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Sample records for rapid molecular typing

  1. SCAR makers and multiplex PCR-based rapid molecular typing of Lentinula edodes strains.

    Science.gov (United States)

    Wu, Xueqian; Li, Haibo; Zhao, Weiwei; Fu, Lizhong; Peng, Huazheng; He, Liang; Cheng, Junwen; Wei, Hailong; Wu, Qingqi

    2010-11-01

    Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.

  2. MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex.

    Directory of Open Access Journals (Sweden)

    Carolina Firacative

    Full Text Available BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this

  3. Transmission Electron Microscopy study of Cu-containing spinel-type In2S3 nanocrystals prepared by rapid pyrolysis of a single molecular precursor

    OpenAIRE

    Quiroga-González, Enrique; Bensch, Wolfgang; Duppel, Viola; Kienle, Lorenz

    2010-01-01

    Abstract Cu-containing spinel-type In2S3 nanocrystals have been prepared by rapid pyrolysis (RP) from a single source inorganic-organic hybrid molecular precursor with an initial Cu:In:S ratio of 1:9:16.5. The precursor was synthesized in a one-step hydrothermal reaction with high yield. After a treatment of the precursor for 10 s at 1000 ?C a powdered material was obtained. The X-ray powder pattern exhibits broad reflections indicative for the spinel-type and crystallites in the n...

  4. Rapid typing of Coxiella burnetii.

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    Heidie M Hornstra

    Full Text Available Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels among multispacer sequence typing (MST loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies.

  5. A rapid molecular approach for chromosomal phasing.

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    John F Regan

    Full Text Available Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information, and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours, scalable (to hundreds of samples, and effective at long genomic distances (200 kb.

  6. Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates

    Science.gov (United States)

    Kreizinger, Zsuzsa; Sulyok, Kinga Mária; Pásztor, Alexandra; Erdélyi, Károly; Felde, Orsolya; Povazsán, János; Kőrösi, László; Gyuranecz, Miklós

    2015-01-01

    Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity. PMID:26207635

  7. Molecular Typing of Nocardia Species

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    Seyyed Saeed Eshraghi

    2012-03-01

    Full Text Available Identification of clinically significant Nocardia species is essential for the definitive diagnosis, predict antimicrobial susceptibility, epidemiological purposes, and for an effective treatment. Conventional identification of Nocardia species in routine medical laboratories which is based on phenotypic (cellular morphology, colonial characteristics, biochemical and enzymatic profiles, and chemotaxonomic characteristics is often laborious, and time-consuming. The procedure requires expertise, and newer species can be difficult to differentiate with accuracy from other related species. Alternative methods of identification, such as high performance liquid chromatography (HPLC and molecular biology techniques allow a better characterization of species. The taxonomy of the genus Nocardia has been dramatically been revised during the last decade and more than 30 valid human clinical significance species of Nocardia have been reported. The use of molecular approaches, including 16S rRNA gene sequencing, restriction fragment length polymorphism (RFLP or PCR restriction endonuclease analysis has been the focus of recent investigations to distinguish the isolates of Nocardia from other actinomycetes genera. The methods have revolutionized the characterization of the Nocardiae by providing rapid, sensitive, and accurate identification procedures. The present review describes the currently known medically important pathogenic species of Nocardia.

  8. Molecular integrals for slater type orbitals using coulomb sturmians

    DEFF Research Database (Denmark)

    Avery, James Emil; Avery, John Scales

    2014-01-01

    The use of Slater type orbitals in molecular calculations is hindered by the slowness of integral evaluation. In the present paper, we introduce a method for overcoming this problem by expanding STO's in terms of Coulomb Sturmians, for which the problem of evaluating molecular integrals rapidly has...

  9. Molecular Typing of Borrelia burgdorferi

    Science.gov (United States)

    Wang, Guiqing; Liveris, Dionysios; Mukherjee, Priyanka; Jungnick, Sabrina; Margos, Gabriele; Schwartz, Ira

    2015-01-01

    Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens. PMID:25082003

  10. Rapid molecular technique to distinguish Fusarium species

    CSIR Research Space (South Africa)

    Lodolo, EJ

    1993-03-01

    Full Text Available The nuclear DNA (nDNA) of different isolates of three closely related, toxin-producing Fusarium species, F. moniliforme, F. nygamai and F. napiforme, was compared to ascertain the sensitivity of a molecular method to distinguish these three species...

  11. Rapid Development of Intestinal Type Gastric Adenocarcinoma

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    Young S. Oh

    2011-04-01

    Full Text Available Intestinal type gastric adenocarcinoma is felt to develop over a protracted time period through a series of defined steps. Several potential risk factors for the development of gastric cancer have been identified, including a family history of gastric cancer and Helicobacter pylori infection. We present the case of a patient with neither risk factor who progressed in a 14 month time frame from histologically normal gastric mucosa to early stage intestinal type gastric adenocarcinoma in the setting of diffuse gastric intestinal metaplasia and atrophic gastritis. This patient’s presentation conflicts with our current understanding of the development of intestinal type gastric adenocarcinoma.

  12. Rapid sampling of molecular motions with prior information constraints.

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    Barak Raveh

    2009-02-01

    Full Text Available Proteins are active, flexible machines that perform a range of different functions. Innovative experimental approaches may now provide limited partial information about conformational changes along motion pathways of proteins. There is therefore a need for computational approaches that can efficiently incorporate prior information into motion prediction schemes. In this paper, we present PathRover, a general setup designed for the integration of prior information into the motion planning algorithm of rapidly exploring random trees (RRT. Each suggested motion pathway comprises a sequence of low-energy clash-free conformations that satisfy an arbitrary number of prior information constraints. These constraints can be derived from experimental data or from expert intuition about the motion. The incorporation of prior information is very straightforward and significantly narrows down the vast search in the typically high-dimensional conformational space, leading to dramatic reduction in running time. To allow the use of state-of-the-art energy functions and conformational sampling, we have integrated this framework into Rosetta, an accurate protocol for diverse types of structural modeling. The suggested framework can serve as an effective complementary tool for molecular dynamics, Normal Mode Analysis, and other prevalent techniques for predicting motion in proteins. We applied our framework to three different model systems. We show that a limited set of experimentally motivated constraints may effectively bias the simulations toward diverse predicates in an outright fashion, from distance constraints to enforcement of loop closure. In particular, our analysis sheds light on mechanisms of protein domain swapping and on the role of different residues in the motion.

  13. Molecular Typing of Treponema pallidum in Denmark

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Cowan, Susan; Gerstoft, Jan

    2016-01-01

    The aim of this nationwide study is to determine the strain type diversity among patients diagnosed with syphilis by PCR during a 4-year period in Denmark. Epidemiological data, including HIV status, for all patients were obtained from the Danish national syphilis registration system. Molecular......%) of the patients reported acquiring syphilis in Denmark. Among patients with concurrent HIV, 9 full strain types were identified and no difference in strain type was found by HIV status (p = 0.197). In conclusion, the majority of patients were infected in Denmark and the HIV-infected syphilis patients were...

  14. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

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    Tongjit Thanchomnang

    Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

  15. Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

    Science.gov (United States)

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

  16. Molecular typing of Chinese Streptococcus pyogenes isolates.

    Science.gov (United States)

    You, Yuanhai; Wang, Haibin; Bi, Zhenwang; Walker, Mark; Peng, Xianhui; Hu, Bin; Zhou, Haijian; Song, Yanyan; Tao, Xiaoxia; Kou, Zengqiang; Meng, Fanliang; Zhang, Menghan; Bi, Zhenqiang; Luo, Fengji; Zhang, Jianzhong

    2015-06-01

    Streptococcus pyogenes causes human infections ranging from mild pharyngitis and impetigo to serious diseases including necrotizing fasciitis and streptococcal toxic shock syndrome. The objective of this study was to compare molecular emm typing and pulsed field gel electrophoresis (PFGE) with multiple-locus variable-number tandem-repeat analysis (MLVA) for genotyping of Chinese S. pyogenes isolates. Molecular emm typing and PFGE were performed using standard protocols. Seven variable number tandem repeat (VNTR) loci reported in a previous study were used to genotype 169 S. pyogenes geographically-diverse isolates from China isolated from a variety of disease syndromes. Multiple-locus variable-number tandem-repeat analysis provided greater discrimination between isolates when compared to emm typing and PFGE. Removal of a single VNTR locus (Spy2) reduced the sensitivity by only 0.7%, which suggests that Spy2 was not informative for the isolates screened. The results presented support the use of MLVA as a powerful epidemiological tool for genotyping S. pyogenes clinical isolates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. A simple and rapid molecular method for Leptospira species identification

    NARCIS (Netherlands)

    Ahmed, Ahmed; Anthony, Richard M.; Hartskeerl, Rudy A.

    2010-01-01

    Serological and DNA-based classification systems only have little correlation. Currently serological and molecular methods for characterizing Leptospira are complex and costly restricting their world-wide distribution and use. Ligation mediated amplification combined with microarray analysis

  18. Molecular evolution of type VI intermediate filament proteins

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    Vincent Michel

    2007-09-01

    Full Text Available Abstract Background Tanabin, transitin and nestin are type VI intermediate filament (IF proteins that are developmentally regulated in frogs, birds and mammals, respectively. Tanabin is expressed in the growth cones of embryonic vertebrate neurons, whereas transitin and nestin are found in myogenic and neurogenic cells. Another type VI IF protein, synemin, is expressed in undifferentiated and mature muscle cells of birds and mammals. In addition to an IF-typical α-helical core domain, type VI IF proteins are characterized by a long C-terminal tail often containing distinct repeated motifs. The molecular evolution of type VI IF proteins remains poorly studied. Results To examine the evolutionary history of type VI IF proteins, sequence comparisons, BLAST searches, synteny studies and phylogenic analyses were performed. This study provides new evidence that tanabin, transitin and nestin are indeed orthologous type VI IF proteins. It demonstrates that tanabin, transitin and nestin genes share intron positions and sequence identities, have a similar chromosomal context and display closely related positions in phylogenic analyses. Despite this homology, fast evolution rates of their C-terminal extremity have caused the appearance of repeated motifs with distinct biological activities. In particular, our in silico and in vitro analyses of their tail domain have shown that (avian transitin, but not (mammalian nestin, contains a repeat domain displaying nucleotide hydrolysis activity. Conclusion These analyses of the evolutionary history of the IF proteins fit with a model in which type VI IFs form a branch distinct from NF proteins and are composed of two major proteins: synemin and nestin orthologs. Rapid evolution of the C-terminal extremity of nestin orthologs could be responsible for their divergent functions.

  19. Using current molecular techniques for rapid differentiation of ...

    African Journals Online (AJOL)

    Typhoid fever is responsible for the deaths of many people annually. However, conventional and timeconsuming detection methods for Salmonella Typhi still dominate. By using a molecular based approach, it was possible to identify Salmonella Typhi by amplifying two specific genes (viaB and tyv) and by using RFLP ...

  20. Introducing molecular selectivity in rapid impedimetric sensing of phthalates

    KAUST Repository

    Zia, Asif I.

    2014-05-01

    This research article reports a real-time and non-invasive detection technique for phthalates in liquids by Electrochemical Impedance Spectroscopy (EIS), incorporating molecular imprinting technique to introduce selectivity for the phthalate molecule in the detection system. A functional polymer with Bis (2-ethylhexyl) phthalate (DEHP) template was immobilized on the sensing surface of the inter-digital (ID) capacitive sensor with sputtered gold sensing electrodes fabricated over a native layer of silicon dioxide on a single crystal silicon substrate. Various concentrations (10 to 200 ppm) of DEHP in deionized MilliQ water were exposed to the sensor surface functionalized with molecular imprinted polymer (MIP) in order to capture the analyte molecule, hence introducing molecular selectivity to the testing system. Impedance spectra were obtained using EIS in order to determine sample conductance for evaluation of phthalate concentration in the solution. Electrochemical Spectrum Analyzer algorithm was used to deduce equivalent circuit and equivalent component parameters from the experimentally obtained impedance spectra employing Randle\\'s cell model curve fitting technique. Experimental results confirmed that the immobilization of the functional polymer on sensing surface introduces selectivity for phthalates in the sensing system. The results were validated by testing the samples using High Performance Liquid Chromatography (HPLC-DAD). © 2014 IEEE.

  1. Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay

    Science.gov (United States)

    Kamphee, Hatairat; Chaiprasert, Angkana; Prammananan, Therdsak; Wiriyachaiporn, Natpapas; Kanchanatavee, Airin; Dharakul, Tararaj

    2015-01-01

    Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection. PMID:26355296

  2. Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay.

    Directory of Open Access Journals (Sweden)

    Hatairat Kamphee

    Full Text Available Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance, while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance. The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb DNA control. The optimized conditions were validated with the H37Rv wild-type (WT Mtb isolate and Mtb isolates with known mutations (MT within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible and MT (drug resistant Mtb isolates, with the least limit of detection (LOD being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.

