified. However, a huge number of these mutations remain uncharacterized for inhibitor sensitivity or resistance. A large-scale computational analysis of clinically reported 298 point mutants of EGFR kinase domain has been performed, and drug sensitivity profiles for each mutant toward seven kinase inhibitors has been determined by molecular docking. In addition, the relative inhibitor binding affinity toward each drug as compared with that of adenosine triphosphate was calculated for each mutant. The inhibitor sensitivity profiles predicted in this study for a set of previously characterized mutants correlated well with the published clinical, experimental, and computational data. Both the single and compound mutations displayed differential inhibitor sensitivity toward first- and next-generation kinase inhibitors. The present study provides predicted drug sensitivity profiles for a large panel of uncommon EGFR mutations toward multiple inhibitors, which may help clinicians in deciding mutant-specific treatment strategies. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  2. Analysis of I-125 IMP and its metabolites using a high performance liquid chromatography

    International Nuclear Information System (INIS)

    Satoh, Motohiro; Ishikawa, Nobuyoshi; Takeda, Tohoru; Jin, Wu; Kuramoto, Kenmei; Itai, Yuji; Yoshizawa, Takashi; Nakajima, Kotaro.

    1991-01-01

    The biodistribution of N-isopropyl-p-[I-123]iodoamphetamine (IMP) and its metabolites was examined in rabbits and Mongolian gerbils. Arterial sampling was performed at one, 5, 15, and 30 min, and one, 3, and 6 hr after bolus iv injection of IMP for the hemodynamic investigation. Similarly, the cerebral hemisphere, lung, liver, and blood samples were collected at 15 min and 3 hr for analyzing IMP metabolites. Activity count in blood was gradually increased from 15 min to 3 hr after iv injection, and thereafter decreased. Relative fraction of IMP in plasma was gradually increased to a plateau value of 80% at one hr. Octanol extraction ratio was decreased to 24.3% at 3 hr, although it was 100% immediately after iv injection. Early (15 min) and delayed (3 hr) analysis using high performance liquid chromatography (HPLC) revealed p-iodoamphetamine (PIA) and p-iodobenzoic acid (PIB) as major metabolites of IMP. Although IMP accounted for the majority on both early and delayed HPLC, the quantity of PIA in the normal hemisphere and lung was significantly increased on delayed HPLC, compared to early HPLC. For the liver, the quantities of both PIA and PIB were larger than IMP on both early and delayed HPLC. The proportion of metabolites also became greater in whole blood than IMP on delayed HPLC. Early HPLC reveald no significant difference in composition of IMP, PIA, and PIB between the normal and ischemic hemispheres. Delayed HPLC revealed a greater proportion of PIA in the ischemic than the normal hemisphere, but this was not statistically significant. (N.K.)

  3. Analysis of essential oil of eaglewood tree (Aquilaria agallocha Roxb. by gas chromatography mass spectrometry

    Directory of Open Access Journals (Sweden)

    Mohammad Nazrul Islam Bhuiyan

    2009-06-01

    Full Text Available The study was carried out to find out the differences in composition of oils obtained from healthy, naturally infected and artificially screws wounds eaglewood (Aquilaria agallocha Roxb. using gas chromatography mass spectrometry (GC-MS analysis. Natural healthy plants agar contained octacosane (19.83%, naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl-, [1R-(1.alpha.,7.beta.,8a.alpha.]- (12.67%, 5-isobutyramido-2-methyl pyrimidine (13.52%, caryophyllene oxide (11.25% and (.+-.-cadinene (5.46%. Natural infected plants agar (super agar contained cycloheptane, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl-1-vinyl- (46.17%, caryophyllene oxide (33.00% and 7-Isopropenyl-4a-methyl-1-methylenedecahydronaphthalene (20.83%. Artificially screw injected plants agar contained diisooctyl phthalate (71.97%, 1H-Cycloprop[e]azulen-4-ol, decahydro-1,1,4,7-tetramethyl-, [1ar-(1a.alpha.,4.beta.,4a.beta., 7.alpha., 7a.beta., 7b.alpha.]- (9.16%, hexadecanoic acid (7.05%, naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl-, [1R-(1.alpha.,7.beta.,8a.alpha.]- (6.45% and aristolene (5.36%. This study showed a marked difference in the oil compositions among the treatments with regards to their quality.

  4. Liquid chromatography/mass spectrometry analysis of exhaled leukotriene B4 in asthmatic children

    Directory of Open Access Journals (Sweden)

    Barnes Peter J

    2005-10-01

    Full Text Available Abstract Background The role of leukotriene (LT B4, a potent inflammatory mediator, in atopic asthmatic and atopic nonasthmatic children is largely unknown. The lack of a gold standard technique for measuring LTB4 in exhaled breath condensate (EBC has hampered its quantitative assessment in this biological fluid. We sought to measure LTB4 in EBC in atopic asthmatic children and atopic nonasthmatic children. Exhaled nitric oxide (NO was measured as an independent marker of airway inflammation. Methods Fifteen healthy children, 20 atopic nonasthmatic children, 25 steroid-naïve atopic asthmatic children, and 22 atopic asthmatic children receiving inhaled corticosteroids were studied. The study design was of cross-sectional type. Exhaled LTB4 concentrations were measured using liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS with a triple quadrupole mass spectrometer. Exhaled NO was measured by chemiluminescence with a single breath on-line method. LTB4 values were expressed as the total amount (in pg of eicosanoid expired in the 15-minute breath test. Kruskal-Wallis test was used to compare groups. Results Compared with healthy children [87.5 (82.5–102.5 pg, median and interquartile range], exhaled LTB4 was increased in steroid-naïve atopic asthmatic [255.1 (175.0–314.7 pg, p 4 than steroid-naïve asthmatics [125.0 (25.0–245.0 pg vs 255.1 (175.0–314.7 pg, p Conclusion In contrast to exhaled NO concentrations, exhaled LTB4 values are selectively elevated in steroid-naïve atopic asthmatic children, but not in atopic nonasthmatic children. Although placebo control studies are warranted, inhaled corticosteroids seem to reduce exhaled LTB4 in asthmatic children. LC/MS/MS analysis of exhaled LTB4 might provide a non-invasive, sensitive, and quantitative method for airway inflammation assessment in asthmatic children.

  5. Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover

    International Nuclear Information System (INIS)

    Ju, H.-J.; Ye, C.-M.; Verchot-Lubicz, Jeanmarie

    2008-01-01

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion to accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocate to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway

  6. Mutational analysis of the PTPN11 gene in Egyptian patients with Noonan syndrome.

    Science.gov (United States)

    Essawi, Mona L; Ismail, Manal F; Afifi, Hanan H; Kobesiy, Maha M; El Kotoury, Ahmed; Barakat, Maged M

    2013-11-01

    Noonan syndrome (NS) is inherited as an autosomal dominant disorder with dysmorphic facies, short stature, and cardiac defects, which can be caused by missense mutations in the protein tyrosine phosphatase nonreceptor type 11 (PTPN11) gene, which encodes src homology region 2 domain containing tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factors and cytokines. The current study aimed to study the molecular characterization of the PTPN11 gene among Egyptian patients with Noonan syndrome. Eleven exons of the PTPN11 gene were amplified and screened by single stranded conformational polymorphism (SSCP). DNA samples showing band shift in SSCP were subjected to sequencing. Mutational analysis of the PTPN11 gene revealed T→C transition at position 854 in exon 8, predicting Phe285Ser substitution within PTP domain of SHP-2 protein, in one NS patient and -21C→T polymorphism in intron 7 in four other cases. Knowing that NS is phenotypically heterogeneous, molecular characterization of the PTPN11 gene should serve to establish NS diagnosis in patients with atypical features, although lack of a mutation does not exclude the possibility of NS. Copyright © 2012. Published by Elsevier B.V.

  7. Capillary electrophoresis fragment analysis and clone sequencing in detection of dynamic mutations of spinocerebellar ataxia

    Directory of Open Access Journals (Sweden)

    Yuan-yuan CHEN

    2018-04-01

    Full Text Available Objective To estimate the accuracy and stability of capillary electrophoresis fragment analysis and clone sequencing in detecting dynamic mutations of spinocerebellar ataxia (SCA. Methods Capillary electrophoresis fragment analysis and clone sequencing were used in detecting trinucleotide repeated sequence of 14 SCA patients (3 cases of SCA2, 2 cases of SCA7, 7 cases of SCA8 and 2 cases of SCA17. Results Capillary electrophoresis fragment analysis of 3 SCA2 cases showed the expanded cytosine-adenine-guanine (CAG repeats were 31, 30 and 32, and the copy numbers of 3 clone sequencing for 3 colonies in each case were 37/40/40, 37/38/39 and 38/39/40 respectively. Capillary electrophoresis fragment analysis of 2 SCA7 cases showed the expanded CAG repeats were 57 and 34, and the copy numbers of repeats were 69, 74, 75 in 3 colonies of one case, and was 45 in the other case. For the 7 SCA8 cases with the expanded cytosine-thymine-adenine (CTA/cytosine-thymine-guanine (CTG repeats of 99, 111, 104, 92, 89, 104 and 75, the results of clone sequencing were 97, 116, 104, 90, 90, 102 and 76 respectively. For 2 SCA17 cases with the short/expanded CAG repeats of 37/50 and 36/45, the results of clone sequencing were 51/50/52 and 45/44 for 3 and 2 colonies. Conclusions Although the higher mobility of polymerase chain reaction (PCR products containing dynamic mutation in the capillary electrophoresis fragment analysis might cause the deviation for analysis of copy numbers, the deviation was predictable and the results were repeatable. The clone sequencing results showed obvious instability, especially for SCA2 and SCA7 genes, which might owing to their simple CAG repeats. Consequently, clone sequencing is not suited for detection of dynamic mutation, not to mention the quantitative criteria of dynamic mutation sequencing. DOI: 10.3969/j.issn.1672-6731.2018.03.008

  8. Analysis of new psychoactive substances in human urine by ultra-high performance supercritical fluid and liquid chromatography: Validation and comparison.

    Science.gov (United States)

    Borovcová, Lucie; Pauk, Volodymyr; Lemr, Karel

    2018-05-01

    New psychoactive substances represent serious social and health problem as tens of new compounds are detected in Europe annually. They often show structural proximity or even isomerism, which complicates their analysis. Two methods based on ultra high performance supercritical fluid chromatography and ultra high performance liquid chromatography with mass spectrometric detection were validated and compared. A simple dilute-filter-and-shoot protocol utilizing propan-2-ol or methanol for supercritical fluid or liquid chromatography, respectively, was proposed to detect and quantify 15 cathinones and phenethylamines in human urine. Both methods offered fast separation (chromatography. Limits of detection in urine ranged from 0.01 to 2.3 ng/mL, except for cathinone (5 ng/mL) in supercritical fluid chromatography. Nevertheless, this technique distinguished all analytes including four pairs of isomers, while liquid chromatography was unable to resolve fluoromethcathinone regioisomers. Concerning matrix effects and recoveries, supercritical fluid chromatography produced more uniform results for different compounds and at different concentration levels. This work demonstrates the performance and reliability of supercritical fluid chromatography and corroborates its applicability as an alternative tool for analysis of new psychoactive substances in biological matrixes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The Analysis Mutation Of The CARD 15 Gene Variants In Chronic Periodontis

    OpenAIRE

    Bahruddin Thalib, Dr.drg. M.Kes,Sp.Pros.

    2014-01-01

    As Conclusion, CARD 15 gene mutation with chronic periodontitis was found to have heterozygote mutation and homozygote mutation variants, and also found genetics variation that changed the composition of C??? T nucleotide at codon 802 in exon 4 amino acid changed from alanine to valine. Purpose of This study was to determine the variant of card 15 gene mutation with periodontitis chronic.

  10. Genetic counseling in Usher syndrome: linkage and mutational analysis of 10 Colombian families.

    Science.gov (United States)

    Tamayo, M L; Lopez, G; Gelvez, N; Medina, D; Kimberling, W J; Rodríguez, V; Tamayo, G E; Bernal, J E

    2008-01-01

    Usher Syndrome (US), an autosomal recessive disease, is characterized by retinitis pigmentosa (RP), vestibular dysfunction, and congenital sensorineural deafness. There are three recognized clinical types of the disorder. In order to improve genetic counseling for affected families, we conducted linkage analysis and DNA sequencing in 10 Colombian families with confirmed diagnosis of US (4 type I and 6 type II). Seventy-five percent of the US1 families showed linkage to locus USH1B, while the remaining 25% showed linkage to loci USH1B and USH1C. Among families showing linkage to USH1B we found two different mutations in the MYO7A gene: IVS42-26insTTGAG in exon 43 (heterozygous state) and R634X (CGA-TGA) in exon 16 (homozygous state). All six US2 families showed linkage to locus USH2A. Of them, 4 had c.2299delG mutation (1 homozygote state and 3 heterozygous); in the remaining 2 we did not identify any pathologic DNA variant. USH2A individuals with a 2299delG mutation presented a typical and homogeneous retinal phenotype with bilateral severe hearing loss, except for one individual with a heterozygous 2299delG mutation, whose hearing loss was asymmetric, but more profound than in the other cases. The study of these families adds to the genotype-phenotype characterization of the different types and subtypes of US and facilitates genetic counseling in these families. We would like to emphasize the need to perform DNA studies as a prerequisite for genetic counseling in affected families.