  3. Intraoperative Molecular Imaging for Rapid Assessment of Tumor Margins

    Science.gov (United States)

    2011-09-01

    approach in animal models using a the MRI- FMT imaging system (Task 5). A description of the primary accomplishments and ongoing efforts follows...guided fluorescence molecular tomography ( FMT ) of ( ) ( ) ( ) tBP k NTNTT etCBP kRktCRtC + − ∗    + ++= 12121 2 1 9 two fluorescent probes in...administration, mice were imaged for an hour at approximately two minutes per frame using an MR-coupled FMT system. The imaging system is a spectrometer

  4. Producing infectious enterovirus type 71 in a rapid strategy

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    Qin E-De

    2010-06-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 is an etiologic agent of hand-foot-and-mouth disease (HFMD, and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF, which is flanked by untranslated regions at both the 5' and 3' ends. Results A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice. Conclusions Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method.

  5. Surprisingly Rapid Orbital Evolution: A Compendium of Solar Type Binaries

    Science.gov (United States)

    Samec, Ronald George

    2015-08-01

    Solar type binaries are believed to be undergoing steady but slow angular momentum losses due to magnetic braking (Réville et al. 2015, Jiang et al. 2014) as stellar winds leave radially away on semi-rigid (out to the Alfvén radius) bipolar field lines: There is an outward radial flow of ions along the rotating magnetic fields. This is happening simultaneously as the gravitationally locked binary rotates about its center of mass. The stream of ions spiral outward resulting in a resistant torque, causing a decay in the orbital radius along with a period decrease due to Kepler’s laws. My past studies have included more than 25 binaries that appear to be undergoing magnetic braking. I have extended the number of systems to 75+ in this group by perusing the literature of modern precision synthetic light curve studies. Several interesting facts arise including their surprisingly rapid orbital evolution, much faster than would be suggested by the theory. Further results are presented in this study.

  6. Meselect – A rapid and effective method for the separation of the main leaf tissue types

    Directory of Open Access Journals (Sweden)

    Julia Svozil

    2016-11-01

    Full Text Available Individual tissues of complex eukaryotic organisms have specific gene expression programs that control their functions. Therefore, tissue-specific molecular information is required to increase our understanding of tissue-specific processes. Established methods in plants to obtain specific tissues or cell types from their organ or tissue context typically require the enzymatic degradation of cell walls followed by fluorescence-activated cell sorting (FACS using plants engineered for localized expression of green fluorescent protein (GFP. This has facilitated the acquisition of valuable data, mainly on root cell type-specific transcript and protein expression. However, FACS of different leaf cell types is difficult because of chlorophyll autofluorescence that interferes with the sorting process. Furthermore, the cell wall composition is different in each cell type. This results in long incubation times for refractory cell types, and cell sorting itself can take several hours. To overcome these limitations, we developed Meselect (mechanical separation of leaf compound tissues, a rapid and effective method for the separation of leaf epidermal, vascular and mesophyll tissues. Meselect is a novel combination of mechanical separation and rapid protoplasting, which benefits from the unique cell wall composition of the different tissue types. Meselect has several advantages over cell sorting: it does not require expensive equipment such as a cell sorter and does not depend on specific fluorescent reporter lines, the use of blenders as well as the inherent mixing of different cell types and of intact and damaged cells can be avoided, and the time between wounding of the leaf and freezing of the sample is short. The efficacy and specificity of the method to enrich the different leaf tissue types has been confirmed using Arabidopsis leaves, but it has also been successfully used for leaves of other plants such as tomato or cassava. The method is therefore

  7. Detection of Streptococcus pyogenes using rapid visual molecular assay.

    Science.gov (United States)

    Zhao, Xiangna; He, Xiaoming; Li, Huan; Zhao, Jiangtao; Huang, Simo; Liu, Wei; Wei, Xiao; Ding, Yiwei; Wang, Zhaoyan; Zou, Dayang; Wang, Xuesong; Dong, Derong; Yang, Zhan; Yan, Xiabei; Huang, Liuyu; Du, Shuangkui; Yuan, Jing

    2015-09-01

    Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. An Extended Multilocus Sequence Typing (MLST Scheme for Rapid Direct Typing of Leptospira from Clinical Samples.

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    Sabrina Weiss

    2016-09-01

    Full Text Available Rapid typing of Leptospira is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST data direct from patient specimens while minimising costs for subsequent sequencing.An existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK and the Lao Peoples Democratic Republic (Lao PDR. Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine PCR positive for pathogenic Leptospira spp. at least one allele was amplified in 22 samples (50% and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%. No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25% and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was L. interrogans (10/14 and 7/8 from UK and Lao PDR, respectively. All other species were detected in samples from only one country (Lao PDR: L. borgpetersenii [1/8]; UK: L. kirschneri [1/14], L. santarosai [1/14], L. weilii [2/14].Typing information of pathogenic Leptospira spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.

  9. Rapid Prototyping of Chemical Microsensors Based on Molecularly Imprinted Polymers Synthesized by Two-Photon Stereolithography.

    Science.gov (United States)

    Gomez, Laura Piedad Chia; Spangenberg, Arnaud; Ton, Xuan-Anh; Fuchs, Yannick; Bokeloh, Frank; Malval, Jean-Pierre; Tse Sum Bui, Bernadette; Thuau, Damien; Ayela, Cédric; Haupt, Karsten; Soppera, Olivier

    2016-07-01

    Two-photon stereolithography is used for rapid prototyping of submicrometre molecularly imprinted polymer-based 3D structures. The structures are evaluated as chemical sensing elements and their specific recognition properties for target molecules are confirmed. The 3D design capability is exploited and highlighted through the fabrication of an all-organic molecularly imprinted polymeric microelectromechanical sensor. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Click-Chemistry-Mediated Rapid Microbubble Capture for Acute Thrombus Ultrasound Molecular Imaging.

    Science.gov (United States)

    Wang, Tuantuan; Yuan, Chuxiao; Dai, Bingyang; Liu, Yang; Li, Mingxi; Feng, Zhenqiang; Jiang, Qing; Xu, Zhihong; Zhao, Ningwei; Gu, Ning; Yang, Fang

    2017-07-18

    Bioorthogonal coupling chemistry has been studied as a potentially advantageous approach for molecular imaging because it offers rapid, efficient, and strong binding, which might also benefit stability, production, and chemical conjugation. The inverse-electron-demand Diels-Alder reaction between a 1,2,4,5-tetrazine and trans-cyclooctene (TCO) is an example of a highly selective and rapid bioorthogonal coupling reaction that has been used successfully to prepare targeted molecular imaging probes. Here we report a fast, reliable, and highly sensitive approach, based on a two-step pretargeting bioorthogonal approach, to achieving activated-platelet-specific CD62p-targeted thrombus ultrasound molecular imaging. Tetrazine-modified microbubbles (tetra-MBs) could be uniquely and rapidly captured by subsequent click chemistry of thrombus tagged with a trans-cyclooctene-pretreated CD62p antibody. Moreover, such tetra-MBs showed great long-term stability under physiological conditions, thus offering the ability to monitor thrombus changes in real time. We demonstrated for the first time that a bioorthogonal targeting molecular ultrasound imaging strategy based on tetra-MBs could be a simple but powerful tool for rapid diagnosis of acute thrombosis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Nanoscale tailor-made membranes for precise and rapid molecular sieve separation.

    Science.gov (United States)

    Wang, Jing; Zhu, Junyong; Zhang, Yatao; Liu, Jindun; Van der Bruggen, Bart

    2017-03-02

    The precise and rapid separation of different molecules from aqueous, organic solutions and gas mixtures is critical to many technologies in the context of resource-saving and sustainable development. The strength of membrane-based technologies is well recognized and they are extensively applied as cost-effective, highly efficient separation techniques. Currently, empirical-based approaches, lacking an accurate nanoscale control, are used to prepare the most advanced membranes. In contrast, nanoscale control renders the membrane molecular specificity (sub-2 nm) necessary for efficient and rapid molecular separation. Therefore, as a growing trend in membrane technology, the field of nanoscale tailor-made membranes is highlighted in this review. An in-depth analysis of the latest advances in tailor-made membranes for precise and rapid molecule sieving is given, along with an outlook to future perspectives of such membranes. Special attention is paid to the established processing strategies, as well as the application of molecular dynamics (MD) simulation in nanoporous membrane design. This review will provide useful guidelines for future research in the development of nanoscale tailor-made membranes with a precise and rapid molecular sieve separation property.

  12. Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

    Science.gov (United States)

    Antonov, Valery A; Tkachenko, Galina A; Altukhova, Viktoriya V; Savchenko, Sergey S; Zinchenko, Olga V; Viktorov, Dmitry V; Zamaraev, Valery S; Ilyukhin, Vladimir I; Alekseev, Vladimir V

    2008-12-01

    Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

  13. Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips

    Science.gov (United States)

    2010-05-12

    Chlamydia (manuscript in preparation) and performed pilot studies on Staphylococcus aureus and Streptoccus pneumoniae (Data S4 and Text S2). Another potential...Found at: doi:10.1371/journal.pone.0010595.s009 (0.00 MB TXT) Text S2 mMLST sequencing of S. aureus and S. pneumoniae . Found at: doi:10.1371...provided S. aureus and S. pneumoniae strains. This publication made use of the Bacillus cereus Multi Locus Sequence Typing website (http://pubmlst.org

  14. Molecular integrals for exponential-type orbitals using hyperspherical harmonics

    DEFF Research Database (Denmark)

    Avery, James Emil; Avery, John Scales

    2015-01-01

    Exponential-type orbitals are better suited to calculations of molecular electronic structure than are Gaussians, since ETO's can accurately represent the behavior of molecular orbitals near to atomic nuclei, as well as their long-distance exponential decay. Orbitals based on Gaussians fail in both...... these respects. Nevertheless, Gaussian technology continues to dominate computational quantum chemistry, because of the ease with which difficult molecular integrals may be evaluated when Gaussians are used as a basis.In the present chapter, we hope to contribute to a new movement in quantum chemistry, in which......-dimensional hypersphere. Using this projection, Fock was able to show that the Fourier transforms of Coulomb Sturmian basis functions are very simply related to four-dimensional hyperspherical harmonics.With the help of Fock's relationships and the theory of hyperspherical harmonics we are able to evaluate molecular...

  15. Molecular diagnosis of multiple endocrine neoplasia type 2A ...

    African Journals Online (AJOL)

    Molecular diagnosis of multiple endocrine neoplasia type 2A. RJ Pegoraro, DJ Hacking, RH Buck, L Rom, PA Lanning, GMB Berger. Abstract. Objective. To identify by means of genetic analyses individuals who are at risk of developing medullary thyroid cancer that is a component of multiple endocrine neoplasia. Subjects.

  16. Acanthamoeba keratitis: improving the Scottish diagnostic service for the rapid molecular detection of Acanthamoeba species.

    Science.gov (United States)

    Alexander, Claire Low; Coyne, Michael; Jones, Brian; Anijeet, Deepa

    2015-07-01

    Acanthamoeba species are responsible for causing the potentially sight-threatening condition, Acanthamoeba keratitis, which is commonly associated with contact lens use. In this report, we highlight the challenges faced using conventional laboratory identification methods to identify this often under-reported pathogen, and discuss the reasons for introducing the first national service in Scotland for the rapid and sensitive molecular identification of Acanthamoeba species. By comparing culture and molecular testing data from a total of 63 patients (n = 80 samples) throughout Scotland presenting with ocular eye disease, we describe the improvement in detection rates where an additional four positive cases were identified using a molecular assay versus culture. The testing of a further ten patients by confocal imaging is also presented. This report emphasizes the importance of continuing to improve clinical laboratory services to ensure a prompt, correct diagnosis and better prognosis, in addition to raising awareness of this potentially debilitating opportunistic pathogen.

  17. Quantum state specific reactant preparation in a molecular beam by rapid adiabatic passage

    Energy Technology Data Exchange (ETDEWEB)

    Chadwick, Helen, E-mail: helen.chadwick@epfl.ch; Hundt, P. Morten; Reijzen, Maarten E. van; Yoder, Bruce L.; Beck, Rainer D. [Laboratoire de Chimie Physique Moléculaire, Ecole Polytechnique Fédérale de Lausanne, Lausanne (Switzerland)

    2014-01-21

    Highly efficient preparation of molecules in a specific rovibrationally excited state for gas/surface reactivity measurements is achieved in a molecular beam using tunable infrared (IR) radiation from a single mode continuous wave optical parametric oscillator (cw-OPO). We demonstrate that with appropriate focusing of the IR radiation, molecules in the molecular beam crossing the fixed frequency IR field experience a Doppler tuning that can be adjusted to achieve complete population inversion of a two-level system by rapid adiabatic passage (RAP). A room temperature pyroelectric detector is used to monitor the excited fraction in the molecular beam and the population inversion is detected and quantified using IR bleaching by a second IR-OPO. The second OPO is also used for complete population transfer to an overtone or combination vibration via double resonance excitation using two spatially separated RAP processes.

  18. Association of genotypes with infection types and antifungal susceptibilities in Candida albicans as revealed by recent molecular typing strategies

    Science.gov (United States)

    Bai, Feng-Yan

    2014-01-01

    Candida albicans is a commensal microorganism in the mucosa of healthy individuals, but is also the most common opportunistic fungal pathogen of humans. It causes from benign infections such as oral and vaginal candidiasis to fatal, systematic diseases in immunocompromised or critically ill patients. In addition to improved therapy, the rapid and accurate identification of the disease-causing strains is crucial for diagnosis, clinical treatment and epidemiological studies of candidiasis. A variety of methods for strain typing of C. albicans have been developed. The most commonly used methods with the focus on recently developed molecular typing or DNA-fingerprinting strategies and the recent findings in the association of specific and genetically similar genotypes with certain infection types and the correlation between azole susceptibilities and certain genotypes of C. albicans from China are reviewed. PMID:24772369

  19. Increased Sensitivity of a New Coagglutination Test for Rapid Identification of Haemophilus influenzae Type b

    OpenAIRE

    Grasso, Robert J.; West, Loyd A.; Holbrook, Nikki J.; Halkias, Demetrios G.; Paradise, Lois J.; Friedman, Herman

    1981-01-01

    A newly developed rapid coagglutination test for identifying Haemophilus influenzae type b organisms isolated from clinical specimens correlated 100% with the slide agglutination test but was 100- to 200-fold more sensitive.

  20. Multidrug-resistant tuberculosis: Rapid molecular detection with MTBDRplus® assay in clinical samples

    Directory of Open Access Journals (Sweden)

    Rita Macedo

    2009-05-01

    Full Text Available Nowadays, the greatest concern of tuberculosis control programmes is the appearance of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Rapid determination of drug resistance in clinical samples, with Mycobacterium tuberculosis complex (MTC, is the prerequisite for initiating effective chemotherapy, ensuring successful treatment of the patient and preventing further spread of drugresistant isolates.The aim of our study was to determine the sensitivity of the new MTBDRplus® assay in comparison to culture, identification and classic DST, directly from smear-positive clinical specimens.A total of 68 smear-positive sputum specimens were processed by both the classical mycobacteriological methods and the molecular assay, MTBDRplus®.MTBDRplus® assay allowed an accurate identification of MTC species by detection of the specific band in all samples, from which we also isolated and identified MTC strains by culture methods. In the samples from which we isolated susceptible strains (63.2%, wild type patterns were found using MTBDRplus® assay. The samples from which we isolated resistant strains (36.8% showed specific mutations associated with the correspondent resistant phenotype.Our study indicated that this assay allows rapid detection of resistance, always in agreement with classic methods. Resumo: Uma das principais problematicas no controlo da tuberculose e o aparecimento de casos de tuberculose multirresistente (TB-MR e tuberculose extensivamente resistente (TB-XDR. A deteccao precoce da resistencia a farmacos, directamente a partir de amostras respiratorias, e essencial para que se assegure o tratamento atempado, adequado e eficaz da tuberculose, bem como para prevenir a disseminacao destes casos de especial gravidade.O nosso objectivo foi avaliar a sensibilidade e comparar os resultados obtidos com um metodo de genetica molecular disponivel comercialmente – MTBDRplus® – e o isolamento

  1. Rapid Molecular detection of citrus brown spot disease using ACT gene in Alternaria alternata

    Directory of Open Access Journals (Sweden)

    Hamid Moghimi

    2017-06-01

    Full Text Available Introduction:Using rapid detection methods is important for detection of plant pathogens and also prevention through spreading pests in agriculture. Citrus brown spot disease caused by pathogenic isolates of Alternaria alternata is a common disease in Iran. Materials and methods: In this study, for the first time a PCR based molecular method was used for rapid diagnosis of brown spot disease. Nine isolates of A. Alternata were isolated in PDA medium from different citrus gardens. The plant pathogenic activity was examined in tangerine leaves for isolates. Results showed that these isolates are the agents of brown spot disease. PCR amplification of specific ACT-toxin gene was performed for DNA extracted from A. alternata isolates, with 11 different fungal isolates as negative controls and 5 DNA samples extracted from soil. Results: Results showed that A. alternata, the causal agent of brown spot disease, can be carefully distinguished from other pathogenic agents by performing PCR amplification with specific primers for ACT toxin gene. Also, the results from Nested-PCR method confirmed the primary reaction and the specificity of A. alternata for brown spot disease. PCR results to control samples of the other standard fungal isolates, showed no amplification band. In addition, PCR with the DNA extracted from contaminated soils confirmed the presence of ACT toxin gene. Discussion and conclusion: Molecular procedure presented here can be used in rapid identification and prevention of brown spot infection in citrus gardens all over the country.

  2. New and rapid procedure for the isolation of ultra-high molecular weight eukaryotic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Longmire, J.L.; Lewis, A.; Meincke, L.J.; Hildebrand, C.E.

    1986-05-01

    The authors have developed a novel procedure that permits the rapid extraction and isolation of ultra-high molecular weight DNA from avian or mammalian cells using dialysis against a solution of polyethylene glycol (PEG). Cells harvested by centrifugation and washed twice in ice-cold Ca/sup + +/- and Mg/sup + +/-free phosphate buffered saline were resuspended in 5 ml 0.01 M Tris-Cl (pH 8.0); 0.001 M EDTA (TE); sodium dodecyl sulfate and proteinase K were added to final concentrations of 0.1% and 0.1 mg/ml, respectively. After incubation at 37/sup 0/C overnight, the viscous solution was transferred to a mini-collodian bag and concentrated by dialysis against 4-5 changes of 20% PEG in TE over a period of 5 hours at RT. Concentrated samples were desalted by dialysis against fresh TE for two 15 minute intervals. DNA obtained using this procedure gives A/sub 260//A/sub 280/ consistently >1.8. Analysis of DNA size using pulsed field gel electrophoresis revealed a distribution of fragments >500 Kb in length. Further measurements examined were (1) restriction enzyme digestibility, (2) ligation efficiency of restricted DNA, and (3) cloning efficiency using the lambda vector Ch21A. This novel methodology offers a valuable alternative protocol for rapid purification of ultrahigh molecular weight DNA for various applications in molecular biology.

  3. A taxonomy of rapid reviews links report types and methods to specific decision-making contexts.

    Science.gov (United States)

    Hartling, Lisa; Guise, Jeanne-Marie; Kato, Elisabeth; Anderson, Johanna; Belinson, Suzanne; Berliner, Elise; Dryden, Donna M; Featherstone, Robin; Mitchell, Matthew D; Motu'apuaka, Makalapua; Noorani, Hussein; Paynter, Robin; Robinson, Karen A; Schoelles, Karen; Umscheid, Craig A; Whitlock, Evelyn

    2015-12-01

    Describe characteristics of rapid reviews and examine the impact of methodological variations on their reliability and validity. We conducted a literature review and interviews with organizations that produce rapid reviews or related products to identify methods, guidance, empiric evidence, and current practices. We identified 36 rapid products from 20 organizations (production time, 5 minutes to 8 months). Methods differed from systematic reviews at all stages. As time frames increased, methods became more rigorous; however, restrictions on database searching, inclusion criteria, data extracted, and independent dual review remained. We categorized rapid products based on extent of synthesis. "Inventories" list what evidence is available. "Rapid responses" present best available evidence with no formal synthesis. "Rapid reviews" synthesize the quality of and findings from the evidence. "Automated approaches" generate meta-analyses in response to user-defined queries. Rapid products rely on a close relationship with end users and support specific decisions in an identified time frame. Limited empiric evidence exists comparing rapid and systematic reviews. Rapid products have tremendous methodological variation; categorization based on time frame or type of synthesis reveals patterns. The similarity across rapid products lies in the close relationship with the end user to meet time-sensitive decision-making needs. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Ion torrent personal genome machine sequencing for genomic typing of Neisseria meningitidis for rapid determination of multiple layers of typing information.

    Science.gov (United States)

    Vogel, Ulrich; Szczepanowski, Rafael; Claus, Heike; Jünemann, Sebastian; Prior, Karola; Harmsen, Dag

    2012-06-01

    Neisseria meningitidis causes invasive meningococcal disease in infants, toddlers, and adolescents worldwide. DNA sequence-based typing, including multilocus sequence typing, analysis of genetic determinants of antibiotic resistance, and sequence typing of vaccine antigens, has become the standard for molecular epidemiology of the organism. However, PCR of multiple targets and consecutive Sanger sequencing provide logistic constraints to reference laboratories. Taking advantage of the recent development of benchtop next-generation sequencers (NGSs) and of BIGSdb, a database accommodating and analyzing genome sequence data, we therefore explored the feasibility and accuracy of Ion Torrent Personal Genome Machine (PGM) sequencing for genomic typing of meningococci. Three strains from a previous meningococcus serogroup B community outbreak were selected to compare conventional typing results with data generated by semiconductor chip-based sequencing. In addition, sequencing of the meningococcal type strain MC58 provided information about the general performance of the technology. The PGM technology generated sequence information for all target genes addressed. The results were 100% concordant with conventional typing results, with no further editing being necessary. In addition, the amount of typing information, i.e., nucleotides and target genes analyzed, could be substantially increased by the combined use of genome sequencing and BIGSdb compared to conventional methods. In the near future, affordable and fast benchtop NGS machines like the PGM might enable reference laboratories to switch to genomic typing on a routine basis. This will reduce workloads and rapidly provide information for laboratory surveillance, outbreak investigation, assessment of vaccine preventability, and antibiotic resistance gene monitoring.

  5. Rapid microbial mineralization of toluene and 1,3-dimethylbenzene in the absence of molecular oxygen.

    Science.gov (United States)

    Zeyer, J; Kuhn, E P; Schwarzenbach, R P

    1986-10-01

    Up to 0.4 mM 1,3-dimethylbenzene (m-xylene) was rapidly mineralized in a laboratory aquifer column operated in the absence of molecular oxygen with nitrate as an electron acceptor. Under continuous flow conditions, the degradation rate constant (pseudo-first order) was >0.45 h. Based on a carbon mass balance with [ring-C]m-xylene and a calculation of the electron balance, m-xylene was shown to be quantitatively (80%) oxidized to CO(2) with a concomitant reduction of nitrate. The mineralization of m-xylene in the column also took place after reducing the redox potential, E', of the inflowing medium with sulfide to sediments, sludge digestors, and groundwater infiltration zones from landfills and polluted rivers are not necessarily persistent but may be mineralized in the absence of molecular oxygen.

  6. Microbiological diagnosis and molecular typing of Legionella strains during an outbreak of legionellosis in Southern Germany.

    Science.gov (United States)

    Essig, Andreas; von Baum, Heike; Gonser, Theodor; Haerter, Georg; Lück, Christian

    2016-02-01

    An explosive outbreak of Legionnaires' disease with 64 reported cases occurred in Ulm/Neu-Ulm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 min reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An Atlas of the Molecular Types

    Directory of Open Access Journals (Sweden)

    Massimo Cogliati

    2013-01-01

    Full Text Available Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. The main etiological agents of cryptococcosis are the two sibling species Cryptococcus neoformans and Cryptococcus gattii that present numerous differences in geographical distribution, ecological niches, epidemiology, pathobiology, clinical presentation and molecular characters. Genotyping of the two Cryptococcus species at subspecies level supplies relevant information to understand how this fungus has spread worldwide, the nature of its population structure, and how it evolved to be a deadly pathogen. At present, nine major molecular types have been recognized: VNI, VNII, VNB, VNIII, and VNIV among C. neoformans isolates, and VGI, VGII, VGIII, and VGIV among C. gattii isolates. In this paper all the information available in the literature concerning the isolation of the two Cryptococcus species has been collected and analyzed on the basis of their geographical origin, source of isolation, level of identification, species, and molecular type. A detailed analysis of the geographical distribution of the major molecular types in each continent has been described and represented on thematic maps. This study represents a useful tool to start new epidemiological surveys on the basis of the present knowledge.

  8. Molecular Typing of Neisseria gonorrhoeae Isolates by Opa-Typing and Ribotyping in New Delhi, India

    Directory of Open Access Journals (Sweden)

    Pejvak Khaki

    2009-01-01

    Full Text Available Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of N. gonorrhoeae isolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes, AvaII and HincII, and opa-typing with TaqI and HpaII for epidemiological characterization of gonococcal population. Ribotyping with AvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed with HincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed with TaqI-digested PCR product while opa-typing with HpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9% between epidemiologically unrelated isolates. In conclusion, the molecular methods such as opa-typing and ribotyping can be used for epidemiological characterization of gonococcal strains.