  11. Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

    Science.gov (United States)

    Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

    2017-06-02

    Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

  12. Applicability of multisyringe chromatography coupled to cold-vapor atomic fluorescence spectrometry for mercury speciation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Guzman-Mar, J.L.; Hinojosa-Reyes, L. [Department of Chemistry Sciences, Universidad Autonoma de Nuevo Leon, Cd. Universitaria, Pedro de Alba s/n, C.P. 66451 San Nicolas de los Garza, Nuevo Leon (Mexico); Serra, A.M. [Department of Chemistry, University of the Balearic Islands, E-07122 Palma de Mallorca (Spain); Hernandez-Ramirez, A. [Department of Chemistry Sciences, Universidad Autonoma de Nuevo Leon, Cd. Universitaria, Pedro de Alba s/n, C.P. 66451 San Nicolas de los Garza, Nuevo Leon (Mexico); Cerda, V., E-mail: victor.cerda@uib.es [Department of Chemistry, University of the Balearic Islands, E-07122 Palma de Mallorca (Spain)

    2011-12-05

    Graphical abstract: An automatic system, based on the applicability of multisyringe chromatography (MSC) coupled to cold-vapor atomic fluorescence spectrometry (CV/AFS) detection is developed for mercury speciation. Highlights: Black-Right-Pointing-Pointer The on-line coupling of MSC to CV/AFS was developed for mercury speciation analysis. Black-Right-Pointing-Pointer The speciation of MeHg{sup +}, Hg{sup 2+} and EtHg{sup +} was achieved on a RP C18 monolithic column. Black-Right-Pointing-Pointer The hyphenated system provided higher sample throughput compared to HPLC-CV/AFS. Black-Right-Pointing-Pointer The limits of detection for mercury species were comparable or better than those reported by HPLC-CV/AFS. Black-Right-Pointing-Pointer The developed method also provided low instrumental and operational costs. - Abstract: In this paper, a novel automatic approach for the speciation of inorganic mercury (Hg{sup 2+}), methylmercury (MeHg{sup +}) and ethylmercury (EtHg{sup +}) using multisyringe chromatography (MSC) coupled to cold-vapor atomic fluorescence spectrometry (CV/AFS) was developed. For the first time, the separation of mercury species was accomplished on a RP C18 monolithic column using a multi-isocratic elution program. The elution protocol involved the use of 0.005% 2-mercapthoethanol in 240 mM ammonium acetate (pH 6)-acetonitrile (99:1, v/v), followed by 0.005% 2-mercapthoethanol in 240 mM ammonium acetate (pH 6)-acetonitrile (90:10, v/v). The eluted mercury species were then oxidized under post-column UV radiation and reduced using tin(II) chloride in an acidic medium. Subsequently, the generated mercury metal were separated from the reaction mixture and further atomized in the flame atomizer and detected by AFS. Under the optimized experimental conditions, the limits of detection (3{sigma}) were found to be 0.03, 0.11 and 0.09 {mu}g L{sup -1} for MeHg{sup +}, Hg{sup 2+} and EtHg{sup +}, respectively. The relative standard deviation (RSD, n = 6) of the

  13. Effect-directed analysis via hyphenated high-performance thin-layer chromatography for bioanalytical profiling of sunflower leaves.

    Science.gov (United States)

    Móricz, Ágnes M; Ott, Péter G; Yüce, Imanuel; Darcsi, András; Béni, Szabolcs; Morlock, Gertrud E

    2018-01-19

    High-performance thin-layer chromatography (HPTLC) coupled with effect-directed analysis was used for non-targeted screening of sunflower leaf extract for components exhibiting antioxidant, antibacterial and/or cholinesterase enzyme inhibitory effects. The active compounds were characterized by HPTLC-electrospray ionization-high resolution mass spectrometry (ESI-HRMS) and HPTLC-Direct Analysis in Real Time (DART)-MS/MS. The latter ambient ionization technique (less soft than ESI) resulted in oxidation and fragmentation products and characteristic fragment ions. NMR spectroscopy after targeted isolation via preparative normal phase flash chromatography and semi-preparative reversed phase high-performance liquid chromatography supported the identification of two diterpenes to be (-)-kaur-16-en-19-oic acid and 15-α-angeloyloxy-ent-kaur-16-en-19-oic acid. Both compounds found to be multi-potent as they inhibited acetylcholinesterase and butyrylcholinesterase and showed antibacterial effects against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri bacteria. Kaurenoic acid was also active against the Gram-negative pepper pathogenic Xanthomonas euvesicatoria bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1.

    Science.gov (United States)

    Nakanishi, Hiroshi; Ohtsubo, Masafumi; Iwasaki, Satoshi; Hotta, Yoshihiro; Takizawa, Yoshinori; Hosono, Katsuhiro; Mizuta, Kunihiro; Mineta, Hiroyuki; Minoshima, Shinsei

    2010-12-01

    Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70-80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

  15. The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

    Science.gov (United States)

    Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

    2012-02-18

    As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Separation of Berberine Hydrochloride and Tetrahydropalmatine and Their Quantitative Analysis with Thin Layer Chromatography Involved with Ionic Liquids

    Directory of Open Access Journals (Sweden)

    Jing Lu

    2015-01-01

    Full Text Available [BMIM]OH was used in mobile and stationary phase of thin layer chromatography (TLC to analyze berberine hydrochloride and tetrahydropalmatine for the first time. Supported imidazole ionic liquid with hydroxide ion on silica gel (SiO2·Im+·OH− was synthesized through simple procedure and characterized by Fourier transform infrared spectroscopy (FT-IR, elemental analysis, and scanning electron microscope (SEM. Moreover, on the plates prepared by SiO2·Im+·OH−, the contents of the above alkaloids in the Chinese patent medicine (CPM of “Stomacheasy” capsule were successfully determined by TLC scanner. The key conditions and chromatographic behaviors were also investigated in detail. According to similar ways, ionic liquids (ILs also can be used in other planar chromatographies in two modes. This study is expected to be helpful in expanding the application of IL and its bonded silica gel in TLC separation field.

  17. Mutational analysis of Peroxiredoxin IV: exclusion of a positional candidate for multinodular goitre

    Directory of Open Access Journals (Sweden)

    Bonifazi Emanuela

    2002-07-01

    Full Text Available Abstract Background Multinodular goitre (MNG is a common disorder characterised by an enlargement of the thyroid, occurring as a compensatory response to hormonogenesis impairment. The incidence of MNG is dependent on sex (female:male ratio 5:1 and several reports have documented a genetic basis for the disease. Last year we mapped a MNG locus to chromosome Xp22 in a region containing the peroxiredoxin IV (Prx-IV gene. Since Prx-IV is involved in the removal of H2O2 in thyroid cells, we hypothesize that mutations in Prx-IV gene are involved in pathogenesis of MNG. Methods Four individuals (2 affected, 2 unrelated unaffected were sequenced using automated methods. All individuals were originated from the original three-generation Italian family described in previous studies. A Southern blot analysis using a Prx-IV full-length cDNA as a probe was performed in order to exclude genomic rearrangements and/or intronic mutations. In addition a RT-PCR of PRX-IV was performed in order to investigate expression alterations. Results No causative mutations were found. Two adjacent nucleotide substitutions were detected within introns 1 and 4. These changes were also detected in unaffected individuals, suggesting that they were innocuous polymorphisms. No gross genomic rearrangements and/or restriction fragment alterations were observed on Southern analysis. Finally, using RT-PCR from tissue-specific RNA, no differences of PRX-IV expression-levels were detected between affected and unaffected samples. Conclusions Based on sequence and genomic analysis, Prx-IV is very unlikely to be the MNG2 gene.

  18. The HIVToolbox 2 web system integrates sequence, structure, function and mutation analysis.

    Directory of Open Access Journals (Sweden)

    David P Sargeant

    Full Text Available There is enormous interest in studying HIV pathogenesis for improving the treatment of patients with HIV infection. HIV infection has become one of the best-studied systems for understanding how a virus can hijack a cell. To help facilitate discovery, we previously built HIVToolbox, a web system for visual data mining. The original HIVToolbox integrated information for HIV protein sequence, structure, functional sites, and sequence conservation. This web system has been used for almost 40,000 searches. We report improvements to HIVToolbox including new functions and workflows, data updates, and updates for ease of use. HIVToolbox2, is an improvement over HIVToolbox with new functions. HIVToolbox2 has new functionalities focused on HIV pathogenesis including drug-binding sites, drug-resistance mutations, and immune epitopes. The integrated, interactive view enables visual mining to generate hypotheses that are not readily revealed by other approaches. Most HIV proteins form multimers, and there are posttranslational modification and protein-protein interaction sites at many of these multimerization interfaces. Analysis of protease drug binding sites reveals an anatomy of drug resistance with different types of drug-resistance mutations regionally localized on the surface of protease. Some of these drug-resistance mutations have a high prevalence in specific HIV-1 M subtypes. Finally, consolidation of Tat functional sites reveals a hotspot region where there appear to be 30 interactions or posttranslational modifications. A cursory analysis with HIVToolbox2 has helped to identify several global patterns for HIV proteins. An initial analysis with this tool identifies homomultimerization of almost all HIV proteins, functional sites that overlap with multimerization sites, a global drug resistance anatomy for HIV protease, and specific distributions of some DRMs in specific HIV M subtypes. HIVToolbox2 is an open-access web application available at

  19. EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)

    Science.gov (United States)

    Method MS014 describes procedures for solvent extraction of aldicarb, bromadiolone, carbofuran, oxamyl and methomyl from water samples, followed by analysis using liquid chromatography tandem mass spectrometry (LC-MS-MS).

  20. High Resolution Melting (HRM) analysis for mutation screening of RGSL1,RGS16 and RGS8 in breast cancer

    DEFF Research Database (Denmark)

    Wiechec, Emilia; Wiuf, Carsten Henrik; Overgaard, Jens

    2011-01-01

    coding exons of RGSL1, RGS16, and RGS8 in tumors from 200 breast cancer patients. All sequence variants detected by HRM resulted in abnormal shape of the melting curves. The identified mutations and known single nucleotide polymorphisms (SNP) were subsequently confirmed by sequencing, and distribution...... cancer patients. In addition, a total of seven known SNPs were identified in this study. Genotype distributions were not significantly different between breast cancer patients and controls. CONCLUSIONS AND IMPACT: Identification of novel mutations within RGSL1 provides a new insight...... into the pathophysiology of breast cancer. Moreover, the HRM analysis represents a reliable and highly sensitive method for mutation scanning of multiple exons....

  1. Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: a gradient of severity in cognitive impairments.

    Science.gov (United States)

    Leblond, Claire S; Nava, Caroline; Polge, Anne; Gauthier, Julie; Huguet, Guillaume; Lumbroso, Serge; Giuliano, Fabienne; Stordeur, Coline; Depienne, Christel; Mouzat, Kevin; Pinto, Dalila; Howe, Jennifer; Lemière, Nathalie; Durand, Christelle M; Guibert, Jessica; Ey, Elodie; Toro, Roberto; Peyre, Hugo; Mathieu, Alexandre; Amsellem, Frédérique; Rastam, Maria; Gillberg, I Carina; Rappold, Gudrun A; Holt, Richard; Monaco, Anthony P; Maestrini, Elena; Galan, Pilar; Heron, Delphine; Jacquette, Aurélia; Afenjar, Alexandra; Rastetter, Agnès; Brice, Alexis; Devillard, Françoise; Assouline, Brigitte; Laffargue, Fanny; Lespinasse, James; Chiesa, Jean; Rivier, François; Bonneau, Dominique; Regnault, Beatrice; Zelenika, Diana; Delepine, Marc; Lathrop, Mark; Sanlaville, Damien; Schluth-Bolard, Caroline; Edery, Patrick; Perrin, Laurence; Tabet, Anne Claude; Schmeisser, Michael J; Boeckers, Tobias M; Coleman, Mary; Sato, Daisuke; Szatmari, Peter; Scherer, Stephen W; Rouleau, Guy A; Betancur, Catalina; Leboyer, Marion; Gillberg, Christopher; Delorme, Richard; Bourgeron, Thomas

    2014-09-01

    SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.

  2. Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: a gradient of severity in cognitive impairments.

    Directory of Open Access Journals (Sweden)

    Claire S Leblond

    2014-09-01

    Full Text Available SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD, but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene, and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04% and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.

  3. Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men

    Directory of Open Access Journals (Sweden)

    Dinić Jelena

    2007-01-01

    Full Text Available Background/Aim. Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. Methods. This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE method. Results. Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions. Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations. Conclusion. This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine diagnostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recommended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and

  4. Computational analysis of KRAS mutations: implications for different effects on the KRAS p.G12D and p.G13D mutations.

    Directory of Open Access Journals (Sweden)

    Chih-Chieh Chen

    Full Text Available BACKGROUND: The issue of whether patients diagnosed with metastatic colorectal cancer who harbor KRAS codon 13 mutations could benefit from the addition of anti-epidermal growth factor receptor therapy remains under debate. The aim of the current study was to perform computational analysis to investigate the structural implications of the underlying mutations caused by c.38G>A (p.G13D on protein conformation. METHODS: Molecular dynamics (MD simulations were performed to understand the plausible structural and dynamical implications caused by c.35G>A (p.G12D and c.38G>A (p.G13D. The potential of mean force (PMF simulations were carried out to determine the free energy profiles of the binding processes of GTP interacting with wild-type (WT KRAS and its mutants (MT. RESULTS: Using MD simulations, we observed that the root mean square deviation (RMSD increased as a function of time for the MT c.35G>A (p.G12D and MT c.38G>A (p.G13D when compared with the WT. We also observed that the GTP-binding pocket in the c.35G>A (p.G12D mutant is more open than that of the WT and the c.38G>A (p.G13D proteins. Intriguingly, the analysis of atomic fluctuations and free energy profiles revealed that the mutation of c.35G>A (p.G12D may induce additional fluctuations in the sensitive sites (P-loop, switch I and II regions. Such fluctuations may promote instability in these protein regions and hamper GTP binding. CONCLUSIONS: Taken together with the results obtained from MD and PMF simulations, the present findings implicate fluctuations at the sensitive sites (P-loop, switch I and II regions. Our findings revealed that KRAS mutations in codon 13 have similar behavior as KRAS WT. To gain a better insight into why patients with metastatic colorectal cancer (mCRC and the KRAS c.38G>A (p.G13D mutation appear to benefit from anti-EGFR therapy, the role of the KRAS c.38G>A (p.G13D mutation in mCRC needs to be further investigated.