  9. MATCH: An Atom- Typing Toolset for Molecular Mechanics Force Fields

    Science.gov (United States)

    Yesselman, Joseph D.; Price, Daniel J.; Knight, Jennifer L.; Brooks, Charles L.

    2011-01-01

    We introduce a toolset of program libraries collectively titled MATCH (Multipurpose Atom-Typer for CHARMM) for the automated assignment of atom types and force field parameters for molecular mechanics simulation of organic molecules. The toolset includes utilities for the conversion from multiple chemical structure file formats into a molecular graph. A general chemical pattern-matching engine using this graph has been implemented whereby assignment of molecular mechanics atom types, charges and force field parameters is achieved by comparison against a customizable list of chemical fragments. While initially designed to complement the CHARMM simulation package and force fields by generating the necessary input topology and atom-type data files, MATCH can be expanded to any force field and program, and has core functionality that makes it extendable to other applications such as fragment-based property prediction. In the present work, we demonstrate the accurate construction of atomic parameters of molecules within each force field included in CHARMM36 through exhaustive cross validation studies illustrating that bond increment rules derived from one force field can be transferred to another. In addition, using leave-one-out substitution it is shown that it is also possible to substitute missing intra and intermolecular parameters with ones included in a force field to complete the parameterization of novel molecules. Finally, to demonstrate the robustness of MATCH and the coverage of chemical space offered by the recent CHARMM CGENFF force field (Vanommeslaeghe, et al., JCC., 2010, 31, 671–690), one million molecules from the PubChem database of small molecules are typed, parameterized and minimized. PMID:22042689

  10. Fabrication of Polymerase Chain Reaction Plastic Lab-on-a-Chip Device for Rapid Molecular Diagnoses.

    Science.gov (United States)

    Trinh, Kieu The Loan; Zhang, Hainan; Kang, Dong-Jin; Kahng, Sung-Hyun; Tall, Ben D; Lee, Nae Yoon

    2016-05-01

    We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately.

  11. Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samples.

    Science.gov (United States)

    Northcott, Paul A; Shih, David J H; Remke, Marc; Cho, Yoon-Jae; Kool, Marcel; Hawkins, Cynthia; Eberhart, Charles G; Dubuc, Adrian; Guettouche, Toumy; Cardentey, Yoslayma; Bouffet, Eric; Pomeroy, Scott L; Marra, Marco; Malkin, David; Rutka, James T; Korshunov, Andrey; Pfister, Stefan; Taylor, Michael D

    2012-04-01

    The diagnosis of medulloblastoma likely encompasses several distinct entities, with recent evidence for the existence of at least four unique molecular subgroups that exhibit distinct genetic, transcriptional, demographic, and clinical features. Assignment of molecular subgroup through routine profiling of high-quality RNA on expression microarrays is likely impractical in the clinical setting. The planning and execution of medulloblastoma clinical trials that stratify by subgroup, or which are targeted to a specific subgroup requires technologies that can be economically, rapidly, reliably, and reproducibly applied to formalin-fixed paraffin embedded (FFPE) specimens. In the current study, we have developed an assay that accurately measures the expression level of 22 medulloblastoma subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology. Comparison of the nanoString assay with Affymetrix expression array data on a training series of 101 medulloblastomas of known subgroup demonstrated a high concordance (Pearson correlation r = 0.86). The assay was validated on a second set of 130 non-overlapping medulloblastomas of known subgroup, correctly assigning 98% (127/130) of tumors to the appropriate subgroup. Reproducibility was demonstrated by repeating the assay in three independent laboratories in Canada, the United States, and Switzerland. Finally, the nanoString assay could confidently predict subgroup in 88% of recent FFPE cases, of which 100% had accurate subgroup assignment. We present an assay based on nanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.

  12. Molecular simulation and experimental studies of a mesoporous ZSM-5 type molecular sieve.

    Science.gov (United States)

    Liu, Baoyu; Wu, Yongbiao; Liu, Defei; Wu, Ying; Xi, Hongxia; Qian, Yu

    2013-02-28

    The mesoporous zeolite is a novel porous material possessing mesopores as well as the inherent micropores of zeolites. This material can exhibit the dual merits of two different pore structures and enable zeolites to have maximum structural functions. During the past few decades, various synthetic strategies have been well developed. However, up to now, there has only been a few attempts to model mesoporous zeolites. In this paper, the structural properties of a mesoporous ZSM-5 type molecular sieve, which has mesopore walls that are made up of ZSM-5 zeolite-like frameworks, were studied using an atomistic model. The full-atom model of the mesoporous ZSM-5 type molecular sieve was constructed using a molecular modeling technique. The structure model was characterized by estimating the nitrogen accessible solvent surface area, small-angle and wide-angle X-ray diffraction patterns, toluene and benzene adsorption. It was found that these simulated results match well with the experimental data. Furthermore, the present approach can be extended to construct other micro-mesoporous molecular sieve structure models in the future.

  13. Longitudinal analysis of the temporal evolution of Acinetobacter baumannii strains in Ohio, USA, by using rapid automated typing methods.

    Directory of Open Access Journals (Sweden)

    Brooke K Decker

    Full Text Available Genotyping methods are essential to understand the transmission dynamics of Acinetobacter baumannii. We examined the representative genotypes of A. baumannii at different time periods in select locations in Ohio, using two rapid automated typing methods: PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS, a form of multi-locus sequence typing (MLST, and repetitive-sequence-based-PCR (rep-PCR. Our analysis included 122 isolates from 4 referral hospital systems, in 2 urban areas of Ohio. These isolates were associated with outbreaks at 3 different time periods (1996, 2000 and 2005-2007. Type assignments of PCR/ESI-MS and rep-PCR were compared to each other and to worldwide (WW clone types. The discriminatory power of each method was determined using the Simpson's index of diversity (DI. We observed that PCR/ESI-MS sequence type (ST 14, corresponding to WW clone 3, predominated in 1996, whereas ST 12 and 14 co-existed in the intermediate period (2000 and ST 10 and 12, belonging to WW clone 2, predominated more recently in 2007. The shift from WW clone 3 to WW clone 2 was accompanied by an increase in carbapenem resistance. The DI was approximately 0.74 for PCR/ESI-MS, 0.88 for rep-PCR and 0.90 for the combination of both typing methods. We conclude that combining rapid automated typing methods such as PCR/ESI-MS and rep-PCR serves to optimally characterize the regional molecular epidemiology of A. baumannii. Our data also sheds light on the changing sequence types in an 11 year period in Northeast Ohio.

  14. Application of Molecular Cytogenetic Technique for Rapid Prenatal Diagnosis of Aneuploidies in Iranian Population

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    Habib Nasiri

    2009-06-01

    Full Text Available Objective: Classic cell culture and karyotyping is routinely used for prenatal detection of different chromosomal abnormalities. Molecular cytogenetic techniques have also recently been developed and used for this purpose. Quantitative florescence PCR using short tandem repeat (STR markers has more potential for high throughput diagnosis. Marker heterozygosity in short tandem repeats (STR is of critical importance in the clinical applicablity of this method. Materials and Methods: Different STR markers on chromosomes 13, 18, 21, X and Y  were analysed from  amniotic samples to detect related disorders such as Down, Edward, Patau,  Klinefelter sundromes , as well as sex chromosomes numerical abnormalities . Results: In our population some markers (D18S976, DXS6854, D21S11, and D21S1411 showed alleles with sizes out of expected ranges. But others occupied narrower range of predicted distribution. Most markers have enough heterozygosity (66.3-94.7 to be used for prenatal diagnosis. Furthermore, results obtained from full karyotype for all samples were in concordance with results of molecular cytogenetic testing. Conclusion: It is concluded that, in urgent situations, if proper markers used, molecular cytogenetic testing (QF-PCR could be a useful method for rapid prenatal diagnosis (PND in populations with high rate of consanguinity such as Iran.  

  15. Using a new molecular genetic of genotype and liquid culture medium for rapid diagnosis tb

    Directory of Open Access Journals (Sweden)

    Ганна Іванівна Барбова

    2015-10-01

    Full Text Available This paper presents the results of molecular genetic test system GenoType multyresistentens MTBDRplus. It was established that the presence of mutations associated with resistance to isoniazid, only 93.1 % of cases of MBT to isoniazid during the test in a liquid medium. Work carried out under the National Programme to combat tuberculosisMaterials and methods. We investigated the clinical sputum samples from patients with pulmonary tuberculosis. The applied system GenoType. Principle DNA strip technology GenoType is that the DNA-coated strip specific test that are complementary to the derived PCR amplicon. After the single-stranded amplicon denaturation associated with tests on strip (hybridize, and visualized in a sequential enzymatic reaction with streptavydynom and alkaline phosphatase. Evaluation of hybridization is performed automatically. For culturing sputum liquid culture medium used - Middlebrook broth 7N9 VASTES MGIT system.Results and discussion. The results of molecular genetic studies of samples of sputum-concentrated and concentrated by a system GenoType not differed (P>0.05. Diagnostic value of two methods (molecular and genetic – system GenoType and phenotype – VASTES MGIT 960 system was very high (100%. Two systems have tested positive in the study 756 (95.5 % Mycobacterium strains that were identified in the system VASTES MGIT 960, formed Cord Factor and the results were positive identification test ID MTB MGIT they attributed to Mycobacterium tuberculosis complex. 36 (4.5 % samples from positive MGIT tubes were negative. As a result of molecular-genetic identification of nontuberculous mycobacteria complex it was found that 18 (2.3 % strains of mycobacteria belonging to the M. avium-intracellulare, 12 (1.5 % mycobacterial cultures were attributed to M. kansasii, 6 (0, 7 % cultures were identified as M. fortuitum. The results of the molecular study of MS on Mycobacterium resistance profile INN + RIF coincided in 95.5 % (894

  16. wzi Gene Sequencing, a Rapid Method for Determination of Capsular Type for Klebsiella Strains

    Science.gov (United States)

    Passet, Virginie; Haugaard, Anita Björk; Babosan, Anamaria; Kassis-Chikhani, Najiby; Struve, Carsten; Decré, Dominique

    2013-01-01

    Pathogens of the genus Klebsiella have been classified into distinct capsular (K) types for nearly a century. K typing of Klebsiella species still has important applications in epidemiology and clinical microbiology, but the serological method has strong practical limitations. Our objective was to evaluate the sequencing of wzi, a gene conserved in all capsular types of Klebsiella pneumoniae that codes for an outer membrane protein involved in capsule attachment to the cell surface, as a simple and rapid method for the prediction of K type. The sequencing of a 447-nucleotide region of wzi distinguished the K-type reference strains with only nine exceptions. A reference wzi sequence database was created by the inclusion of multiple strains representing K types associated with high virulence and multidrug resistance. A collection of 119 prospective clinical isolates of K. pneumoniae were then analyzed in parallel by wzi sequencing and classical K typing. Whereas K typing achieved typeability for 81% and discrimination for 94.4% of the isolates, these figures were 98.1% and 98.3%, respectively, for wzi sequencing. The prediction of K type once the wzi allele was known was 94%. wzi sequencing is a rapid and simple method for the determination of the K types of most K. pneumoniae clinical isolates. PMID:24088853

  17. Molecular typing of Trichophyton rubrum clinical isolates from Poland.

    Science.gov (United States)

    Hryncewicz-Gwóźdź, Anita; Jagielski, Tomasz; Sadakierska-Chudy, Anna; Dyląg, Mariusz; Pawlik, Krzysztof; Baran, Eugeniusz; Szepietowski, Jacek C

    2011-11-01

    The aim of this study was to investigate the intraspecific diversity of Trichophyton rubrum clinical isolates. Thirty clinical isolates of T. rubrum were selected for molecular typing by PCR amplification of two tandemly repetitive elements (TRS-1 and TRS-2) of the rDNA and randomly amplified polymorphic DNA (RAPD) analysis with primers designated 1 and 6. The assignment to the species T. rubrum was achieved by nested PCR of ITS1. Five PCR types were produced from the TRS-1 and three from the TRS-2 locus. Thirteen and 23 individual profiles were obtained by RAPD, with primer 1 and 6 respectively. At the phylogenetic level, 26 (87%) isolates were allocated into four clusters, with each cluster comprising isolates of over 80% similarity. The reproducibility of TRS typing was 100%, whereas that of RAPD was 40% and 30%, when using primer 1 and 6 respectively. Neither correlation between the morphological characteristics and the TRS-1-TRS-2 or RAPD genotype nor between TRS-1-TRS-2 and RAPD genotyping was observed. Although both the TRS amplification and RAPD analysis possess the ability to discriminate between T. rubrum strains, the TRS typing method is particularly valuable as its results are much more reproducible, more easily interpreted and recorded than those generated by RAPD. © 2011 Blackwell Verlag GmbH.

  18. Comparison of various molecular methods for rapid differentiation of intestinal bifidobacteria at the species, subspecies and strain level.

    Science.gov (United States)

    Jarocki, Piotr; Podleśny, Marcin; Komoń-Janczara, Elwira; Kucharska, Jagoda; Glibowska, Agnieszka; Targoński, Zdzisław

    2016-07-22

    Members of the genus Bifidobacterium are anaerobic Gram-positive Actinobacteria, which are natural inhabitants of human and animal gastrointestinal tract. Certain bifidobacteria are frequently used as food additives and probiotic pharmaceuticals, because of their various health-promoting properties. Due to the enormous demand on probiotic bacteria, manufacture of high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. Additionally, isolation of new industrial bacteria from various environments may lead to multiple isolations of the same strain, therefore, it is important to apply rapid, low-cost and effective procedures differentiating bifidobacteria at the intra-species level. The identification of new isolates using microbiological and biochemical methods is difficult, but the accurate characterization of isolated strains may be achieved using a polyphasic approach that includes classical phenotypic methods and molecular procedures. However, some of these procedures are time-consuming and cumbersome, particularly when a large group of new isolates is typed, while some other approaches may have too low discriminatory power to distinguish closely related isolates obtained from similar sources. This work presents the evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level. Our experiments included 17 reference strains and showed that in comparison to ARDRA, genotypic fingerprinting procedures (RAPD and rep-PCR) seemed to be less reproducible, however, they allowed to differentiate the tested microorganisms even at the intra-species level. In general, RAPD and rep-PCR have similar discriminatory power, though, in some instances more than one oligonucleotide needs to be used in random amplified polymorphic DNA analysis. Moreover, the results also

  19. A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

    Science.gov (United States)

    Saroj, Dina B; Dengeti, Shrinivas N; Aher, Supriya; Gupta, Anil K

    2015-06-01

    Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.

  20. Molecular dynamics simulation studies of structural and dynamical properties of rapidly quenched Al

    Energy Technology Data Exchange (ETDEWEB)

    Shen, B.; Liu, C. Y.; Jia, Y.; Yue, G. Q.; Ke, F. S.; Zhao, H. B.; Chen, L. Y.; Wang, S. Y.; Wang, C. Z.; Ho, K. M.

    2014-01-01

    The structural and dynamical properties of rapidly quenched Al are studied by molecular dynamics simulations. The pair-correlation function of high temperature liquid Al agrees well with the experimental results. Different cooling rates are applied with high cooling rates leading to glass formation, while low cooling rates leading to crystallization. The local structures are characterized by Honeycutt–Andersen indices and Voronoi tessellation analysis. The results show that for high cooling rates, the local structures of the liquid and glassy Al are predominated by icosahedral clusters, together with considerable amount of face-centered cubic and hexagonal close packed short-range orders. These short-range order results are further confirmed using the recently developed atomic cluster alignment method. Moreover, the atomic cluster alignment clearly shows the crystal nucleation process in supercooled liquid of Al. Finally, the mean square displacement for the liquid is also analyzed, and the corresponding diffusion coefficient as a function of temperature is calculated.

  1. Fabrication of Polymerase Chain Reaction Plastic Lab-on-a-Chip Device for Rapid Molecular Diagnoses

    Science.gov (United States)

    2016-01-01

    Purpose: We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. Methods: A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. Results: A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. Conclusions: In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately. PMID:27230459

  2. Nanotools and molecular techniques to rapidly identify and fight bacterial infections.

    Science.gov (United States)

    Dinarelli, S; Girasole, M; Kasas, S; Longo, G

    2017-07-01

    Reducing the emergence and spread of antibiotic-resistant bacteria is one of the major healthcare issues of our century. In addition to the increased mortality, infections caused by multi-resistant bacteria drastically enhance the healthcare costs, mainly because of the longer duration of illness and treatment. While in the last 20years, bacterial identification has been revolutionized by the introduction of new molecular techniques, the current phenotypic techniques to determine the susceptibilities of common Gram-positive and Gram-negative bacteria require at least two days from collection of clinical samples. Therefore, there is an urgent need for the development of new technologies to determine rapidly drug susceptibility in bacteria and to achieve faster diagnoses. These techniques would also lead to a better understanding of the mechanisms that lead to the insurgence of the resistance, greatly helping the quest for new antibacterial systems and drugs. In this review, we describe some of the tools most currently used in clinical and microbiological research to study bacteria and to address the challenge of infections. We discuss the most interesting advancements in the molecular susceptibility testing systems, with a particular focus on the many applications of the MALDI-TOF MS system. In the field of the phenotypic characterization protocols, we detail some of the most promising semi-automated commercial systems and we focus on some emerging developments in the field of nanomechanical sensors, which constitute a step towards the development of rapid and affordable point-of-care testing devices and techniques. While there is still no innovative technique that is capable of completely substituting for the conventional protocols and clinical practices, many exciting new experimental setups and tools could constitute the basis of the standard testing package of future microbiological tests. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Cost and Impact on Patient Length of Stay of Rapid Molecular Testing for Clostridium difficile.

    Science.gov (United States)

    Sewell, Bernadette; Rees, Eugene; Thomas, Ian; Ch'ng, Chin Lye; Isaac, Mike; Berry, Nidhika

    2014-12-01

    A study was performed to assess the cost of a rapid molecular assay (PCR) for diagnosis of Clostridium difficile infection (CDI) and the impact of its routine use on patient length of stay (LOS) in comparison with cell culture cytotoxin neutralization assay (CCNA). From March 2011 to September 2011, Xpert(®) C. difficile (Cepheid, Sunnyvale, CA, USA) PCR was used on patients with suspicion of CDI in two acute care hospitals in Abertawe Bro Morgannwg University Health Board, Swansea, Wales, UK. Test results were used for patient management. LOS and time to reportable result were compared for negative and positive prospective patients tested by PCR and historic control patients tested by CCNA during March 2010 to September 2010. Tests were priced using micro-costing and a cost comparison analysis was undertaken. In total, 506 patients were included. Time to reportable result for PCR samples was 1.53 h compared to 46.54 h for CCNA negatives and 22.45 h for CCNA positives. Patients tested by CCNA stayed 4.88 days longer in hospital compared to PCR patients if they tested positive and 7.03 days if tests were negative. The mean reduction in LOS observed in our study has the potential to generate cost savings of up to £2,292.62 for every patient with suspected CDI, if samples were to be tested routinely with PCR instead of CCNA. A rapid molecular test for C. difficile in an acute hospital setting produced quick results that led to a decrease in LOS compared to historic CCNA control patients. This could result in considerable savings through reduced excess inpatient days.

  4. Using ultra-rapid insulin analogs in children and adolescents with type 1 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    О.V. Bolshova

    2017-11-01

    Full Text Available Background. The purpose of the study was a retrospective comparative analysis of using insulin analogues of the prolonged and ultra-short action and human genetically engineered insulins of middle and short action in children and adolescents with type 1 diabetes mellitus (DM. Materials and methods. The influence of ultra-rapid insulin analog in comparison with human rapid-action insulin on the course of type 1 DM in 100 children and adolescents was studied. It was applied as basal-bolus regimen of insulin therapy. Analysis of parameters which reflect criteria of insulin therapy effectiveness, positive effect of ultra-rapid insulin analog on the course of DM has been performed. Results. Application of ultra-rapid insulin analog before each meal improved parameters of pre- and postprandial glycemia, decreased the range of fluctuations of blood sugar during the day, reduced and maintained HbA1c level without augmentation of frequency and intensity of hypoglycaemia, and also decreased the level of noctural hypoglycaemia. Conclusions. The ultra-rapid insulin analog is the drug of choice for the effective use in insulin pumps.

  5. The Use of MoStBioDat for Rapid Screening of Molecular Diversity

    Directory of Open Access Journals (Sweden)

    Agata Kurczyk

    2009-09-01

    Full Text Available MoStBioDat is a uniform data storage and extraction system with an extensive array of tools for structural similarity measures and pattern matching which is essential to facilitate the drug discovery process. Structure-based database screening has recently become a common and efficient technique in early stages of the drug development, shifting the emphasis from rational drug design into the probability domain of more or less random discovery. The virtual ligand screening (VLS, an approach based on high-throughput flexible docking, samples a virtually infinite molecular diversity of chemical libraries increasing the concentration of molecules with high binding affinity. The rapid process of subsequent examination of a large number of molecules in order to optimize the molecular diversity is an attractive alternative to the traditional methods of lead discovery. This paper presents the application of the MoStBioDat package not only as a data management platform but mainly in substructure searching. In particular, examples of the applications of MoStBioDat are discussed and analyzed.

  6. Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety.

    Science.gov (United States)

    Mangal, Manisha; Bansal, Sangita; Sharma, Satish K; Gupta, Ram K

    2016-07-03

    Food safety is a global health concern. For the prevention and recognition of problems related to health and safety, detection of foodborne pathogen is of utmost importance at all levels of food production chain. For several decades, a lot of research has been targeted at the development of rapid methodology as reducing the time needed to complete pathogen detection tests has been the primary goal of food microbiologists. With the result, food microbiology laboratories now have a wide array of detection methods and automated technologies such as enzyme immunoassay, polymerase chain reaction, and microarrays, which can cut test times considerably. Nucleic acid amplification strategies and advances in amplicon detection methodologies have been the key factors in the progress of molecular microbiology. A comprehensive literature survey has been carried out to give an overview in the field of foodborne pathogen detection. In this paper, we describe the conventional methods, as well as recent developments in food pathogen detection, identification, and quantification, with a major emphasis on molecular detection methods.

  7. Rapid Characterization of Molecular Chemistry, Nutrient Make-Up and Microlocation of Internal Seed Tissue

    Energy Technology Data Exchange (ETDEWEB)

    Yu,P.; Block, H.; Niu, Z.; Doiron, K.

    2007-01-01

    Wheat differs from corn in biodegradation kinetics and fermentation characteristics. Wheat exhibits a relatively high rate (23% h{sup 01}) and extent (78% DM) of biodegradation, which can lead to metabolic problems such as acidosis and bloat in ruminants. The objective of this study was to rapidly characterize the molecular chemistry of the internal structure of wheat (cv. AC Barrie) and reveal both its structural chemical make-up and nutrient component matrix by analyzing the intensity and spatial distribution of molecular functional groups within the intact seed using advanced synchrotron-powered Fourier transform infrared (FTIR) microspectroscopy. The experiment was performed at the U2B station of the National Synchrotron Light Source at Brookhaven National Laboratory, New York, USA. The wheat tissue was imaged systematically from the pericarp, seed coat, aleurone layer and endosperm under the peaks at {approx}1732 (carbonyl C{double_bond}O ester), 1515 (aromatic compound of lignin), 1650 (amide I), 1025 (non-structural CHO), 1550 (amide II), 1246 (cellulosic material), 1160, 1150, 1080, 930, 860 (all CHO), 3350 (OH and NH stretching), 2928 (CH{sub 2} stretching band) and 2885 cm{sup -1} (CH{sub 3} stretching band). Hierarchical cluster analysis and principal component analysis were applied to analyze the molecular FTIR spectra obtained from the different inherent structures within the intact wheat tissues. The results showed that, with synchrotron-powered FTIR microspectroscopy, images of the molecular chemistry of wheat could be generated at an ultra-spatial resolution. The features of aromatic lignin, structural and non-structural carbohydrates, as well as nutrient make-up and interactions in the seeds, could be revealed. Both principal component analysis and hierarchical cluster analysis methods are conclusive in showing that they can discriminate and classify the different inherent structures within the seed tissue. The wheat exhibited distinguishable

  8. A low complexity rapid molecular method for detection of Clostridium difficile in stool.

    Directory of Open Access Journals (Sweden)

    Cathal J McElgunn

    Full Text Available Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE. The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB. The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+, n = 111; tcdB(-, n= 107. The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+, n = 47; tcdB(-, n= 28 HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH, and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+, n = 25; tcdB(-, n= 2 were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

  9. Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

    Science.gov (United States)

    Kano, Rui; Okubo, Miki; Siew, Han Hock; Kamata, Hiroshi; Hasegawa, Atsuhiko

    2015-04-01

    Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia. © 2015 Blackwell Verlag GmbH.

  10. Molecular pathogenesis of long QT syndrome type 1

    Directory of Open Access Journals (Sweden)

    Jie Wu, PhD

    2016-10-01

    Full Text Available Long QT syndrome type 1 (LQT1 is a subtype of a congenital cardiac syndrome caused by mutation in the KCNQ1 gene, which encodes the α-subunit of the slow component of delayed rectifier K+ current (IKs channel. Arrhythmias in LQT1 are characterized by prolongation of the QT interval on ECG, as well as the occurrence of life-threatening cardiac events, frequently triggered by adrenergic stimuli (e.g., physical or emotional stress. During the past two decades, much advancement has been made in understanding the molecular pathogenesis underlying LQT1. Uncovering the genotype-phenotype correlations in LQT1 is of clinical importance to better understand the gene-specific differences that may influence the propensity for developing life-threatening arrhythmias under specific conditions. Elucidation of these mechanisms will also help to improve the diagnosis and management of this cardiac disorder based on gene-specific considerations. This review describes the current medical consensus and recent developments regarding the molecular pathogenesis of LQT1 and provides a novel insight into the adrenergic regulation of this disease.