  5. Mutation screening and association analysis of six candidate genes for autism on chromosome 7q

    DEFF Research Database (Denmark)

    Bonora, E.; Lamb, J.A.; Barnby, G.

    2005-01-01

    in the genes CUTL1, LAMB1 and PTPRZ1. Analysis of genetic variants provided evidence for association with autism for one of the new missense changes identified in LAMB1; this effect was stronger in a subgroup of affected male sibling pair families, implying a possible specific sex-related effect......Genetic studies have provided evidence for an autism susceptibility locus (AUTS1) on chromosome 7q. Screening for mutations in six genes mapping to 7q, CUTL1, SRPK2, SYPL, LAMB1, NRCAM and PTPRZ1 in 48 unrelated individuals with autism led to the identification of several new coding variants...

  6. Value of TIRADS, BSRTC and FNA-BRAF V600E mutation analysis in differentiating high-risk thyroid nodules.

    Science.gov (United States)

    Zhang, Yu-zhi; Xu, Ting; Cui, Dai; Li, Xiao; Yao, Qing; Gong, Hai-yan; Liu, Xiao-yun; Chen, Huan-huan; Jiang, Lin; Ye, Xin-hua; Zhang, Zhi-hong; Shen, Mei-ping; Duan, Yu; Yang, Tao; Wu, Xiao-hong

    2015-11-24

    The thyroid imaging reporting and data system (TIRADS) and Bethesda system for reporting thyroid cytopathology (BSRTC) have been used for interpretation of ultrasound and fine-needle aspiration cytology (FNAC) results of thyroid nodules. BRAF(V600E) mutation analysis is a molecular tool in diagnosing thyroid carcinoma. Our objective was to compare the diagnostic value of these methods in differentiating high-risk thyroid nodules. Total 220 patients with high-risk thyroid nodules were recruited in this prospective study. They all underwent ultrasound, FNAC and BRAF(V600E) mutation analysis. The sensitivity and specificity of TIRADS were 73.1% and 88.4%. BSRTC had higher specificity (97.7%) and similar sensitivity (77.6%) compared with TIRADS. The sensitivity and specificity of BRAF(V600E) mutation (85.1%, 100%) were the highest. The combination of BSRTC and BRAF(V600E) mutation analysis significantly increased the efficiency, with 97.8% sensitivity, 97.7% specificity. In patients with BSRTC I-III, the mutation rate of BRAF(V600E) was 64.5% in nodules with TIRADS 4B compared with 8.4% in nodules with TIRADS 3 or 4A (P value in differentiating high-risk thyroid nodules. The TIRADS is useful in selecting high-risk patients for FNAB and patients with BSRTC I-III for BRAF(V600E) mutation analysis.

  7. Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications.

    Science.gov (United States)

    Hage, David S

    2017-06-01

    The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically related binding agent, are 2 methods that can be used to study these interactions. This review presents various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. © 2016 American Association for Clinical Chemistry.

  8. Gas chromatography/sniffing port analysis of aroma compounds released under mouth conditions

    NARCIS (Netherlands)

    Ruth, van S.M.; Roozen, J.P.

    2000-01-01

    The release of aroma compounds from rehydrated French beans in an artificial mouth system and in the mouths of 12 assessors was studied by gas chromatography combined with flame ionisation detection and sniffing port detection. In an artificial mouth system, volatile compounds were isolated under

  9. Gas chromatography-mass spectrometry (GC-MS) analysis of extractives of naturally durable wood

    Science.gov (United States)

    G.T. Kirker; A.B. Blodgett; S.T. Lebow; C.A. Clausen

    2011-01-01

    A preliminary study to evaluate naturally durable wood species in an above ground field trial using Gas Chromatography-Mass Spectrometry (GC-MS) detected differences in fatty acid extractives between species and within the same species over time. Fatty acids were extracted with chloroform: methanol mixture then methylated with sodium methoxide and fractionated using...

  10. Gas Chromatography/mass Spectrometry Analysis of Exhaled Leukotrienes in Asthmatic Patients

    Czech Academy of Sciences Publication Activity Database

    Čáp, P.; Chládek, J.; Pehal, F.; Malý, Marek; Petrů, V.; Barnes, P.J.; Montuschi, P.

    2004-01-01

    Roč. 59, č. 6 (2004), s. 465-470 ISSN 0040-6376 Source of funding: V - iné verejné zdroje Keywords : asthma * breath condensate * gas chromatography/mass spectrometry * leukotrienes Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 5.040, year: 2004

  11. Gas Chromatography-Mass Spectroscopic (GC-MS) Analysis of n ...

    African Journals Online (AJOL)

    1Department of Pharmacognosy and Phytotherapy, 2Department of Pharmaceutics and Pharmaceutical Technology, University of Port Harcourt, Nigeria ... tuber-regium (synonym Pleurotus tuber regium) using gas chromatography-mass spectroscopic (GC- ... Department of Plant Science and Biotechnology,. University of ...

  12. Simultaneous analysis of small organic acids and humic acids using high performance size exclusion chromatography

    NARCIS (Netherlands)

    Qin, X.P.; Liu, F.; Wang, G.C.; Weng, L.P.

    2012-01-01

    An accurate and fast method for simultaneous determination of small organic acids and much larger humic acids was developed using high performance size exclusion chromatography. Two small organic acids, i.e. salicylic acid and 2,3-dihydroxybenzoic acid, and one purified humic acid material were used

  13. Analysis of Currently Available Analgesic Tablets by Modern Liquid Chromatography: An Undergraduate Laboratory Introduction to HPLC.

    Science.gov (United States)

    Kagel, R. A.; Farwell, S. O.

    1983-01-01

    Background information, procedures, and results, are provided for an undergraduate experiment in which analgesic tablets are analyzed using liquid chromatography. The experiment, an improved, modified version of the Waters Associates Inc. experiment, is simple to prepare, requiring little glassware and minimal sample manipulation by students. (JN)

  14. Analysis of 2-methylthio-derivatives of isoprenoid cytokinins by liquid chromatography-tandem mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Tarkowski, Petr; Václavíková, Kateřina; Novák, Ondřej; Pertry, I.; Hanuš, Jan; Whenham, R.; Vereecke, D.; Šebela, M.; Strnad, Miroslav

    2010-01-01

    Roč. 680, 1-2 (2010), s. 86-91 ISSN 0003-2670 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * Rhodococcus fascians * High-performance liquid chromatography Subject RIV: CE - Biochemistry Impact factor: 4.310, year: 2010

  15. Mutation analysis of the ERCC4/FANCQ gene in hereditary breast cancer.

    Directory of Open Access Journals (Sweden)

    Sandra Kohlhase

    Full Text Available The ERCC4 protein forms a structure-specific endonuclease involved in the DNA damage response. Different cancer syndromes such as a subtype of Xeroderma pigmentosum, XPF, and recently a subtype of Fanconi Anemia, FA-Q, have been attributed to biallelic ERCC4 gene mutations. To investigate whether monoallelic ERCC4 gene defects play some role in the inherited component of breast cancer susceptibility, we sequenced the whole ERCC4 coding region and flanking untranslated portions in a series of 101 Byelorussian and German breast cancer patients selected for familial disease (set 1, n = 63 or for the presence of the rs1800067 risk haplotype (set 2, n = 38. This study confirmed six known and one novel exonic variants, including four missense substitutions but no truncating mutation. Missense substitution p.R415Q (rs1800067, a previously postulated breast cancer susceptibility allele, was subsequently screened for in a total of 3,698 breast cancer cases and 2,868 controls from Germany, Belarus or Russia. The Gln415 allele appeared protective against breast cancer in the German series, with the strongest effect for ductal histology (OR 0.67; 95%CI 0.49; 0.92; p = 0.003, but this association was not confirmed in the other two series, with the combined analysis yielding an overall Mantel-Haenszel OR of 0.94 (95% CI 0.81; 1.08. There was no significant effect of p.R415Q on breast cancer survival in the German patient series. The other three detected ERCC4 missense mutations included two known rare variants as well as a novel substitution, p.E17V, that we identified on a p.R415Q haplotype background. The p.E17V mutation is predicted to be probably damaging but was present in just one heterozygous patient. We conclude that the contribution of ERCC4/FANCQ coding mutations to hereditary breast cancer in Central and Eastern Europe is likely to be small.

  16. Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

    Science.gov (United States)

    Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

    2016-11-01

    Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for

  17. Mutation analysis of β-thalassemia in East-Western Indian population: a recent molecular approach

    Science.gov (United States)

    Shah, Parth S; Shah, Nidhi D; Ray, Hari Shankar P; Khatri, Nikunj B; Vaghasia, Ketan K; Raval, Rutvik J; Shah, Sandip C; Rao, Mandava V

    2017-01-01

    Background β-Thalassemia is the most prevalent genetic disorder in India. Its traits and coinheritance vary from mild to severe conditions, resulting in thalassemia minor, intermediate, and major, depending upon many factors. Purpose The objective of this study was to identify the incidence of β-thalassemia traits, their coinheritance, and mutations, as well as to support the patients already diagnosed with β-thalassemia in East-Western Indian population for better management. Patients and methods Seventy-five referral cases for β-thalassemia were analyzed for various β-thalassemia traits, heterozygosity, and homozygosity conditions. Blood phenotypic parameters using cell counter and capillary electrophoresis were investigated. Analyses of eight common mutations of thalassemia in India were carried out using polymerase chain reaction-amplification refractory mutation system, end point polymerase chain reaction, and DNA sequencing methods. Results Of these (75) referral cases from East-Western Indian region, 68 were positive for β-thalassemia (90.67%). The majority of case types were of β-thalassemia minor (49, 65.33%), followed by HbE traits (6, 8.0%) and β-thalassemia major, including heterozygous and homozygous (5, 6.66%; 4, 5.33%) types and then HbE homozygous (2, 2.66%), as well as one each of the HbE/β-thalassemia and HbD/β-thalassemia (1, 1.34%) combination. Mutation analysis also revealed that the highest frequency of mutation was c.92+5G>C (41, 60.29%) followed by deletion 619bp (9, 13.23%) and c.79G>A (8, 11.76%) in our study group. Five cases (nos. 24, 27, 33, 58, and 71) exhibited coinheritance between β0/β+ (2), β0/β D (1), and c.124_127delTTCT/β+ or β0(2) affecting the Rajasthani and Gujarati populations in our study of the Western region of India. Conclusion We strongly recommend these Western populations for genetic screening before adopting reproductive technologies and interracial marital relations. PMID:28546763

  18. Hydroxylapatite chromatography.

    Science.gov (United States)

    Broadhurst, A V

    2001-05-01

    Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low-pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step-gradient elution.

  19. BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis.

    Science.gov (United States)

    Chen, Dong; Huang, Jun-Fu; Liu, Kai; Zhang, Li-Qun; Yang, Zhao; Chuai, Zheng-Ran; Wang, Yun-Xia; Shi, Da-Chuan; Huang, Qing; Fu, Wei-Ling

    2014-01-01

    Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC. We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model. 25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation. This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.

  20. BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Dong Chen

    Full Text Available BACKGROUND: Colorectal cancer (CRC is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF plays an important role in the mitogen-activated protein kinase (MAPK signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC. METHODS: We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs with 95% confidence intervals (CIs for each study using a fixed effects or random effects model. RESULTS: 25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955. The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI, CpG island methylator phenotype (CIMP. This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1 methylation. CONCLUSIONS: This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.

  1. Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

    Science.gov (United States)

    2012-11-01

    REPORT Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis 14. ABSTRACT 16. SECURITY CLASSIFICATION OF... Bacillus subtilis spores with a gerP mutation triggered spore germination via nutrient germinant receptors (GRs) slowly, although this defect was...gerP, Bacillus subtilis , dipicolinic acid Xuan Y. Butzin, Anthony J. Troiano, William H. Coleman, Keren K. Griffiths, Christopher J. Doona, Florence E

  2. Mutation analysis of the COL1A1 and COL1A2 genes in Vietnamese patients with osteogenesis imperfecta.

    Science.gov (United States)

    Ho Duy, Binh; Zhytnik, Lidiia; Maasalu, Katre; Kändla, Ivo; Prans, Ele; Reimann, Ene; Märtson, Aare; Kõks, Sulev

    2016-08-12

    The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI. Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish's osteogenesis imperfecta mutation database. The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G > A (p.Gly821Ser) in four unrelated patients and one, c.2005G > A (p.Ala669Thr), in two unrelated patients. Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.

  3. Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations.

    Science.gov (United States)

    Leenen, C H M; Geurts-Giele, W R R; Dubbink, H J; Reddingius, R; van den Ouweland, A M; Tops, C M J; van de Klift, H M; Kuipers, E J; van Leerdam, M E; Dinjens, W N M; Wagner, A

    2011-12-01

    Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives. © 2011 John Wiley & Sons A/S.

  4. Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays

    Energy Technology Data Exchange (ETDEWEB)

    Kurowska, Marzena, E-mail: mkurowsk@us.edu.pl [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland); Labocha-Pawlowska, Anna; Gnizda, Dominika; Maluszynski, Miroslaw; Szarejko, Iwona [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland)

    2012-10-15

    We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M{sub 2} populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117 bp in the MNU population, the average mutation density was estimated as 1/504 kb. Only one nucleotide change was found after an analysis of 3,207,444 bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G > A and C > T substitutions. The similar share of G > A and C > T transitions indicates a lack of bias in the repair of O{sup 6}-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O{sup 6}-meG at the -1 position. Purines formed 81% of nucleotides observed at the -1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.