  11. A Boussinesq-type method for fully nonlinear waves interacting with a rapidly varying bathymetry

    DEFF Research Database (Denmark)

    Madsen, Per A.; Fuhrman, David R.; Wang, Benlong

    2006-01-01

    New equations are derived for fully nonlinear and highly dispersive water waves interacting with a rapidly varying bathymetry. The derivation is an extension of a recent high order Boussinesq type formulation valid on a mildly sloping bottom. It is based on a series expansion from a rapidly...... locally deteriorate, and we provide a guideline for using this technique within acceptable accuracy bounds. Numerical results are given for the linear reflection from a plane shelf, a Gaussian shaped trench, and a symmetric trench with sloped transitions. Furthermore, we simulate the linear class I...

  12. Molecular Targeted Therapy Approaches for BRAF Wild-Type Melanoma.

    Science.gov (United States)

    Johnpulle, Romany A N; Johnson, Douglas B; Sosman, Jeffrey A

    2016-01-01

    Patients with metastatic melanoma have historically had dismal outcomes. The last several years has seen the emergence of effective immune and targeted therapies for metastatic melanoma. Targeted therapies have primarily impacted the 40-50% of patients with BRAF(V600) mutated melanoma. The remainder of patients with advanced melanoma harbor a wide spectrum of mutations other than BRAF(V600) that are associated with unique pathophysiological, prognostic, and therapeutic implications. The treatment of this subset of patients is a challenging problem. In recent years, preclinical and early clinical studies have suggested that inhibitors of mitogen activated protein kinase (MAPK) pathway and parallel signaling networks may have activity in treatment of BRAF(V600) wild-type (WT) melanoma. In this review, we will discuss available and developing therapies for BRAF WT patients with metastatic melanoma, particularly focusing on molecular targeted options for various genetically defined melanoma subsets.

  13. Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

    Science.gov (United States)

    V, Pavana Jyothi; S, Akila; Selvan, Malini K; Naidu, Hariprasad; Raghunathan, Shwethaa; Kota, Sathish; Sundaram, R C Raja; Rana, Samir Kumar; Raj, G Dhinakar; Srinivasan, V A; Mohana Subramanian, B

    2016-12-01

    Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing. Copyright © 2016. Published by Elsevier B.V.

  14. Application tests of a new-type LNG rapid gasification unit

    Directory of Open Access Journals (Sweden)

    Ping Yan

    2017-01-01

    Full Text Available Liquefied natural gas (LNG is stored under low temperature and high pressure. It has to be gasified before it is used. Therefore, LNG gasification unit is essential and it is vital to the high-efficiency utilization of LNG. In this paper, a new-type LNG rapid gasification unit was developed. Adopted in this unit are some innovative technologies authorized with the national patent of invention, such as the umbrella-shape gas flow circle unit, the flue gas circulation system and the water feeding system, which help to guarantee its operation safety and increase its operation efficiency. After it was justified in lab test, the unit for industrial application was designed and manufactured and then tested to verify its design rationality. The results show that the new-type LNG rapid gasification unit meets the design requirements in the aspect of efficiency, exhaust gas loss, radiation loss and fuel gas consumption rate; at a load of 1800–2200 m3/h, its efficiency is over 95%; at a load of 1976.0 m3/h which is close to the design value of 2000 m3/h, its efficiency is 96.34% or even up to 2800 m3/h. This new-type LNG rapid gasification unit is adaptable to a large range of loads and can adapt to the rapid increase of external load. Its fuel gas consumption rate is only 1.5%, which is in the range of energy conservation. It presents the advantages of high heating efficiency, rapid startup, high gasification rate, compact structure, small land occupation and invulnerability to the environment, therefore, it is applicable to the middle and small independent regions which cannot be connected to the natural gas supply pipeline networks due to various reasons.

  15. The rapidly evolving therapies for advanced melanoma--Towards immunotherapy, molecular targeted therapy, and beyond.

    Science.gov (United States)

    Zhu, Ziqiang; Liu, Wei; Gotlieb, Vladimir

    2016-03-01

    The incidence of melanoma in both males and females continues to rise during the past 40 years despite the stable or declining trends for most cancer types. Due to the tremendous advance in immunobiology and molecular biology, breakthroughs in both immunotherapies and molecular targeted therapies have recently revolutionized the standard of care for patients with advanced melanoma. In 2011, US Food and Drug Administration (FDA) approved ipilimumab, an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody for metastatic melanoma therapy. Since then, novel drugs including antibodies to programmed cell death 1 (PD-1) such as pembrolizumab and nivolumab (both approved in 2014), selective BRAF inhibitors such as vemurafenib (approved in 2011), dabrafenib (approved in 2013); and MEK inhibitor trametinib (approved in 2013), have greatly extended the potential of immunotherapy and molecular targeted therapy for advanced melanoma. All of which have been demonstrated a significant increase in overall survival rate, and long-term benefits in multiple large clinical trials. Several new agents and novel therapies are currently under phase III clinical trials with the hope of being approved in the near future. We already entered a golden era in oncology that are providing significant survival improvement. In the meantime, new challenges for clinicians also started to emerge. In this review, we presented the existing evidence for the newest treatments for advanced melanoma, including CTLA-4, PD-1/PD-L1 checkpoint inhibitors and BRAF, MEK inhibitors. We also discussed the strengths, limitations and challenges of using these novel therapies, and potential solutions as well as highlighted the areas requiring further research. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Meteorite Impact-Induced Rapid NH3 Production on Early Earth: Ab Initio Molecular Dynamics Simulation

    Science.gov (United States)

    Shimamura, Kohei; Shimojo, Fuyuki; Nakano, Aiichiro; Tanaka, Shigenori

    2016-12-01

    NH3 is an essential molecule as a nitrogen source for prebiotic amino acid syntheses such as the Strecker reaction. Previous shock experiments demonstrated that meteorite impacts on ancient oceans would have provided a considerable amount of NH3 from atmospheric N2 and oceanic H2O through reduction by meteoritic iron. However, specific production mechanisms remain unclear, and impact velocities employed in the experiments were substantially lower than typical impact velocities of meteorites on the early Earth. Here, to investigate the issues from the atomistic viewpoint, we performed multi-scale shock technique-based ab initio molecular dynamics simulations. The results revealed a rapid production of NH3 within several picoseconds after the shock, indicating that shocks with greater impact velocities would provide further increase in the yield of NH3. Meanwhile, the picosecond-order production makes one expect that the important nitrogen source precursors of amino acids were obtained immediately after the impact. It was also observed that the reduction of N2 proceeded according to an associative mechanism, rather than a dissociative mechanism as in the Haber-Bosch process.

  17. Meteorite Impact-Induced Rapid NH3 Production on Early Earth: Ab Initio Molecular Dynamics Simulation

    Science.gov (United States)

    Shimamura, Kohei; Shimojo, Fuyuki; Nakano, Aiichiro; Tanaka, Shigenori

    2016-01-01

    NH3 is an essential molecule as a nitrogen source for prebiotic amino acid syntheses such as the Strecker reaction. Previous shock experiments demonstrated that meteorite impacts on ancient oceans would have provided a considerable amount of NH3 from atmospheric N2 and oceanic H2O through reduction by meteoritic iron. However, specific production mechanisms remain unclear, and impact velocities employed in the experiments were substantially lower than typical impact velocities of meteorites on the early Earth. Here, to investigate the issues from the atomistic viewpoint, we performed multi-scale shock technique-based ab initio molecular dynamics simulations. The results revealed a rapid production of NH3 within several picoseconds after the shock, indicating that shocks with greater impact velocities would provide further increase in the yield of NH3. Meanwhile, the picosecond-order production makes one expect that the important nitrogen source precursors of amino acids were obtained immediately after the impact. It was also observed that the reduction of N2 proceeded according to an associative mechanism, rather than a dissociative mechanism as in the Haber-Bosch process. PMID:27966594

  18. Reads2Type: a web application for rapid microbial taxonomy identification

    DEFF Research Database (Denmark)

    Saputra, Dhany; Rasmussen, Simon; Larsen, Mette Voldby

    2015-01-01

    Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire g......, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html .......Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire...... genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete...

  19. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    Science.gov (United States)

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

  20. Species identification and molecular typing of human Brucella isolates from Kuwait.

    Science.gov (United States)

    Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

    2017-01-01

    Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

  1. Use of molecular beacons for the rapid analysis of DNA damage induced by exposure to an atmospheric pressure plasma jet

    Energy Technology Data Exchange (ETDEWEB)

    Kurita, Hirofumi, E-mail: kurita@ens.tut.ac.jp, E-mail: mizuno@ens.tut.ac.jp; Miyachika, Saki; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira, E-mail: kurita@ens.tut.ac.jp, E-mail: mizuno@ens.tut.ac.jp [Department of Environmental and Life Sciences, Toyohashi University of Technology, Aichi 441-8580 (Japan)

    2015-12-28

    A rapid method for evaluating the damage caused to DNA molecules upon exposure to plasma is demonstrated. Here, we propose the use of a molecular beacon for rapid detection of DNA strand breaks induced by atmospheric pressure plasma jet (APPJ) irradiation. Scission of the molecular beacon by APPJ irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the DNA strand breaks. The results show that the increase in fluorescence intensity is proportional to the exposure time and the rate of fluorescence increase is proportional to the discharge power. This simple and rapid method allows the estimation of DNA damage induced by exposure to a non-thermal plasma.

  2. Molecular typing of Legionella pneumophila from air-conditioning cooling waters using mip gene, SBT, and FAFLP methods.

    Science.gov (United States)

    Gong, Xiangli; Li, Juntao; Zhang, Ying; Hou, Shuiping; Qu, Pinghua; Yang, Zhicong; Chen, Shouyi

    2017-08-01

    Legionella spp. are important waterborne pathogens. Molecular typing has become an important method for outbreaks investigations and source tracking of Legionnaires. In a survey program conducted by the Guangzhou Center for Disease Control and Prevention, multiple serotypes Legionella pneumophila (L. pneumophila) were isolated from waters in air-conditioning cooling towers in urban Guangzhou region, China between 2008 and 2011. Three genotyping methods, mip (macrophage infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent amplified fragment length polymorphism analysis) were used to type these waterborne L. pneumophila isolates. The three methods were capable of typing all the 134 isolates and a reference strain of L. pneumophila (ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134 isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and FAFLP methods respectively. The mip genotyping and SBT typing are more feasible for inter-laboratory results sharing and comparison of different types of L. pneumophila. The SBT and FAFLP typing methods were rapid with higher discriminatory abilities. Combinations of two or more of the typing methods enables more accurate typing of Legionella isolates for outbreak investigations and source tracking of Legionnaires. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Crop Type Classification Using Vegetation Indices of RapidEye Imagery

    Science.gov (United States)

    Ustuner, M.; Sanli, F. B.; Abdikan, S.; Esetlili, M. T.; Kurucu, Y.

    2014-09-01

    Cutting-edge remote sensing technology has a significant role for managing the natural resources as well as the any other applications about the earth observation. Crop monitoring is the one of these applications since remote sensing provides us accurate, up-to-date and cost-effective information about the crop types at the different temporal and spatial resolution. In this study, the potential use of three different vegetation indices of RapidEye imagery on crop type classification as well as the effect of each indices on classification accuracy were investigated. The Normalized Difference Vegetation Index (NDVI), the Green Normalized Difference Vegetation Index (GNDVI), and the Normalized Difference Red Edge Index (NDRE) are the three vegetation indices used in this study since all of these incorporated the near-infrared (NIR) band. RapidEye imagery is highly demanded and preferred for agricultural and forestry applications since it has red-edge and NIR bands. The study area is located in Aegean region of Turkey. Radial Basis Function (RBF) kernel was used here for the Support Vector Machines (SVMs) classification. Original bands of RapidEye imagery were excluded and classification was performed with only three vegetation indices. The contribution of each indices on image classification accuracy was also tested with single band classification. Highest classification accuracy of 87, 46 % was obtained using three vegetation indices. This obtained classification accuracy is higher than the classification accuracy of any dual-combination of these vegetation indices. Results demonstrate that NDRE has the highest contribution on classification accuracy compared to the other vegetation indices and the RapidEye imagery can get satisfactory results of classification accuracy without original bands.

  4. Nanowire array chips for molecular typing of rare trafficking leukocytes with application to neurodegenerative pathology

    Science.gov (United States)

    Kwak, Minsuk; Kim, Dong-Joo; Lee, Mi-Ri; Wu, Yu; Han, Lin; Lee, Sang-Kwon; Fan, Rong

    2014-05-01

    Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective

  5. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

    Science.gov (United States)

    Peters, Sarah E.; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A.; Luan, Shi-Lu; Chaudhuri, Roy R.; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M.; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Maskell, Duncan J.; Tucker, Alexander W.

    2015-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. PMID:26424843

  6. Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ze-Xiong Xie

    2018-01-01

    Full Text Available Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ∼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLα and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0 genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.

  7. Whole-exome sequencing enables rapid determination of xeroderma pigmentosum molecular etiology.

    Directory of Open Access Journals (Sweden)

    Oscar Ortega-Recalde

    Full Text Available Xeroderma pigmentosum (XP is a rare autosomal recessive disorder characterized by extreme sensitivity to actinic pigmentation changes in the skin and increased incidence of skin cancer. In some cases, patients are affected by neurological alterations. XP is caused by mutations in 8 distinct genes (XPA through XPG and XPV. The XP-V (variant subtype of the disease results from mutations in a gene (XPV, also named POLH which encodes for Polη, a member of the Y-DNA polymerase family. Although the presence and severity of skin and neurological dysfunctions differ between XP subtypes, there are overlapping clinical features among subtypes such that the sub-type cannot be deduced from the clinical features. In this study, in order to overcome this drawback, we undertook whole-exome sequencing in two XP sibs and their father. We identified a novel homozygous nonsense mutation (c.897T>G, p.Y299X in POLH which causes the disease. Our results demonstrate that next generation sequencing is a powerful approach to rapid determination of XP genetic etiology.

  8. DNA-based genetic markers for Rapid Cycling Brassica rapa (Fast Plants type designed for the teaching laboratory.

    Directory of Open Access Journals (Sweden)

    Eryn E. Slankster

    2012-06-01

    Full Text Available We have developed DNA-based genetic markers for rapid-cycling Brassica rapa (RCBr, also known as Fast Plants. Although markers for Brassica rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP based markers and 14 variable number tandem repeat (VNTR-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a nongenic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology.

  9. DNA-Based Genetic Markers for Rapid Cycling Brassica Rapa (Fast Plants Type) Designed for the Teaching Laboratory

    Science.gov (United States)

    Slankster, Eryn E.; Chase, Jillian M.; Jones, Lauren A.; Wendell, Douglas L.

    2012-01-01

    We have developed DNA-based genetic markers for rapid cycling Brassica rapa (RCBr), also known as Fast Plants. Although markers for B. rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP) based markers and 14 variable number tandem repeat (VNTR)-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a non-genic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology. PMID:22675329

  10. Contributions of Different Cloud Types to Feedbacks and Rapid Adjustments in CMIP5*

    Energy Technology Data Exchange (ETDEWEB)

    Zelinka, Mark D. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Program for Climate Model Diagnosis and Intercomparison; Klein, Stephen A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Program for Climate Model Diagnosis and Intercomparison; Taylor, Karl E. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Program for Climate Model Diagnosis and Intercomparison; Andrews, Timothy [Met Office Hadley Center, Exeter (United Kingdom); Webb, Mark J. [Met Office Hadley Center, Exeter (United Kingdom); Gregory, Jonathan M. [Univ. of Reading, Exeter (United Kingdom). National Center for Atmospheric Science; Forster, Piers M. [Univ. of Leeds (United Kingdom)

    2013-07-01

    When using five climate model simulations of the response to an abrupt quadrupling of CO2, the authors perform the first simultaneous model intercomparison of cloud feedbacks and rapid radiative adjustments with cloud masking effects removed, partitioned among changes in cloud types and gross cloud properties. After CO2 quadrupling, clouds exhibit a rapid reduction in fractional coverage, cloud-top pressure, and optical depth, with each contributing equally to a 1.1 W m-2 net cloud radiative adjustment, primarily from shortwave radiation. Rapid reductions in midlevel clouds and optically thick clouds are important in reducing planetary albedo in every model. As the planet warms, clouds become fewer, higher, and thicker, and global mean net cloud feedback is positive in all but one model and results primarily from increased trapping of longwave radiation. As was true for earlier models, high cloud changes are the largest contributor to intermodel spread in longwave and shortwave cloud feedbacks, but low cloud changes are the largest contributor to the mean and spread in net cloud feedback. The importance of the negative optical depth feedback relative to the amount feedback at high latitudes is even more marked than in earlier models. Furthermore, the authors show that the negative longwave cloud adjustment inferred in previous studies is primarily caused by a 1.3 W m-2 cloud masking of CO2 forcing. Properly accounting for cloud masking increases net cloud feedback by 0.3 W m-2 K-1, whereas accounting for rapid adjustments reduces by 0.14 W m-2 K-1 the ensemble mean net cloud feedback through a combination of smaller positive cloud amount and altitude feedbacks and larger negative optical depth feedbacks.

  11. The Salmonella In Silico Typing Resource (SISTR: An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies.

    Directory of Open Access Journals (Sweden)

    Catherine E Yoshida

    Full Text Available For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR, a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST, ribosomal MLST (rMLST, and core genome MLST (cgMLST. We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence

  12. Rapid detection and typing of live bacteria from human joint fluid samples by utilizing an integrated microfluidic system.

    Science.gov (United States)

    Chang, Wen-Hsin; Wang, Chih-Hung; Lin, Chih-Lin; Wu, Jiunn-Jong; Lee, Mel S; Lee, Gwo-Bin

    2015-04-15

    Periprosthetic joint infection (PJI) is one of the most dreading complications that hinder the merits of an arthroplasty. A prerequisite for treatment of the above procedure is rapid detection of live bacteria to prevent its recurrence and proper choice of antibiotics. Conventional culture methods are time-consuming and associated with a high false negative rate. Amplification of bacterial genetic materials requires a tedious process but is associated with a high false positive rate. An integrated microfluidic system capable of molecular diagnosis for detecting live bacteria was reported in our previous work. However, the system could not provide detailed information about infectious bacteria for the subsequent antibiotic choices. Furthermore, it took at least 55min to finish the entire process. In this work, a microfluidic platform using ethidium monoazide (EMA) which can only penetrate into dead bacteria is presented for live bacteria detection and typing within a short period of time (30min for the detection of live bacteria and another 40min for the typing of bacteria strains). We tested the proposed system by using human joint fluid samples and found its limit of detection for bacterial detection equal to 10(2)CFU (colony formation unit) for live bacteria detection with gold nanoparticle probes and 10(2)-10(4)CFU for typing bacteria by an on-chip polymerase chain reaction. The whole procedure of the integrated microfluidic system is automated with little human intervention. Moreover, this is the first time that sequential live bacteria detection and typing are demonstrated on the same microfluidic platform. Based on the promising results, the proposed system may become in the near future an auxiliary tool for immediate medical decision and choice of antibiotics in routine arthroplasties or PJI's. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Next-Generation Sequencing-Aided Rapid Molecular Diagnosis of Occult Macular Dystrophy in a Chinese Family

    OpenAIRE

    Yu-He Qi; Feng-Juan Gao; Fang-Yuan Hu; Sheng-Hai Zhang; Jun-Yi Chen; Wan-Jing Huang; Guo-Hong Tian; Min Wang; De-Kang Gan; Ji-Hong Wu; Ge-Zhi Xu

    2017-01-01

    Purpose: To show early, rapid and accurate molecular diagnosis of occult macular dystrophy (OMD) in a four-generation Chinese family with inherited macular dystrophy.Methods: In the current study, we comprehensively screened 130 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the proband of a four-generation Chinese family that has suffered from maculopathy without a definitive diagnosis for over 10 years. Varian...

  14. Molecular typing of fecal eukaryotic microbiota of human infants and ...

    Indian Academy of Sciences (India)

    Keywords. 18S rRNA library; gastrointestinal tract; micro-eukaryotic diversity ... Insect Molecular Biology Unit, National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411 007, Maharashtra, India; Gastroenterology Unit, Department of P ediatrics, KEM Hospital, Rasta Peth, Pune 411 011, India ...

  15. Fluorescent immunochromatography for rapid and sensitive typing of seasonal influenza viruses.

    Directory of Open Access Journals (Sweden)

    Akira Sakurai

    Full Text Available Lateral flow tests also known as Immunochromatography (IC is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60% and the limit of detection (LOD is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC for subtyping H5 influenza viruses (FLIC-H5. Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB. This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75, specificity 100% (54/54, Type B: sensitivity 100% (90/90, specificity 98.2% (54/55 in nasal swab samples in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13 h than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.

  16. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Directory of Open Access Journals (Sweden)

    Yang Moon-Sik

    2004-09-01

    Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  17. Nitrogen conversion under rapid pyrolysis of two types of aquatic biomass and corresponding blends with coal.

    Science.gov (United States)

    Yuan, Shuai; Chen, Xue-li; Li, Wei-feng; Liu, Hai-feng; Wang, Fu-chen

    2011-11-01

    Rapid pyrolysis of two types of aquatic biomass (blue-green algae and water hyacinth), and their blends with two coals (bituminous and anthracite) was carried out in a high-frequency furnace. Nitrogen conversions during rapid pyrolysis of the two biomass and the interactions between the biomass and coals on nitrogen conversions were investigated. Results show that little nitrogen retained in char after the biomass pyrolysis, and NH(3) yields were higher than HCN. During co-pyrolysis of biomass and coal, interactions between biomass and coal decreased char-N yields and increased volatile-N yields, but the total yields of NH(3)+HCN in volatile-N were decreased in which HCN formations were decreased consistently, while NH(3) formations were only decreased in the high-temperature range but promoted in the low-temperature range. Interactions between blue-green algae and coals are stronger than those between water hyacinth and coal, and interactions between biomass and bituminous are stronger than those between biomass and anthracite. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Rapid Screening of MDR-TB in Cases of Extra Pulmonary Tuberculosis Using Geno Type MTBDRplus.

    Directory of Open Access Journals (Sweden)

    Richa Kumari

    should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.

  19. Rapid Screening of MDR-TB in Cases of Extra Pulmonary Tuberculosis Using Geno Type MTBDRplus.

    Science.gov (United States)

    Kumari, Richa; Tripathi, Rajneesh; Pandey, Alok Prakash; Banerjee, Tuhina; Sinha, Pallavi; Anupurba, Shampa

    2016-01-01

    given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.

  20. Molecular diagnosis of multiple endocrine neoplasia type 2A

    African Journals Online (AJOL)

    1998-01-01

    Jan 1, 1998 ... occurs as a component of either multiple endocrine neoplasia (MEN) type 2A, comprising MTC, phaeochromocytoma and hyperparathyroidism, or the rarer. MEN type 28 characterised by MTC, phaeochromocytoma,. Department of Chemical Pathology, University of Natal, Durban. R J Pegoraro. BSc, PhD.

  1. Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

    Directory of Open Access Journals (Sweden)

    Yang Yang

    2017-01-01

    Full Text Available Porcine circovirus virus type II (PCV2 is the etiology of postweaning multisystemic wasting syndrome (PMWS, porcine dermatitis, nephropathy syndrome (PDNS, and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA assays using a real-time fluorescent detection (PCV2 real-time RPA assay and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

  2. Rapid and Specific Detection of the Escherichia coli Sequence Type 648 Complex within Phylogroup F.

    Science.gov (United States)

    Johnson, James R; Johnston, Brian D; Gordon, David M

    2017-04-01

    The Escherichia coli sequence type 648 complex (STc648) is an emerging lineage within phylogroup F-formerly included within phylogroup D-that is associated with multidrug resistance. Here, we designed and validated a novel multiplex PCR-based assay for STc648 that took advantage of (i) four distinctive single-nucleotide polymorphisms in icd allele 96 and gyrB allele 87, two of the multilocus sequence typing alleles that define ST648; and (ii) the typical absence within STc648 of uidA, an E. coli-specific gene encoding β-glucuronidase. Within a diverse 212-strain validation set that included 109 STs other than STc648, from phylogroups A, B1, B2, C, D, E, and F, the assay exhibited 100% sensitivity (95% confidence interval [CI], 82% to 100%) and specificity (95% CI, 98% to 100%). It functioned similarly well in two distant laboratories that used boiled lysates or DNAzol-purified DNA as the template DNA. Thus, this novel multiplex PCR-based assay should enable any laboratory equipped for diagnostic PCR to rapidly, accurately, and economically screen E. coli isolates for membership in STc648. Copyright © 2017 American Society for Microbiology.

  3. Ligand Replacement Approach to Raman-Responded Molecularly Imprinted Monolayer for Rapid Determination of Penicilloic Acid in Penicillin.