  5. Multi-component determination and chemometric analysis of Paris polyphylla by ultra high performance liquid chromatography with photodiode array detection.

    Science.gov (United States)

    Chen, Pei; Jin, Hong-Yu; Sun, Lei; Ma, Shuang-Cheng

    2016-09-01

    Multi-source analysis of traditional Chinese medicine is key to ensuring its safety and efficacy. Compared with traditional experimental differentiation, chemometric analysis is a simpler strategy to identify traditional Chinese medicines. Multi-component analysis plays an increasingly vital role in the quality control of traditional Chinese medicines. A novel strategy, based on chemometric analysis and quantitative analysis of multiple components, was proposed to easily and effectively control the quality of traditional Chinese medicines such as Chonglou. Ultra high performance liquid chromatography was more convenient and efficient. Five species of Chonglou were distinguished by chemometric analysis and nine saponins, including Chonglou saponins I, II, V, VI, VII, D, and H, as well as dioscin and gracillin, were determined in 18 min. The method is feasible and credible, and enables to improve quality control of traditional Chinese medicines and natural products. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Ocular Phenotype Analysis of a Family With Biallelic Mutations in the BEST1 Gene

    DEFF Research Database (Denmark)

    Sharon, Dror; Al-Hamdani, Sermed; Engelsberg, Karl

    2014-01-01

    in the inner nuclear layer, no light rise in the electro-oculography, and a reduced central but preserved peripheral retinal function by multifocal electroretinography. Full-field electroretinography demonstrated a reduced rod response and inner retina dysfunction. Retinal structure was normal in all 3 family......PURPOSE: To investigate the genetic cause and perform a comprehensive clinical analysis of a Danish family with autosomal recessive bestrophinopathy; to investigate whether Bestrophin may be expressed in normal human retina. DESIGN: Retrospective clinical and molecular genetic analysis...... and immunohistochemical observational study. METHODS: setting: National referral center. participants: A family with 5 individuals and biallelic BEST1 mutations, and enucleated eyes from 2 individuals with nonaffected retinas. observation procedures: Molecular genetic analysis included sequencing of BEST1 and co...

  7. Targeted ultradeep next-generation sequencing as a method for KIT D816V mutation analysis in mastocytosis

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Broesby-Olsen, Sigurd; Vestergaard, Hanne

    2016-01-01

    mutation levels. In this study, we established an NGS-based KIT mutation analysis and analyzed the sensitivity of D816V detection using the Ion Torrent platform. Eighty-two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation...

  8. Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

    Science.gov (United States)

    Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy Te; Basso, Giuseppe

    2016-10-04

    To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

  9. [Mutation analysis of FAH gene in patients with tyrosinemia type 1].

    Science.gov (United States)

    Dou, Li-Min; Fang, Ling-Juan; Wang, Xiao-Hong; Lu, Wei; Chen, Rui; Li, Li-Ting; Zhao, Jing; Wang, Jian-She

    2013-04-01

    . Alpha-fetoprotein 412.8 µg/L, levels of tyrosine in blood and succinylacetone in urine were 835.8 µmol/L and 27.48 µmol/L. Rickets did not improve after administration of calcium and vitamine D3. She is homozygous for the mutation c.1027G > A/c.1027G > A, which predicts G343R. The parents were mutation carriers. Analysis by Clustal X on the alignment of amino acids residual reservation among different species showed that the locative amino acid was highly conserved. Polyphen software predicted G343R was probably damaging (PISC score 3.235). Children with tyrosinemia type 1 can have manifestations of persistent diarrhea or late-onset rickets. Physical examination can reveal hepatosplenomegaly, laboratory tests indicate markedly elevated serum concentration of alpha-fetoprotein and alkaline phosphatase in plasma and succinylacetone in urine, other members in family may have tyrosinemias or parents are consanguineous. Mutations c.455G > A and c.1027G > A can be detected in FAH gene of Chinese children.

  10. Ionic liquids as silica deactivating agents in gas chromatography for direct analysis of primary amines in water.

    Science.gov (United States)

    Krzyżaniak, Agnieszka; Weggemans, Wilko; Schuur, Boelo; de Haan, André B

    2011-12-16

    Analysis of primary amines in aqueous samples remains a challenging analytical issue. The preferred approach by gas chromatography is hampered by interactions of free silanol groups with the highly reactive amine groups, resulting in inconsistent measurements. Here, we report a method for direct analysis of aliphatic amines and diamines in aqueous samples by gas chromatography (GC) with silanol deactivation using ionic liquids (ILs). ILs including trihexyl(tetradecyl)phosphonium bis 2,4,4-(trimethylpentyl)phosphinate (Cyphos IL-104), 1-methyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide [pmim][Tf(2)N] and N″-ethyl-N,N,N',N'-tetramethylguanidinium tris(pentafluoroethyl)trifluorophosphate [etmg][FAP] were tested as deactivating media for the GC liner. Solutions of these ILs in methanol were injected in the system prior to the analysis of primary amines. Butane-1,4-diamine (putrescine, BDA) was used as a reference amine. The best results were obtained using the imidazolium IL [pmim][Tf(2)N]. With this deactivator, excellent reproducibility of the analysis was achieved, and the detection limit of BDA was as low as 1mM. The applicability of the method was proven for the analysis of two different primary amines (C4-C5) and pentane-1,5-diamine. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Current trends in green liquid chromatography for the analysis of pharmaceutically active compounds in the environmental water compartments.

    Science.gov (United States)

    Shaaban, Heba; Górecki, Tadeusz

    2015-01-01

    Green analytical chemistry is an aspect of green chemistry which introduced in the late nineties. The main objectives of green analytical chemistry are to obtain new analytical technologies or to modify an old method to incorporate procedures that use less hazardous chemicals. There are several approaches to achieve this goal such as using environmentally benign solvents and reagents, reducing the chromatographic separation times and miniaturization of analytical devices. Traditional methods used for the analysis of pharmaceutically active compounds require large volumes of organic solvents and generate large amounts of waste. Most of them are volatile and harmful to the environment. With the awareness about the environment, the development of green technologies has been receiving increasing attention aiming at eliminating or reducing the amount of organic solvents consumed everyday worldwide without loss in chromatographic performance. This review provides the state of the art of green analytical methodologies for environmental analysis of pharmaceutically active compounds in the aquatic environment with special emphasis on strategies for greening liquid chromatography (LC). The current trends of fast LC applied to environmental analysis, including elevated mobile phase temperature, as well as different column technologies such as monolithic columns, fully porous sub-2 μm and superficially porous particles are presented. In addition, green aspects of gas chromatography (GC) and supercritical fluid chromatography (SFC) will be discussed. We pay special attention to new green approaches such as automation, miniaturization, direct analysis and the possibility of locating the chromatograph on-line or at-line as a step forward in reducing the environmental impact of chromatographic analyses. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Thin-layer chromatography and colorimetric analysis of multi-component explosive mixtures

    Science.gov (United States)

    Pagoria, Philip F.; Mitchell, Alexander R.; Whipple, Richard E.; Carman, M. Leslie

    2014-08-26

    A thin-layer chromatography method for detection and identification of common military and peroxide explosives in samples includes the steps of provide a reverse-phase thin-layer chromatography plate; prepare the plate by marking spots on which to deposit the samples by touching the plate with a marker; spot one micro liter of a first standard onto one of the spots, spot one micro liter of a second standard onto another of the spots, and spot samples onto other of spots producing a spotted plate; add eluent to a developing chamber; add the spotted plate to the developing chamber; remove the spotted plate from the developing chamber producing a developed plate; place the developed plate in an ultraviolet light box; add a visualization agent to a dip tank; dip the developed plate in the dip tank and remove the developed plate quickly; and detect explosives by viewing said developed plate.

  13. Association of telomerase reverse transcriptase promoter mutations with clinicopathological features and prognosis of thyroid cancer: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Su X

    2016-11-01

    Full Text Available Xingyun Su,1 Xiaoxia Jiang,1 Weibin Wang,1 Haiyong Wang,1 Xin Xu,2 Aihui Lin,1 Xiaodong Teng,3 Huiling Wu,4 Lisong Teng1 1Department of Surgical Oncology, 2Department of Medical Oncology, 3Department of Pathology, 4Department of Plastic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China Abstract: The clinicopathological and prognostic significance of telomerase reverse transcriptase (TERT promoter mutations have been widely investigated in thyroid cancer; however, the results are still discrepant. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, and the Cochran Library databases for relevant articles prior to April 2016. Mutation rates were synthesized by R statistical software. The odds ratio or standardized mean difference with 95% confidence interval was pooled by Stata. A total of 22 studies with 4,907 cases were included in this meta-analysis. TERT promoter mutations tended to present in aggressive histological types including poorly differentiated thyroid cancer (33.37%, anaplastic thyroid cancer (38.69%, and tall-cell variant papillary thyroid cancer (30.23%. These promoter mutations were likely to exist in older patients and males and were well associated with larger tumor size, extrathyroidal extension, vascular invasion, lymph node metastasis, distant metastasis, advanced tumor stage, disease recurrence/persistence, and mortality. In addition, TERT promoter mutations (especially C228T tended to coexist with BRAFV600E mutation, which indicated more aggressive tumor behavior. Therefore, TERT promoter mutations may be promising biomarkers for early diagnosis, risk stratification, prognostic prediction, and management of thyroid cancer. Keywords: TERT promoter mutations, thyroid cancer, clinicopathological features, prognosis, BRAFV600E mutation

  14. Genotype-phenotype analysis of a rare type of osteogenesis imperfecta in four Chinese families with WNT1 mutations.

    Science.gov (United States)

    Liu, Yi; Song, Lijie; Ma, Doudou; Lv, Fang; Xu, Xiaojie; Wang, Jianyi; Xia, Weibo; Jiang, Yan; Wang, Ou; Song, Yuwen; Xing, Xiaoping; Asan; Li, Mei

    2016-10-01

    Osteogenesis imperfecta (OI) is a rare inherited disease characterized by increased bone fragility and vulnerability to fractures. Recently, WNT1 is identified as a new candidate gene for OI, here we detect pathogenic mutations in WNT1 and analyze the genotype-phenotype association in four Chinese families with OI. We designed a targeted next generation sequencing panel with known fourteen OI-related genes. We applied the approach to detect pathogenic mutations in OI patients and confirmed the mutations with Sanger sequencing and cosegregation analysis. Clinical fractures, bone mineral density (BMD) and the other clinical manifestations were evaluated. We also observed the effects of bisphosphonates in OI patients with WNT1 mutations. Four compound heterozygous mutations (c.110T>C; c.505 G>T; c. 385G>A; c.506 G>A) in WNT1 were detected in three unrelated families. These four mutations had not been reported yet. A recurrent homozygous mutation (c.506dupG) was identified in the other two families. These patients had moderate to severe OI, white to blue sclera, absence of dentinogenesis imperfecta and no brain malformation. We did not observe clear genotype-phenotype correlation in WNT1 mutated OI patients. Though bisphosphonates increased BMD in WNT1 related OI patients, height did not increase and fracture continued. We reported four novel heterozygous variants and confirmed a previous reported WNT1 mutation in four Chinese families with a clinical diagnosis of OI. Our study expanded OI spectrum and confirmed moderate to severe bone fragility induced by WNT1 defects. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Analysis of oak tannins by liquid chromatography-electrospray ionisation mass spectrometry.

    Science.gov (United States)

    Mämmelä, P; Savolainen, H; Lindroos, L; Kangas, J; Vartiainen, T

    2000-09-01

    Extractable tannins were analysed by liquid chromatography-electrospray ionisation mass spectrometry in two oak species, North American white oak (Quercus alba) and European red oak (Quercus robur). They mainly included various glucose gallic and ellagic acid esters. The structures were partially determined, and they included grandinin/roburin E, castalagin/vescalagin, gallic acid, valoneic acid bilactone, monogalloyl glucose, digalloyl glucose, trigalloyl glucose, ellagic acid rhamnose, quercitrin and ellagic acid.

  16. Enantioselective analysis of drugs: contributions of high-performance liquid chromatography and capillary electrophoresis

    OpenAIRE

    Bonato, Pierina Sueli; Jabor, Valquíria Aparecida Polisel; Gaitani, Cristiane Masetto de

    2005-01-01

    The demand for analytical methods suitable for accurate and reproducible determination of drug enantiomers has increased significantly in the last years. High-performance liquid chromatography (HPLC) using chiral stationary phases and capillary electrophoresis (CE) are the most important techniques used for this purpose. In this paper, the fundamental aspects of chiral separations using both techniques are presented. Some important aspects for the development of enantioselective methods, part...

  17. Quantitative analysis of cytokinins in plants by liquid chromatography single-quadrupole mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Novák, Ondřej; Tarkowski, Petr; Tarkowská, Danuše; Doležal, Karel; Lenobel, René; Strnad, Miroslav

    2003-01-01

    Roč. 480, č. 2 (2003), s. 207ů218 ISSN 0003-2670 R&D Projects: GA ČR GA522/01/0275 Grant - others:Volkswagen Stiftung(DE) I/76 865 Institutional research plan: CEZ:AV0Z5038910; CEZ:MSM 153100008 Keywords : Liquid chromatography mass spectrometry * Cytokinins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.210, year: 2003

  18. Fingerprint analysis of Resina Draconis by ultra-performance liquid chromatography

    OpenAIRE

    Xue, Yudi; Zhu, Lin; Yi, Tao

    2017-01-01

    Background Resina Draconis, a bright red resin derived from Dracaena cochinchinensis, is a traditional medicine used in China. To improve its quality control approach, an ultra-performance liquid chromatography (UPLC) fingerprint method was developed for rapidly evaluating the quality of Resina Draconis. Methods The precision, repeatability and stability of the proposed UPLC method were validated in the study. Twelve batches of Resina Draconis samples from various sources were analyzed by the...

  19. Methods of analysis-Determination of pesticides in sediment using gas chromatography/mass spectrometry

    Science.gov (United States)

    Hladik, Michelle; McWayne, Megan M.

    2012-01-01

    A method for the determination of 119 pesticides in environmental sediment samples is described. The method was developed by the U.S. Geological Survey (USGS) in support of the National Water Quality Assessment (NAWQA) Program. The pesticides included in this method were chosen through prior prioritization. Herbicides, insecticides, and fungicides along with degradates are included in this method and span a variety of chemical classes including, but not limited to, chloroacetanilides, organochlorines, organophosphates, pyrethroids, triazines, and triazoles. Sediment samples are extracted by using an accelerated solvent extraction system (ASE®, and the compounds of interest are separated from co-extracted matrix interferences (including sulfur) by passing the extracts through high performance liquid chromatography (HPLC) with gel-permeation chromatography (GPC) along with the use of either stacked graphitized carbon and alumina solid-phase extraction (SPE) cartridges or packed Florisil®. Chromatographic separation, detection, and quantification of the pesticides from the sediment-sample extracts are done by using gas chromatography with mass spectrometry (GC/MS). Recoveries in test sediment samples fortified at 10 micrograms per kilogram (μg/kg) dry weight ranged from 75 to 102 percent; relative standard deviations ranged from 3 to 13 percent. Method detection limits (MDLs), calculated by using U.S. Environmental Protection Agency procedures (40 CFR 136, Appendix B), ranged from 0.6 to 3.4 μg/kg dry weight.

  20. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    Energy Technology Data Exchange (ETDEWEB)

    Lasa, A.; Baiget, M.; Gallano, P. [Hospital Sant Pau, Barcelona (Spain)

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  1. Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

    Directory of Open Access Journals (Sweden)

    Pablo Cordero

    2015-11-01

    Full Text Available Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2 and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10% and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

  2. High-performance liquid chromatography with fluorescence detection for the rapid analysis of pheophytins and pyropheophytins in virgin olive oil.

    Science.gov (United States)

    Li, Xueqi; Woodman, Michael; Wang, Selina C

    2015-08-01

    Pheophytins and pyropheophytin are degradation products of chlorophyll pigments, and their ratios can be used as a sensitive indicator of stress during the manufacturing and storage of olive oil. They increase over time depending on the storage condition and if the oil is exposed to heat treatments during the refining process. The traditional analysis method includes solvent- and time-consuming steps of solid-phase extraction followed by analysis by high-performance liquid chromatography with ultraviolet detection. We developed an improved dilute/fluorescence method where multi-step sample preparation was replaced by a simple isopropanol dilution before the high-performance liquid chromatography injection. A quaternary solvent gradient method was used to include a fourth strong solvent wash on a quaternary gradient pump, which avoided the need to premix any solvents and greatly reduced the oil residues on the column from previous analysis. This new method not only reduces analysis cost and time but shows reliability, repeatability, and improved sensitivity, especially important for low-level samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Identification of di(ethylhexyl) phthalate as impurity in the analysis by using chromatography gas tandem mass spectrometry

    Science.gov (United States)

    Pusfitasari, Eka Dian; Hendarsyah, Hendris; Salahuddin, Ariani, Novita

    2017-01-01

    Di(ethylhexyl) phthalate (DEHP) is a plasticizer commonly used in plastics. Physically DEHP has a low vapor pressure. DEHP can seep into the liquid in direct contact with the plastic wrapping materials, and typically can occur rapidly if extractable into food or non-polar solvents, such as oil, once the food is packaged in PVC packaging materials. DEHP has been analyzed by using gas chromatography which has a high sensitivity level. If the equipment used for the analysis is made from plastic containing DEHP, then it may be possible that DEHP can be extracted and appear on the outcome of the injection. It can interfere with the process of analysis, especially for the analysis of food samples. This study has identified the present of DEHP in the blank injection performed by Gas Chromatography tandem Mass Spectrometry with Selected Ion Monitoring mode (SIM). Researchers are required to verify whether the gas chromatographic system used is ready for the analysis process. In addition, the comparison and calculation of the intensity of the ion fragmentation spectra generated by mass spectrometry detector can be used for the qualitative determination to ensure the presence of the target compound. In this study is also discussed the differences between the high-intensity fragmentation of DEHP and dioctyl phthalate (DOP).

  4. NUTRIGENOMIC ANALYSIS OF C677T MUTATION OF MTHFR GENE IN SLOVAK POPULATION

    Directory of Open Access Journals (Sweden)

    Jozef Bulla

    2011-04-01

    Full Text Available Total of 124 individuals originated from Slovak Republic has been nutrignomically analysed. Analysis was focused to mutation C677T of MTHFR gene detection and analysis of mutant genotypes frequency. Observed frequency of allele 677C was 0.6998 and allelic frequency of mutant variant 677T was 0.3992. Genotype frequency of mutant heterozygotes with 71% activity of MTHFR enzyme was 0,391 and mutant homozygotes with 33% MTHFR enzyme activity was 0.153. Result shows 64% of Slovak has decreased activity of enzyme MTHFR, and 14.3% of Slovak has predisposition to cancer, cardio vascular diseases, loss of fertility and many others complications according to improper nutrition, low folic acid and B12 vitamin intake.  doi:10.5219/136

  5. High-performance liquid chromatography coupled with tandem mass spectrometry technology in the analysis of Chinese Medicine Formulas: A bibliometric analysis (1997-2015).

    Science.gov (United States)

    He, Xi-Ran; Li, Chun-Guang; Zhu, Xiao-Shu; Li, Yuan-Qing; Jarouche, Mariam; Bensoussan, Alan; Li, Ping-Ping

    2017-01-01

    There is a recognized challenge in analyzing traditional Chinese medicine formulas because of their complex chemical compositions. The application of modern analytical techniques such as high-performance liquid chromatography coupled with a tandem mass spectrometry has improved the characterization of various compounds from traditional Chinese medicine formulas significantly. This study aims to conduct a bibliometric analysis to recognize the overall trend of high-performance liquid chromatography coupled with tandem mass spectrometry approaches in the analysis of traditional Chinese medicine formulas, its significance and possible underlying interactions between individual herbs in these formulas. Electronic databases were searched systematically, and the identified studies were collected and analyzed using Microsoft Access 2010, Graph Pad 5.0 software and Ucinet software package. 338 publications between 1997 and 2015 were identified, and analyzed in terms of annual growth and accumulated publications, top journals, forms of traditional Chinese medicine preparations and highly studied formulas and single herbs, as well as social network analysis of single herbs. There is a significant increase trend in using high-performance liquid chromatography coupled with tandem mass spectrometry related techniques in analysis of commonly used forms of traditional Chinese medicine formulas in the last 3 years. Stringent quality control is of great significance for the modernization and globalization of traditional Chinese medicine, and this bibliometric analysis provided the first and comprehensive summary within this field. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Lung cancer mutation profile of EGFR, ALK, and KRAS: Meta-analysis and comparison of never and ever smokers.

    Science.gov (United States)

    Chapman, Aaron M; Sun, Kathie Y; Ruestow, Peter; Cowan, Dallas M; Madl, Amy K

    2016-12-01

    Lung cancer is the leading cause of cancer-related mortality. While the majority of lung cancers are associated with tobacco smoke, approximately 10-15% of U.S. lung cancers occur in never smokers. Evidence suggests that lung cancer in never smokers appears to be a distinct disease caused by driver mutations which are different than the genetic pathways observed with lung cancer in smokers. A meta-analysis of human epidemiologic data was conducted to evaluate the profile of common or therapy-targetable mutations in lung cancers of never and ever smokers. Epidemiologic studies (N=167) representing over 63,000 lung cancer cases were identified and used to calculate summary odds ratios for lung cancer in never and ever smokers containing gene mutations: EGFR, chromosomal rearrangements and fusion of EML4 and ALK, and KRAS. This analysis also considered the effect of histopathology, smoking status, sex, and ethnicity. There were significantly increased odds of presenting the EGFR and ALK-EML4 mutations in 1) adenocarcinomas compared to non-small cell lung cancer and 2) never smokers compared to ever smokers. The prevalence of EGFR mutations was higher in Asian women as compared to women of Caucasian/Mixed ethnicity. As the smoking history increased, there was a decreased odds for exhibiting the EGFR mutation, particularly for cases >30 pack-years. Compared to ever smokers, never smokers had a decreased odds of KRAS mutations among those of Caucasian/Mixed ethnicity (OR=0.22, 95% CI: 0.17-0.29) and those of Asian ethnicity (OR=0.39, 95% CI: 0.30-0.50). Our findings show that key driver mutations and several patient features are highly prevalent in lung cancers of never smokers. These associations may be helpful as patient demographic models are developed to predict successful outcomes of targeted therapeutic interventions NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Somatic mutation load of estrogen receptor-positive breast tumors predicts overall survival: an analysis of genome sequence data.

    Science.gov (United States)

    Haricharan, Svasti; Bainbridge, Matthew N; Scheet, Paul; Brown, Powel H

    2014-07-01

    Breast cancer is one of the most commonly diagnosed cancers in women. While there are several effective therapies for breast cancer and important single gene prognostic/predictive markers, more than 40,000 women die from this disease every year. The increasing availability of large-scale genomic datasets provides opportunities for identifying factors that influence breast cancer survival in smaller, well-defined subsets. The purpose of this study was to investigate the genomic landscape of various breast cancer subtypes and its potential associations with clinical outcomes. We used statistical analysis of sequence data generated by the Cancer Genome Atlas initiative including somatic mutation load (SML) analysis, Kaplan-Meier survival curves, gene mutational frequency, and mutational enrichment evaluation to study the genomic landscape of breast cancer. We show that ER(+), but not ER(-), tumors with high SML associate with poor overall survival (HR = 2.02). Further, these high mutation load tumors are enriched for coincident mutations in both DNA damage repair and ER signature genes. While it is known that somatic mutations in specific genes affect breast cancer survival, this study is the first to identify that SML may constitute an important global signature for a subset of ER(+) tumors prone to high mortality. Moreover, although somatic mutations in individual DNA damage genes affect clinical outcome, our results indicate that coincident mutations in DNA damage response and signature ER genes may prove more informative for ER(+) breast cancer survival. Next generation sequencing may prove an essential tool for identifying pathways underlying poor outcomes and for tailoring therapeutic strategies.

  8. [Children with idiopathic hypogonadotropic hypogonadism: clinical data analysis and mutations analysis of KAL1 and FGFR1 gene].

    Science.gov (United States)

    Qin, Miao; Gong, Chunxiu; Qi, Zhan; Wu, Di; Liu, Min; Gu, Yi; Cao, Bingyan; Li, Wenjing; Liang, Xuejun

    2014-12-01

    delayed puberty, involving only one parent in 6 families, involving both in 2 families and the other one was an uncle having micropenis with a child. Among these 21 cases, only one boy's father had hyposmia and his first emission age was 14-15 years. Eleven patients accompanied abnormal sense of smelling and the olfactory organ abnormalities on MRI, 4 had olfactory organ abnormalities on MRI while they had good smelling function self-reportedly. We got 15 samples (12 KS and 3 nIHH cases) to screen the mutation of KAL1 (14 exons) and FGFR1 (18 exons). A splicing mutation c.1062+1G>A in KAL1 is identified in case 17 with IHH. One novel heterozygous FGFR1 mutation, a single base deletion mutation on the exon 1 c.27delC is identified in case 14. This mutation causes the premature termination codons. This pilot research showed that IHH/KS diagnosis in children depends on clinical manifestation rather than gene analysis. Small penis or cryptorchidism, smelling abnormality and positive familial history may contribute to the KS/HH diagnosis. MRI of olfactory bulb acts as important proof for diagnosis of KS. Mutations in KAL1 and FGFR1 gene are not main causes of Kallmann syndrome.

  9. Genetic analysis of a four generation Indian family with Usher syndrome: a novel insertion mutation in MYO7A.

    Science.gov (United States)

    Kumar, Arun; Babu, Mohan; Kimberling, William J; Venkatesh, Conjeevaram P

    2004-11-24

    Usher syndrome (USH) is a rare autosomal recessive disorder characterized by deafness and retinitis pigmentosa. The purpose of this study was to determine the genetic cause of USH in a four generation Indian family. Peripheral blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to known USH loci, microsatellite markers were selected from the candidate regions of known loci and used to genotype the family. Exon specific intronic primers for the MYO7A gene were used to amplify DNA samples from one affected individual from the family. PCR products were subsequently sequenced to detect mutation. PCR-SSCP analysis was used to determine if the mutation segregated with the disease in the family and was not present in 50 control individuals. All affected individuals had a classic USH type I (USH1) phenotype which included deafness, vestibular dysfunction and retinitis pigmentosa. Pedigree analysis suggested an autosomal recessive mode of inheritance of USH in the family. Haplotype analysis suggested linkage of this family to the USH1B locus on chromosome 11q. DNA sequence analysis of the entire coding region of the MYO7A gene showed a novel insertion mutation c.2663_2664insA in a homozygous state in all affected individuals, resulting in truncation of MYO7A protein. This is the first study from India which reports a novel MYO7A insertion mutation in a four generation USH family. The mutation is predicted to produce a truncated MYO7A protein. With the novel mutation reported here, the total number of USH causing mutations in the MYO7A gene described to date reaches to 75.

  10. Mutation analysis of the PAH gene in phenylketonuria patients from Rio de Janeiro, Southeast Brazil.

    Science.gov (United States)

    Vieira Neto, Eduardo; Laranjeira, Francisco; Quelhas, Dulce; Ribeiro, Isaura; Seabra, Alexandre; Mineiro, Nicole; D M Carvalho, Lilian; Lacerda, Lúcia; G Ribeiro, Márcia

    2018-05-10

    Phenylketonuria (PKU) is an autosomal recessive disease resulting from mutations in the PAH gene. Most of the patients are compound heterozygotes, and genotype is a major factor in determining the phenotypic variability of PKU. More than 1,000 variants have been described in the PAH gene. Rio de Janeiro's population has a predominance of Iberian, followed by African and Amerindian ancestries. It is expected that most PKU variants in this Brazilian state have originated in the Iberian Peninsula. However, rare European, African or pathogenic variants that are characteristic of the admixed population of the state might also be found. A total of 102 patients were included in this study. Genomic DNA was isolated from dried blood spots. Sanger sequencing was used for PAH gene variant identification. Deletions and duplications were also screened using MLPA analysis. Haplotypes were also determined. Nine (8.8%) homozygous and 93 (91.2%) compound heterozygous patients were found. The spectrum included 37 causative mutations. Missense, nonsense, and splicing pathogenic variants corresponded to 63.7%, 2.9%, and 22.6% of the mutant alleles, respectively. Large (1.5%), and small deletions, inframe (5.4%) and with frameshift (3.9%), comprised the remainder. The most frequent pathogenic variants were: p.V388M (12.7%), p.R261Q (11.8%), IVS10-11G>A (10.3%), IVS2+5G>C (6.4%), p.S349P (6.4%), p.R252W (5.4%), p.I65T (4.4%), p.T323del (4.4%), and p.P281L (3.4%). One novel variant was detected: c.934G>T (p.G312C) [rs763115697]. The three most frequent pathogenic variants in our study (34.8% of the alleles) were also the most common in other Brazilian states, Portugal, and Spain (p.V388M, p.R261Q, IVS10-11G>A), corroborating that the Iberian Peninsula is the major source of PAH mutations in Rio de Janeiro. Pathogenic variants that have other geographical origins, such IVS2+5G>C, p.G352Vfs*48, and IVS12+1G>A were also detected. Genetic drift and founder effect may have also played a role

  11. N-linked glycoprotein analysis using dual-extraction ultrahigh-performance liquid chromatography and electrospray tandem mass spectrometry.

    Science.gov (United States)

    Siu, S O; Lam, Maggie P Y; Lau, Edward; Yeung, William S B; Cox, David M; Chu, Ivan K

    2010-01-01

    Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.

  12. Analysis of human milk oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection

    DEFF Research Database (Denmark)

    Lie, Aleksander; Pedersen, Lars Haastrup

    ) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), among others. High-performance anion-exchange chromatography (HPAE) with pulsed amperometric detection (PAD) is an analysis method highly suited for carbohydrates. HPAE with alkaline eluents results in retention of neutral carbohydrates depending...... on the number of charged group in the molecule, pH and concentration of competing anions, while the PAD has sensitivity for carbohydrates in the pmol-range (Lee 1990). As a basis for the development and optimisation of HPAE elution methods, the parameter space was investigated in terms of eluent concentrations...

  13. Analysis of L-serine-O-phosphate in cerebrospinal spinal fluid by derivatization-liquid chromatography/mass spectrometry.

    Science.gov (United States)

    McNaney, Colleen A; Benitex, Yulia; Luchetti, David; Labasi, Jeffrey M; Olah, Timothy V; Morgan, Daniel G; Drexler, Dieter M

    2014-05-01

    L-serine-O-phosphate (L-SOP), the precursor of L-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of L-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine L-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization-LC/MS, d-LC/MS). Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Rapid detection of most frequent Slovenian germ-line mutations in BRCA1 gene using real-time PCR and melting curve analysis

    International Nuclear Information System (INIS)

    Novakovic, S.; Stegel, V.

    2005-01-01

    Background. Detection of inherited mutations in cancer susceptibility genes is of great importance in some types of cancers including the colorectal cancer (mutations of APC gene in familial adenomatous polyposis - FAP, mutations in mismatch repair genes in hereditary nonpolyposis colorectal cancer - HNPCC), malignant melanoma (mutations in CDKN2A and CDK4 genes) and breast cancer (mutations in BRCA1 and BRCA2 genes). Methods. This article presents the technical data for the detection of five mutations in BRCA1 gene in breast cancer patients and their relatives. The mutations - 1806C>T, 300T>G, 300T>A, 310G>A, 5382insC - were determined by the real-time PCR and the melting curve analysis. Results and conclusion. In comparison to direct sequencing, this method proved to be sensitive and rapid enough for the routine daily determination of mutations in DNA isolated from the peripheral blood. (author)

  15. Analysis of benzene, toluene, ethylbenzene and xylenes in soils by headspace and gas chromatography/flame ionization detector

    Directory of Open Access Journals (Sweden)

    Jurandir Pereira Pinto

    2006-02-01

    Full Text Available The constituents of gasoline: benzene, toluene, ethylbenzene and xylenes (BTEX are frequently found in soils due to leaks in fuel storage tanks and they present chronic toxicity. In this work it was developed and validated a methodology of BTEX analysis in soil by gas chromatography/ flame ionization detector and static headspace. The recovery of BTEX in soil samples was evaluated using soils with different textures (sandy and loamy. The analysis method showed good resolution, in a low time of analysis (less than 30 minutes. Limits of quantification of 0.05 mg Kg¯¹ soil for benzene, toluene, ethylbenzene and xylenes are below the guiding values that range from 0.15 to 95 mg Kg¯¹ soil, established to determine soil quality. It was verified that the methodology enables the use of this method for BTEX analysis of soil samples for passive environmental identification of gas stations.

  16. Mutation scanning analysis of genetic variation within and among Echinococcus species: implications and future prospects.

    Science.gov (United States)

    Jabbar, Abdul; Gasser, Robin B

    2013-07-01

    Adult tapeworms of the genus Echinococcus (family Taeniidae) occur in the small intestines of carnivorous definitive hosts and are transmitted to particular intermediate mammalian hosts, in which they develop as fluid-filled larvae (cysts) in internal organs (usually lung and liver), causing the disease echinococcosis. Echinococcus species are of major medical importance and also cause losses to the meat and livestock industries, mainly due to the condemnation of infected offal. Decisions regarding the treatment and control of echinococcosis rely on the accurate identification of species and population variants (strains). Conventional, phenetic methods for specific identification have some significant limitations. Despite advances in the development of molecular tools, there has been limited application of mutation scanning methods to species of Echinococcus. Here, we briefly review key genetic markers used for the identification of Echinococcus species and techniques for the analysis of genetic variation within and among populations, and the diagnosis of echinococcosis. We also discuss the benefits of utilizing mutation scanning approaches to elucidate the population genetics and epidemiology of Echinococcus species. These benefits are likely to become more evident following the complete characterization of the genomes of E. granulosus and E. multilocularis.

  17. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    Science.gov (United States)

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics.

    Science.gov (United States)

    Magliacane, Gilda; Grassini, Greta; Bartocci, Paola; Francaviglia, Ilaria; Dal Cin, Elena; Barbieri, Gianluca; Arrigoni, Gianluigi; Pecciarini, Lorenza; Doglioni, Claudio; Cangi, Maria Giulia

    2015-10-13

    Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

  19. A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

    Science.gov (United States)

    Neuzillet, Yann; Paoletti, Xavier; Ouerhani, Slah; Mongiat-Artus, Pierre; Soliman, Hany; de The, Hugues; Sibony, Mathilde; Denoux, Yves; Molinie, Vincent; Herault, Aurélie; Lepage, May-Linda; Maille, Pascale; Renou, Audrey; Vordos, Dimitri; Abbou, Claude-Clément; Bakkar, Ashraf; Asselain, Bernard; Kourda, Nadia; El Gaaied, Amel; Leroy, Karen; Laplanche, Agnès; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Radvanyi, François

    2012-01-01

    TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12) and OR = 0.99 [0.37-2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

  20. A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

    Directory of Open Access Journals (Sweden)

    Yann Neuzillet

    Full Text Available TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001 or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009. However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12 and OR = 0.99 [0.37-2.7] (p = 0.35, respectively. After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4. These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

  1. Gas chromatography in space

    Science.gov (United States)

    Akapo, S. O.; Dimandja, J. M.; Kojiro, D. R.; Valentin, J. R.; Carle, G. C.

    1999-01-01

    Gas chromatography has proven to be a very useful analytical technique for in situ analysis of extraterrestrial environments as demonstrated by its successful operation on spacecraft missions to Mars and Venus. The technique is also one of the six scientific instruments aboard the Huygens probe to explore Titan's atmosphere and surface. A review of gas chromatography in previous space missions and some recent developments in the current environment of fiscal constraints and payload size limitations are presented.

  2. Supercritical fluid chromatography

    Science.gov (United States)

    Vigdergauz, M. S.; Lobachev, A. L.; Lobacheva, I. V.; Platonov, I. A.

    1992-03-01

    The characteristic features of supercritical fluid chromatography (SCFC) are examined and there is a brief historical note concerning the development of the method. Information concerning the use of supercritical fluid chromatography in the analysis of objects of different nature is presented in the form of a table. The roles of the mobile and stationary phases in the separation process and the characteristic features of the apparatus and of the use of the method in physicochemical research are discussed. The bibliography includes 364 references.

  3. Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Uutela, Päivi; Ketola, Raimo A; Piepponen, Petteri; Kostiainen, Risto

    2009-02-09

    The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI-MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, beta-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5-75fmol) were as much as 2-60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r(2)=0.996-0.999, range=100-8500nmolL(-1)). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC-MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.

  4. Alpha-cardiac myosin heavy chain (MYH6) mutations affecting myofibril formation are associated with congenital heart defects.

    Science.gov (United States)

    Granados-Riveron, Javier T; Ghosh, Tushar K; Pope, Mark; Bu'Lock, Frances; Thornborough, Christopher; Eason, Jacqueline; Kirk, Edwin P; Fatkin, Diane; Feneley, Michael P; Harvey, Richard P; Armour, John A L; David Brook, J

    2010-10-15

    Congenital heart defects (CHD) are collectively the most common form of congenital malformation. Studies of human cases and animal models have revealed that mutations in several genes are responsible for both familial and sporadic forms of CHD. We have previously shown that a mutation in MYH6 can cause an autosomal dominant form of atrial septal defect (ASD), whereas others have identified mutations of the same gene in patients with hypertrophic and dilated cardiomyopathy. In the present study, we report a mutation analysis of MYH6 in patients with a wide spectrum of sporadic CHD. The mutation analysis of MYH6 was performed in DNA samples from 470 cases of isolated CHD using denaturing high-performance liquid chromatography and sequence analysis to detect point mutations and small deletions or insertions, and multiplex amplifiable probe hybridization to detect partial or complete copy number variations. One non-sense mutation, one splicing site mutation and seven non-synonymous coding mutations were identified. Transfection of plasmids encoding mutant and non-mutant green fluorescent protein-MYH6 fusion proteins in mouse myoblasts revealed that the mutations A230P and A1366D significantly disrupt myofibril formation, whereas the H252Q mutation significantly enhances myofibril assembly in comparison with the non-mutant protein. Our data indicate that functional variants of MYH6 are associated with cardiac malformations in addition to ASD and provide a novel potential mechanism. Such phenotypic heterogeneity has been observed in other genes mutated in CHD.

  5. [Clinical investigation and mutation analysis of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia].

    Science.gov (United States)

    Wen, Peng-qiang; Wang, Guo-bing; Chen, Zhan-ling; Liu, Xiao-hong; Cui, Dong; Shang, Yue; Li, Cheng-rong

    2013-12-01

    To analyze the clinical features and SLC25A13 gene mutations of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia. The patient was subjected to physical examination and routine laboratory tests. Blood amino acids and acylcarnitines, and urine organic acids and galactose were analyzed respectively with tandem mass spectrometry and gas chromatographic mass spectrometry. SLC25A13 gene mutation screening was conducted by high resolution melt (HRM) analysis. The petechiae on the patient's face and platelet count (27×10(9)/L, reference range 100×10(9)/L-300×10(9)/L) supported the diagnosis of immunologic thrombocytopenic purpura (ITP). Laboratory tests found that the patient have abnormal coagulation, cardiac enzyme, liver function and liver enzymes dysfunction. Tandem mass spectrometry also found methionine to be increased (286 μmol/L, reference ranges 8-35 μmol/L). The patient did not manifest any galactosemia, citrullinemia and tyrosinemia. Analysis of SLC25A13 gene mutation found that the patient has carried IVS16ins3kb, in addition with abnormal HRM result for exon 6. Direct sequencing of exon 6 revealed a novel mutation c.495delA. The same mutation was not detected in 100 unrelated healthy controls. Further analysis of her family has confirmed that the c.495delA mutation has derived from her farther, and that the IVS16ins3kb was derived from her mother. The clinical features and metabolic spectrum of citrin deficiency can be variable. The poor prognosis and severity of clinical symptoms of the patient may be attributed to the novel c.495delA mutation.

  6. Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection †

    Science.gov (United States)

    Karr, Dale B.; Waters, James K.; Emerich, David W.

    1983-01-01

    Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis. PMID:16346443

  7. The combination of heteroduplex analysis and protein truncation test for exact detection of the APC gene mutations

    International Nuclear Information System (INIS)

    Tomka, M.; Kirchhoff, T.; Stefurkova, V.; Zajac, V.; Kulcsar, L.

    1998-01-01

    Familial adenomatous polyposis (FAP) is usually associated with mutation in the adenomatous polyposis coli (APC) gene. To examine the occurrence of these mutations in the number of FAP suspected families from the whole Slovakia effectively, we have applied heteroduplex analysis (HDA) and protein truncation test (PTT) for the analyses of 2-5 base pair deletions and point mutations of the APC gene. In the analyzed exon 15 of the APC gene determined by the primers 15Efor-15Grev for HDA and 15ET7-15J3 for PTT more than 70% of mutations should be deletions [3, 12], which are detectable by HDA. In our collection of 5 FAP families mutations in the APC gene were found in families 10, 27 and 41 using HDA. By PTT test the formation of truncated APC protein in FAP families 2, 10, 16 and 27 were revealed. The necessity of combination of at least HDA and PTT techniques for exact detection of APC mutations in analyzed APC region is discussed. (authors)

  8. Analysis of Gel Permeation Chromatography From Irradiation Copolymer Grafting of Methylmethacrylate on to Natural Rubber

    International Nuclear Information System (INIS)

    Hendrana, Sunit; Purwanto, Indratmoko Hari; Karyaningsih, Ipit; Utama, MargaHerwinarni

    2004-01-01

    Grafting of methyl methacrylate (MMA) onto natural rubber was carried out by γ-irradiation using 60 Co source at dose rate 1 KGy/h and total dose of 5 KGy. Gel permeation chromatography (GPC) was used to analyse the grafting. The GPC's chromatogram, molecular weight and molecular weight distribution data indicate that grafting of MMA onto natural rubber and homo polymerization of MMA are the reaction mostly occurs. Meanwhile, the presence of natural rubber radical with a chain end natural rubber radical affect the PMMA homopolymer occurred

  9. [Gas chromatography in quantitative analysis of hydrocyanic acid and its salts in cadaveric blood].

    Science.gov (United States)

    Iablochkin, V D

    2003-01-01

    A direct gas chromatography method was designed for the quantitative determination of cyanides (prussic acid) in cadaveric blood. Its sensitivity is 0.05 mg/ml. The routine volatile products, including substances, which emerge due to putrefaction of organic matters, do not affect the accuracy and reproducibility of the method; the exception is H-propanol that was used as the internal standard. The method was used in legal chemical expertise related with acute cyanide poisoning (suicide) as well as with poisoning of products of combustion of nonmetals (foam-rubber). The absolute error does not exceed 10% with a mean quadratic deviation of 0.0029-0.0033 mg.

  10. Mosaicism in segmental darier disease: an in-depth molecular analysis quantifying proportions of mutated alleles in various tissues

    DEFF Research Database (Denmark)

    Harboe, Theresa Larriba; Willems, Patrick; Jespersgaard, Cathrine

    2011-01-01

    Darier disease is an autosomal dominant genodermatosis caused by germline mutations in the ATP2A2 gene. Clinical expression is variable, including rare segmental phenotypes thought to be caused by postzygotic mosaicism. Genetic counseling of segmental Darier patients is complex, as risk of transm......Darier disease is an autosomal dominant genodermatosis caused by germline mutations in the ATP2A2 gene. Clinical expression is variable, including rare segmental phenotypes thought to be caused by postzygotic mosaicism. Genetic counseling of segmental Darier patients is complex, as risk...... of transmitting a nonsegmental phenotype to offspring is of unknown magnitude. We present the first in-depth molecular analysis of a mosaic patient with segmental disease, quantifying proportions of mutated and normal alleles in various tissues. Pyrosequence analysis of DNA from semen, affected and normal skin......, peripheral leukocytes and hair revealed an uneven distribution of the mutated allele, from 14% in semen to 37% in affected skin. We suggest a model for segmental manifestation expression where a threshold number of mutated cells is needed for manifestation development. We further recommend molecular analysis...

  11. Qualitative and Quantitative Analysis of Volatile Components of Zhengtian Pills Using Gas Chromatography Mass Spectrometry and Ultra-High Performance Liquid Chromatography.

    Science.gov (United States)

    Liu, Cui-Ting; Zhang, Min; Yan, Ping; Liu, Hai-Chan; Liu, Xing-Yun; Zhan, Ruo-Ting

    2016-01-01

    Zhengtian pills (ZTPs) are traditional Chinese medicine (TCM) which have been commonly used to treat headaches. Volatile components of ZTPs extracted by ethyl acetate with an ultrasonic method were analyzed by gas chromatography mass spectrometry (GC-MS). Twenty-two components were identified, accounting for 78.884% of the total components of volatile oil. The three main volatile components including protocatechuic acid, ferulic acid, and ligustilide were simultaneously determined using ultra-high performance liquid chromatography coupled with diode array detection (UHPLC-DAD). Baseline separation was achieved on an XB-C18 column with linear gradient elution of methanol-0.2% acetic acid aqueous solution. The UHPLC-DAD method provided good linearity (R (2) ≥ 0.9992), precision (RSD components, protocatechuic acid, ferulic acid, and ligustilide, in 13 batches of ZTPs, which is suitable for discrimination and quality assessment of ZTPs.

  12. The characterization of petroleum and creosote-contaminated soils: Class component analysis by thin layer chromatography with flame ionization detection

    International Nuclear Information System (INIS)

    Pollard, S.J.T.; Hrudey, S.E.; Fuhr, B.J.; Alex, R.F.; Holloway, L.R.

    1992-01-01

    The assessment and reclamation of coal tar, creosote and petroleum-contaminated sites is emerging as a major challenge to industry and the federal and provincial Canadian governments. Contaminants frequently include polynuclear aromatic compounds (PAH), several high molecular weight analogues of which are documented carcinogens. Adaptation of thin layer chromatography with flame ionization detection for the analysis of hydrocarbon-contaminated soils is described. The method is directly applicable to the analysis of oily waste extracts from petroleum and creosote wood preservative site soils and is capable of distinguishing between the saturate, aromatic and polar components of waste residues. The method provides a rapid, low cost class component analysis of heavy hydrocarbon waste extracts and is particularly useful for estimating the extent of weathering experienced by chronically exposed hydrocarbon wastes in the soil environment. As such, it is useful as a screening tool for a preliminary assessment of the biotreatability or inherent recalcitrance of hydrocarbon waste mixtures. 11 refs., 5 figs

  13. Acid monomer analysis in waterborne polymer systems by targeted labeling of carboxylic acid functionality, followed by pyrolysis - gas chromatography.

    Science.gov (United States)

    Brooijmans, T; Okhuijsen, R; Oerlemans, I; Schoenmakers, P J; Peters, R

    2018-05-14

    Pyrolysis - gas chromatography - (PyGC) is a common method to analyse the composition of natural and synthetic resins. The analysis of acid functionality in, for example, waterborne polyacrylates and polyurethanes polymers has proven to be difficult due to solubility issues, inter- and intramolecular interaction effects, lack of detectability in chromatographic analysis, and lack of thermal stability. Conventional analytical techniques, such as PyGC, cannot be used for the direct detection and identification of acidic monomers, due to thermal rearrangements that take place during pyrolysis. To circumvent this, the carboxylic acid groups are protected prior to thermal treatment by reaction with 2-bromoacetophenone. Reaction conditions are investigated and optimised wrt. conversion measurements. The aproach is applied to waterborne polyacryalates and the results are discussed. This approach enables identification and (semi)quantitative analysis of different acid functionalities in waterborne polymers by PyGC. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Molecularly imprinted membrane extraction combined with high-performance liquid chromatography for selective analysis of cloxacillin from shrimp samples.

    Science.gov (United States)

    Du, Wei; Sun, Min; Guo, Pengqi; Chang, Chun; Fu, Qiang

    2018-09-01

    Nowadays, the abuse of antibiotics in aquaculture has generated considerable problems for food safety. Therefore, it is imperative to develop a simple and selective method for monitoring illegal use of antibiotics in aquatic products. In this study, a method combined molecularly imprinted membranes (MIMs) extraction and liquid chromatography was developed for the selective analysis of cloxacillin from shrimp samples. The MIMs was synthesized by UV photopolymerization, and characterized by scanning electron microscope, Fourier transform infrared spectra, thermo-gravimetric analysis and swelling test. The results showed that the MIMs exhibited excellent permselectivity, high adsorption capacity and fast adsorption rate for cloxacillin. Finally, the method was utilized to determine cloxacillin from shrimp samples, with good accuracies and acceptable relative standard deviation values for precision. The proposed method was a promising alternative for selective analysis of cloxacillin in shrimp samples, due to the easy-operation and excellent selectivity. Copyright © 2018. Published by Elsevier Ltd.

  15. Amino acid δ13C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry

    DEFF Research Database (Denmark)

    Raghavan, Maanasa; McCullagh, James S. O.; Lynnerup, Niels

    2010-01-01

    We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (delta(13)C) of individual amino acids in hair proteins and bone collagen using the LC-IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS......). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound-specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context...... by the delta(13)C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used...

  16. Comprehensive lipidomic analysis of human plasma using multidimensional liquid- and gas-phase separations: Two-dimensional liquid chromatography-mass spectrometry vs. liquid chromatography-trapped-ion-mobility-mass spectrometry.

    Science.gov (United States)

    Baglai, Anna; Gargano, Andrea F G; Jordens, Jan; Mengerink, Ynze; Honing, Maarten; van der Wal, Sjoerd; Schoenmakers, Peter J

    2017-12-29

    Recent advancements in separation science have resulted in the commercialization of multidimensional separation systems that provide higher peak capacities and, hence, enable a more-detailed characterization of complex mixtures. In particular, two powerful analytical tools are increasingly used by analytical scientists, namely online comprehensive two-dimensional liquid chromatography (LC×LC, having a second-dimension separation in the liquid phase) and liquid chromatography-ion mobility-spectrometry (LC-IMS, second dimension separation in the gas phase). The goal of the current study was a general assessment of the liquid-chromatography-trapped-ion-mobility-mass spectrometry (LC-TIMS-MS) and comprehensive two-dimensional liquid chromatography-mass spectrometry (LC×LC-MS) platforms for untargeted lipid mapping in human plasma. For the first time trapped-ion-mobility spectrometry (TIMS) was employed for the separation of the major lipid classes and ion-mobility-derived collision-cross-section values were determined for a number of lipid standards. The general effects of a number of influencing parameters have been inspected and possible directions for improvements are discussed. We aimed to provide a general indication and practical guidelines for the analyst to choose an efficient multidimensional separation platform according to the particular requirements of the application. Analysis time, orthogonality, peak capacity, and an indicative measure for the resolving power are discussed as main characteristics for multidimensional separation systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Mutational analysis a joint framework for Cauchy problems in and beyond vector spaces

    CERN Document Server

    Lorenz, Thomas

    2010-01-01

    Ordinary differential equations play a central role in science and have been extended to evolution equations in Banach spaces. For many applications, however, it is difficult to specify a suitable normed vector space. Shapes without a priori restrictions, for example, do not have an obvious linear structure. This book generalizes ordinary differential equations beyond the borders of vector spaces with a focus on the well-posed Cauchy problem in finite time intervals. Here are some of the examples: - Feedback evolutions of compact subsets of the Euclidean space - Birth-and-growth processes of random sets (not necessarily convex) - Semilinear evolution equations - Nonlocal parabolic differential equations - Nonlinear transport equations for Radon measures - A structured population model - Stochastic differential equations with nonlocal sample dependence and how they can be coupled in systems immediately - due to the joint framework of Mutational Analysis. Finally, the book offers new tools for modelling.

  18. Mutation analysis of GLDC, AMT and GCSH in cataract captive-bred vervet monkeys (Chlorocebus aethiops).

    Science.gov (United States)

    Chauke, Chesa G; Magwebu, Zandisiwe E; Sharma, Jyoti R; Arieff, Zainunisha; Seier, Jürgen V

    2016-08-01

    Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive inborn error of glycine metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. This study describes the first screening of NKH in cataract captive-bred vervet monkeys (Chlorocebus aethiops). Glycine dehydrogenase (GLDC), aminomethyltransferase (AMT) and glycine cleavage system H protein (GCSH) were prioritized. Mutation analysis of the complete coding sequence of GLDC and AMT revealed six novel single-base substitutions, of which three were non-synonymous missense and three were silent nucleotide changes. Although deleterious effects of the three amino acid substitutions were not evaluated, one substitution of GLDC gene (S44R) could be disease-causing because of its drastic amino acid change, affecting amino acids conserved in different primate species. This study confirms the diagnosis of NKH for the first time in vervet monkeys with cataracts. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Analysis of Essential Oil in Jerusalem Artichoke (Helianthus tuberosus L.) Leaves and Tubers by Gas Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Helmi, Zead; Al Azzam, Khaldun Mohammad; Tsymbalista, Yuliya; Ghazleh, Refat Abo; Shaibah, Hassan; Aboul-Enein, Hassan

    2014-12-01

    To investigate, for the first time, the chemical composition of essential oil of the tubers and leaves of Jerusalem artichoke (Helianthus tuberosus L.), a species of sunflower native to eastern North America, growing in Ukraine. A hydrodistillation apparatus was used for the extraction of volatile components and then it was analysed by gas chromatography equipped with a split-splitless injector (split ratio, 1:50) and flame ionization detector (FID). The oil was analyzed under linear temperature programming applied at 4°C/min from 50°C - 340°C. Temperatures of the injector and FID detector were maintained at 280°C and 300°C, respectively. The chemical analysis of the oil was carried out using gas chromatography coupled to mass spectrometry (GC-MS), to determine the chemical composition of the volatile fraction. The essential oils content ranged from 0.00019 to 0.03486 and 0.00011 to 0.00205 (g/100g), in leaves and tubers, respectively. The qualitative and quantitative analysis led to the identification of 17 components in both species samples. The major component found in leaves and tubers was (-)-β-bisabolene with 70.7% and 63.1%, respectively. Essential oil profile of Jerusalem artichoke species showed significant differences between leaves and tubers species. Additionally, the leaves of Jerusalem artichoke are a promising source of natural β-bisabolene.

  20. Characterisation of capillary ionic liquid columns for gas chromatography-mass spectrometry analysis of fatty acid methyl esters.

    Science.gov (United States)

    Zeng, Annie Xu; Chin, Sung-Tong; Nolvachai, Yada; Kulsing, Chadin; Sidisky, Leonard M; Marriott, Philip J

    2013-11-25

    Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as gas chromatography (GC) stationary phases offer unique GC separation especially in the analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution behaviour of FAME on several commercialised IL capillary columns including phosphonium based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82, SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated in gas chromatography-mass spectrometry (GC-MS) analysis. The phases were further characterised by using a linear solvation energy relationship (LSER) approach according to the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution temperatures of saturated FAME increased as their McReynolds' polarity value decreased, except for IL60. ECL values increased markedly as the stationary phase polarity increased, particularly for the polyunsaturated FAME. The LSER study indicated a lowest l/e value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with higher peak capacity within a carbon number isomer group. s and e descriptors calculated from LSER were validated by excellent correlation with dipole moments and lowest unoccupied molecular orbital (LUMO) energies, with R(2) values of 0.99 and 0.92 respectively, calculated using GAUSSIAN. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins.

    Science.gov (United States)

    Ren, Jiangtao; Beckner, Matthew A; Lynch, Kyle B; Chen, Huang; Zhu, Zaifang; Yang, Yu; Chen, Apeng; Qiao, Zhenzhen; Liu, Shaorong; Lu, Joann J

    2018-05-15

    A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Analysis of neonicotinoids by gas chromatography coupled to nuclide 63Ni - Electron Capture Detector - GC/ECD

    International Nuclear Information System (INIS)

    Amaral, Priscila O.; Leao, Claudio; Redigolo, Marcelo M.; Crepaldi, Caike; Bustillos, Oscar V.

    2015-01-01

    Recently, several reports have been published discussing reduction in bee population which polymerizes cultures around the world this phenomenon is known as Colony Collapse Disorder (CCD). The phenomenon describes the lack of worker honeybees in the colony despite having pups and food. The causes of this problem are unknown but there are studies that claim that reduction of population of bees is linked to poisoning through insecticides specifically neonicotinoids. Among this type of pesticide are imidacloprid (C 9 H 10 ClN 5 O 2 ), clothianidin (C 6 H 8 ClN 5 O 2 S) and thiamethoxam (C 8 H 10 ClN 5 O 3 S). This paper presents the analysis of neonicotinoids - clothianidin, imidacloprid and thiamethoxam - by the technique of gas chromatography coupled to nuclide 63 Ni electron capture detector (GC/ECD). The electron capture detector (ECD) is a gas chromatography detector that has been used for the detection of organic halogens, nitriles, nitrates and organometallic compounds. The ECD detector ionizes the analytes by the beta particles from the nuclide sources 63 Ni within carrier gas N 2 . The electrons produced in this process are collected and create a current that are amplified and generates a chromatographic peak. Methodology and details of the analysis are present in this work. (author)

  3. Analysis of neonicotinoids by gas chromatography coupled to nuclide {sup 63}Ni - Electron Capture Detector - GC/ECD

    Energy Technology Data Exchange (ETDEWEB)

    Amaral, Priscila O.; Leao, Claudio; Redigolo, Marcelo M.; Crepaldi, Caike; Bustillos, Oscar V., E-mail: priscilaoamaral@gmail.com, E-mail: claudio.leao@usp.br, E-mail: marceloredigolo@gmail.com, E-mail: caike1995@gmail.com, E-mail: ovega@ipen.bremails [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2015-07-01

    Recently, several reports have been published discussing reduction in bee population which polymerizes cultures around the world this phenomenon is known as Colony Collapse Disorder (CCD). The phenomenon describes the lack of worker honeybees in the colony despite having pups and food. The causes of this problem are unknown but there are studies that claim that reduction of population of bees is linked to poisoning through insecticides specifically neonicotinoids. Among this type of pesticide are imidacloprid (C{sub 9}H{sub 10}ClN{sub 5}O{sub 2}), clothianidin (C{sub 6}H{sub 8}ClN{sub 5}O{sub 2}S) and thiamethoxam (C{sub 8}H{sub 10}ClN{sub 5}O{sub 3}S). This paper presents the analysis of neonicotinoids - clothianidin, imidacloprid and thiamethoxam - by the technique of gas chromatography coupled to nuclide {sup 63}Ni electron capture detector (GC/ECD). The electron capture detector (ECD) is a gas chromatography detector that has been used for the detection of organic halogens, nitriles, nitrates and organometallic compounds. The ECD detector ionizes the analytes by the beta particles from the nuclide sources {sup 63}Ni within carrier gas N{sub 2}. The electrons produced in this process are collected and create a current that are amplified and generates a chromatographic peak. Methodology and details of the analysis are present in this work. (author)

  4. Comprehensive analysis of chemical constituents in Xingxiong injection by high performance liquid chromatography coupled with mass spectrometry.

    Science.gov (United States)

    Guo, Long; Dou, Li-Li; Duan, Li; Liu, Ke; Bi, Zhi-Ming; Li, Ping; Liu, E-Hu

    2015-09-01

    Xingxiong injection (XXI) is a widely used Chinese herbal formula prepared by the folium ginkgo extract and ligustrazine for the treatment of cardiovascular and cerebrovascular diseases. Compared with the pharmacological studies, chemical analysis and quality control studies on this formula are relatively limited. In the present study, a high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was applied to comprehensive analysis of constituents in XXI. According to the fragmentation rules and previous reports, thirty ginkgo flavonoids, four ginkgo terpene lactones, and one alkaloid were identified. A high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ MS) method was then applied to quantify ten major constituents in XXI. The method validation results indicated that the developed method had desirable specificity, linearity, precision and accuracy. The total contents of ginkgo flavonoids were about 22.05-25.51 μg·mL(-1) and the ginkgo terpene lactones amounts were about 4.41-8.70 μg·mL(-1) in six batches of XXI samples, respectively. Furthermore, cosine ratio algorithm and distance measurements were employed to evaluate the similarity of XXI samples, and the results demonstrated a high-quality consistency. This work could provide comprehensive information on the quality control of Xingxiong injection, which be helpful in the establishment of a rational quality control standard. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  5. Analysis of perchlorate, thiocyanate, nitrate and iodide in human amniotic fluid using ion chromatography and electrospray tandem mass spectrometry

    International Nuclear Information System (INIS)

    Blount, Benjamin C.; Valentin-Blasini, Liza

    2006-01-01

    Because of health concerns surrounding in utero exposure to perchlorate, we developed a sensitive and selective method for quantifying iodide, as well as perchlorate and other sodium-iodide symporter (NIS) inhibitors in human amniotic fluid using ion chromatography coupled with electrospray ionization tandem mass spectrometry. Iodide and NIS inhibitors were quantified using a stable isotope-labeled internal standards (Cl 18 O 4 - , S 13 CN - and 15 NO 3 - with excellent assay accuracy of 100%, 98%, 99%, 95% for perchlorate, thiocyanate, nitrate and iodide, respectively, in triplicate analysis of spiked amniotic fluid sample). Excellent analytical precision (<5.2% RSD for all analytes) was found when amniotic fluid quality control pools were repetitively analyzed for iodide and NIS-inhibitors. Selective chromatography and tandem mass spectrometry reduced the need for sample cleanup, resulting in a rugged and rapid method capable of routinely analyzing 75 samples/day. Analytical response was linear across the physiologically relevant concentration range for the analytes. Analysis of a set of 48 amniotic fluid samples identified the range and median levels for perchlorate (0.057-0.71, 0.18 μg/L), thiocyanate (<10-5860, 89 μg/L), nitrate (650-8900, 1620 μg/L) and iodide (1.7-170, 8.1 μg/L). This selective, sensitive, and rapid method will help assess exposure of the developing fetus to low levels of NIS-inhibitors and their potential to inhibit thyroid function

  6. Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog.

    Science.gov (United States)

    Solomon, Benjamin D; Bear, Kelly A; Wyllie, Adrian; Keaton, Amelia A; Dubourg, Christele; David, Veronique; Mercier, Sandra; Odent, Sylvie; Hehr, Ute; Paulussen, Aimee; Clegg, Nancy J; Delgado, Mauricio R; Bale, Sherri J; Lacbawan, Felicitas; Ardinger, Holly H; Aylsworth, Arthur S; Bhengu, Ntombenhle Louisa; Braddock, Stephen; Brookhyser, Karen; Burton, Barbara; Gaspar, Harald; Grix, Art; Horovitz, Dafne; Kanetzke, Erin; Kayserili, Hulya; Lev, Dorit; Nikkel, Sarah M; Norton, Mary; Roberts, Richard; Saal, Howard; Schaefer, G B; Schneider, Adele; Smith, Erika K; Sowry, Ellen; Spence, M Anne; Shalev, Stavit A; Steiner, Carlos E; Thompson, Elizabeth M; Winder, Thomas L; Balog, Joan Z; Hadley, Donald W; Zhou, Nan; Pineda-Alvarez, Daniel E; Roessler, Erich; Muenke, Maximilian

    2012-07-01

    Holoprosencephaly (HPE), the most common malformation of the human forebrain, may result from mutations in over 12 genes. Sonic Hedgehog (SHH) was the first such gene discovered; mutations in SHH remain the most common cause of non-chromosomal HPE. The severity spectrum is wide, ranging from incompatibility with extrauterine life to isolated midline facial differences. To characterise genetic and clinical findings in individuals with SHH mutations. Through the National Institutes of Health and collaborating centres, DNA from approximately 2000 individuals with HPE spectrum disorders were analysed for SHH variations. Clinical details were examined and combined with published cases. This study describes 396 individuals, representing 157 unrelated kindreds, with SHH mutations; 141 (36%) have not been previously reported. SHH mutations more commonly resulted in non-HPE (64%) than frank HPE (36%), and non-HPE was significantly more common in patients with SHH than in those with mutations in the other common HPE related genes (pC-terminus (including accounting for the relative size of the coding regions, p=0.00010), no specific genotype-phenotype correlations could be established regarding mutation location. SHH mutations overall result in milder disease than mutations in other common HPE related genes. HPE is more frequent in individuals with truncating mutations, but clinical predictions at the individual level remain elusive.

  7. Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

    Science.gov (United States)

    Minervini, Angela; Francesco Minervini, Crescenzio; Anelli, Luisa; Zagaria, Antonella; Casieri, Paola; Coccaro, Nicoletta; Cumbo, Cosimo; Tota, Giuseppina; Impera, Luciana; Orsini, Paola; Brunetti, Claudia; Giordano, Annamaria; Specchia, Giorgina; Albano, Francesco

    2016-12-27

    In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the "watch and wait" interval and after standard chemotherapy.

  8. Application of partition chromatography method for separation and analysis of actinium radionuclides

    International Nuclear Information System (INIS)

    Sinitsina, G.S.; Shestakova, I.A.; Shestakov, B.I.; Plyushcheva, N.A.; Malyshev, N.A.; Belyatskij, A.F.; Tsirlin, V.A.

    1979-01-01

    The method of partition chromatography is considered with the use of different extractants for the extraction of actinium-227, actinium-225 and actinium-228. It is advisable to extract actinium-227 from the irradiated radium with the help of D2FGFK. The use of 2DEGFK allows us to separate actinium-227 from alkaline and alkaline-earth elements. Amines have a higher radiative stability. An express-method has been developed for the identification of actinium-227 with TOA by its intrinsic α-emission in nonequilibrium preparations of irradiated radium-226 of small activity. Actinium-225 is extracted from uranium-233 with due regard for the fact that U, Th, and Ac are extracted differently by TBP from HNO 3 solutions. With the help of the given procedure one can reach the purifying coefficient of 10 4 . Actinium-228 is extracted from the radiummesothorium preparations by a deposition of decay products, including polonium-210 on the iron hydroxyde. Actinium-228 extraction from the mixture of radium radionuclides is performed by the partition chromatography method on D2EGFK. All the procedures for separation of actinium isotopes by the above methods are described

  9. Comprehensive sample analysis using high performance liquid chromatography with multi-detection

    International Nuclear Information System (INIS)

    Pravadali, Sercan; Bassanese, Danielle N.; Conlan, Xavier A.; Francis, Paul S.; Smith, Zoe M.; Terry, Jessica M.; Shalliker, R. Andrew

    2013-01-01

    Graphical abstract: -- Highlights: •Detection selectivity was assessed with 6 detection modes. •Natural samples show great diversity in detection selectivity. •Complex samples require evaluation using a multifaceted approach to detection. •23/30 known compounds (detected by MS) detected by chemiluminescence, DPPH and UV. -- Abstract: Herein we assess the separation space offered by a liquid chromatography system with an optimised uni-dimensional separation for the determination of the key chemical entities in the highly complex matrix of a tobacco leaf extract. Multiple modes of detection, including UV–visible absorbance, chemiluminescence (acidic potassium permanganate, manganese(IV), and tris(2,2′-bipyridine)ruthenium(III)), mass spectrometry and DPPH radical scavenging were used in an attempt to systematically reduce the data complexity of the sample whilst obtaining a greater degree of molecule-specific information. A large amount of chemical data was obtained, but several limitations i