    Science.gov (United States)

    Zhang, Liying; Jin, Yang; Huang, Xiaoyan; Zhou, Yujie; Du, Shuhu; Zhang, Zhongping

    2015-12-01

    Penicilloic acid (PA) is a degraded byproduct of penicillin and often causes fatal allergies to humans, but its rapid detection in penicillin drugs remains a challenge due to its similarity to the mother structure of penicillin. Here, we reported a ligand-replaced molecularly imprinted monolayer strategy on a surface-enhanced Raman scattering (SERS) substrate for the specific recognition and rapid detection of Raman-inactive PA in penicillin. The bis(phenylenediamine)-Cu(2+)-PA complex was first synthesized and stabilized onto the surface of silver nanoparticle film that was fabricated by a bromide ion-added silver mirror reaction. A molecularly imprinted monolayer was formed by the further modification of alkanethiol around the stabilized complex on the Ag film substrate, and the imprinted recognition site was then created by the replacement of the complex template with Raman-active probe molecule p-aminothiophenol. When PA rebound into the imprinted site in the alkanethiol monolayer, the SERS signal of p-aminothiophenol exhibited remarkable enhancement with a detection limit of 0.10 nM. The imprinted monolayer can efficiently exclude the interference of penicillin and thus provides a selective determination of 0.10‰ (w/w) PA in penicillin, which is about 1 order of magnitude lower than the prescribed residual amount of 1.0‰. The strategy reported here is simple, rapid and inexpensive compared to the traditional chromatography-based methods.

  4. Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

    OpenAIRE

    Bauernfeind, Adolf; Roller, Carsten; Meyer, Detlef; Jungwirth, Renate; Schneider, Ines

    1998-01-01

    A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.

  5. Prevalence and molecular typing of the antiseptic resistance genes ...

    African Journals Online (AJOL)

    Additionally, two predominant PFGE (B and A) and spa types (t037 and t042) were identified along with two major antimicrobial resistance profiles. All of these qacA/B-positive strains strains were pvl-negative by PCR. The qacA/B-positive MRSA strains all contained the group III SCCmec element. These strains were ...

  6. Strain typing methods and molecular epidemiology of Pneumocystis pneumonia

    DEFF Research Database (Denmark)

    Beard, Charles Ben; Roux, Patricia; Nevez, Gilles

    2004-01-01

    Pneumocystis pneumonia (PCP) caused by the opportunistic fungal agent Pneumocystis jirovecii (formerly P. carinii) continues to cause illness and death in HIV-infected patients. In the absence of a culture system to isolate and maintain live organisms, efforts to type and characterize the organism...

  7. Characterization of two types of prostasomes with distinct molecular compositions

    NARCIS (Netherlands)

    Aalberts, M.|info:eu-repo/dai/nl/313888515

    2012-01-01

    During the last years it has become evident that many different cell types can communicate with each other through intercellular transfer of extracellular vesicles. Such cell-derived membrane vesicles function in several physiological processes and also in disease. This thesis describes two

  8. biochemical and molecular diagnosis of glutaric aciduria type 1

    African Journals Online (AJOL)

    2001-12-02

    Dec 2, 2001 ... Glutaric aciduria type 1 (GA-l) is an inborn error of metabolism caused by a deficiency of .... Organic and amino acid levels in urine and plasma from patient LZ. Urine (umol/mmol creatinine) Patient Reference Range. Glutaric acid 520 <2 .... Glutaric aciduria: a 'new' disorder of aminoacid metabolism.

  9. Molecular analysis of glycogen storage disease type Ia in Iranian ...

    Indian Academy of Sciences (India)

    SHEKARI KHANIANI MAHMOUD1,2

    Abstract. Glycogen storage diseases (GSDs) are caused by abnormalities in enzymes that are involved in the regulation of gluconeo- genesis and glycogenolysis. GSD I, an autosomal recessive metabolic disorder, is the most common GSD and has four sub- types. Here, we examined GSD Ia caused by the defective ...

  10. Elevated levels of serum type I collagen C-telopeptide in patients with rapidly destructive osteoarthritis of the hip

    OpenAIRE

    Berger, Christian E.; Kröner, Andreas; Stiegler, Helmar; Leitha, Thomas; Engel, Alfred

    2004-01-01

    We compared type I collagen degradation using serum cross-linking C-terminal telopeptide (ICTP) in 18 patients with rapidly destructive osteoarthrosis and in 20 patients with slowly progressive osteoarthrosis of the hip. The diagnosis was established by clinical examination and radiographic evaluation. Total hip arthroplasty was performed in all patients. Serum levels of ICTP, bone-specific alkaline phosphatase, osteocalcin and N-terminal propeptide were studied. Patients with rapidly destruc...

  11. Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

    Science.gov (United States)

    Bauernfeind, Adolf; Roller, Carsten; Meyer, Detlef; Jungwirth, Renate; Schneider, Ines

    1998-01-01

    A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel. PMID:9705426

  12. Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

    Science.gov (United States)

    2011-08-01

    criteria as described by the National Institutes of Health and the CDC (9). All Fronciselk1 isolates were grown on chocolate agar plates (Remcl, Lenexa...not type the F. tularensis isolates. No viable F. tularensis was recovered from these two tick specimens by culturing on chocolate agar plates, and...discrimination between these two clades was still apparent. In Mycobacterium, Salmonella , and other pathogens the locations of IS elements have been

  13. Neurofibromatosis type 2 (NF2): A clinical and molecular review

    OpenAIRE

    Evans D Gareth R

    2009-01-01

    Abstract Neurofibromatosis type 2 (NF2) is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000) is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular ...

  14. Molecular identification and typing of lactobacilli isolated from kefir grains.

    Science.gov (United States)

    Delfederico, Lucrecia; Hollmann, Axel; Martínez, Mariano; Iglesias, N Gabriel; De Antoni, Graciela; Semorile, Liliana

    2006-02-01

    Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.

  15. Separation of Ethanol-Water MixtureUsing Type-A Zeolite Molecular Sieve

    Directory of Open Access Journals (Sweden)

    Juan Camilo Díaz

    2010-01-01

    Full Text Available This paper presents an experimental procedure for the synthesis of a type-A zeolite molecular sieve, usable in the separation of an ethanol-water mixture. The type-A zeolite molecular sieve is obtained from a reactive mixture formed by a source of alumina, a source of silica and a promoter. Characterization of the molecular sieves obtained in this work was undertaken using X-ray diffraction (XRD, adsorption isotherms and scanning electron microscopy (SEM. The results from the characterization procedures showed that part of the product presented a pore diameter suitable for the separation of an ethanol-water mixture.

  16. Strain typing methods and molecular epidemiology of Pneumocystis pneumonia

    DEFF Research Database (Denmark)

    Beard, Charles Ben; Roux, Patricia; Nevez, Gilles

    2004-01-01

    Pneumocystis pneumonia (PCP) caused by the opportunistic fungal agent Pneumocystis jirovecii (formerly P. carinii) continues to cause illness and death in HIV-infected patients. In the absence of a culture system to isolate and maintain live organisms, efforts to type and characterize the organism...... have relied on polymerase chain reaction-based approaches. Studies using these methods have improved understanding of PCP epidemiology, shedding light on sources of infection, transmission patterns, and potential emergence of antimicrobial resistance. One concern, however, is the lack of guidance...

  17. Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2 ▿

    Science.gov (United States)

    Borman, Andrew M.; Linton, Christopher J.; Oliver, Debra; Palmer, Michael D.; Szekely, Adrien; Johnson, Elizabeth M.

    2010-01-01

    Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures. PMID:20702674

  18. Rapid microwave-assisted synthesis of molecularly imprinted polymers on carbon quantum dots for fluorescent sensing of tetracycline in milk.

    Science.gov (United States)

    Hou, Juan; Li, Huiyu; Wang, Long; Zhang, Ping; Zhou, Tianyu; Ding, Hong; Ding, Lan

    2016-01-01

    In this paper, a novel, selective and eco-friendly sensor for the detection of tetracycline was developed by grafting imprinted polymers onto the surface of carbon quantum dots. A simple microwave-assisted approach was utilized to fabricate the fluorescent imprinted composites rapidly for the first time, which could shorten the polymerization time and simplify the experimental procedure dramatically. The novel composites not only demonstrated excellent fluorescence stability and special binding sites, but also could selectively accumulate target analytes. Under optimal conditions, the relative fluorescence intensity of the composites decreased linearly with increasing the concentration of tetracycline from 20 nM to 14 µM. The detection limit of tetracycline was 5.48 nM. The precision and reproducibility of the proposed sensor were also acceptable. Significantly, the practicality of this ultrasensitive sensor for tetracycline detection in milk was further validated, revealing the advantages of simplicity, sensitivity, selectivity and low cost. This approach combines the high selective adsorption property of molecular imprinted polymers and the sensitivity of fluorescence detection. It is envisioned that the development of fluorescent molecularly imprinted composites will offer a new way of thinking for rapid analysis in complex samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

    Directory of Open Access Journals (Sweden)

    Camille Escadafal

    2014-03-01

    Full Text Available BACKGROUND: Yellow fever (YF is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV, is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY: The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE: The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction and rapid processing time (<20 min. Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for

  20. Elevated levels of high-molecular-weight adiponectin in type 1 diabetes

    DEFF Research Database (Denmark)

    Leth, H.; Andersen, K.K.; Frystyk, J.

    2008-01-01

    BACKGROUND: Several studies have shown that type 1 diabetic patients have elevated total levels of the adipocyte-derived adipocytokine adiponectin. However, adiponectin circulates in three different subforms, and the high-molecular-weight (HMW) subform is believed to be the primary biologically...... active form. The effects of the medium-molecular-weight (MMW) subform and the low-molecular-weight (LMW) subform are still unresolved. PURPOSE: The objective of the study was to investigate the distribution of the three molecular subforms of adiponectin in well-characterized groups of type 1 diabetics...... with varying degrees of nephropathy as well as in healthy control subjects. STUDY POPULATION: Two hundred seven individuals were included: 58 type 1 diabetics with normoalbuminuria, 46 with microalbuminuria, 46 with macroalbuminuria, and 57 matched controls. METHODS: The HMW, MMW, and LMW subforms were...

  1. Role of GenoType(®) Mycobacterium Common Mycobacteria/Additional Species Assay for Rapid Differentiation Between Mycobacterium tuberculosis Complex and Different Species of Non-Tuberculous Mycobacteria.

    Science.gov (United States)

    Singh, Amresh Kumar; Maurya, Anand Kumar; Umrao, Jyoti; Kant, Surya; Kushwaha, Ram Awadh Singh; Nag, Vijaya Laskshmi; Dhole, Tapan N

    2013-07-01

    Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType(®) Mycobacterium common mycobacteria/additional species (CM/AS) assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB) cases. A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France). A total of 219 culture positive clinical isolates (BacT/ALERT(®) MP cultures) were selected for differentiation by p-nitrobenzoic acid (PNB) sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType(®) Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany). Out of 219 BacT/ALERT(®) MP culture positive isolates tested by PNB as 153 MTBC (69.9%) and by GenoType(®) Mycobacterium CM/AS assay as 159 (72.6%) MTBC and remaining 60 (27.4%) were considered as NTM species. The GenoType(®) Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3%) among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3%) among slow growing mycobacteria. The GenoType(®) Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria.

  2. Role of GenoType® Mycobacterium Common Mycobacteria/Additional Species Assay for Rapid Differentiation Between Mycobacterium tuberculosis Complex and Different Species of Non-Tuberculous Mycobacteria

    Science.gov (United States)

    Singh, Amresh Kumar; Maurya, Anand Kumar; Umrao, Jyoti; Kant, Surya; Kushwaha, Ram Awadh Singh; Nag, Vijaya Laskshmi; Dhole, Tapan N

    2013-01-01

    Background: Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType® Mycobacterium common mycobacteria/additional species (CM/AS) assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB) cases. Materials and Methods: A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France). A total of 219 culture positive clinical isolates (BacT/ALERT® MP cultures) were selected for differentiation by p-nitrobenzoic acid (PNB) sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType® Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany). Results: Out of 219 BacT/ALERT® MP culture positive isolates tested by PNB as 153 MTBC (69.9%) and by GenoType® Mycobacterium CM/AS assay as 159 (72.6%) MTBC and remaining 60 (27.4%) were considered as NTM species. The GenoType® Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3%) among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3%) among slow growing mycobacteria. Conclusion: The GenoType® Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria. PMID:24701099

  3. Rapid DNA extraction protocol from stool, suitable for molecular genetic diagnosis of colon cancer.

    Science.gov (United States)

    Abbaszadegan, Mohammad Reza; Velayati, Arash; Tavasoli, Alireza; Dadkhah, Ezzat

    2007-07-01

    Colorectal cancer (CRC) is one of the most common forms of cancers in the world and is curable if diagnosed at the early stage. Analysis of DNA extracted from stool specimens is a recent advantage to cancer diagnostics. Many protocols have been recommended for DNA extraction from stool, and almost all of them are difficult and time consuming, dealing with high amount of toxic materials like phenol. Their results vary due to sample collection method and further purification treatment. In this study, an easy and rapid method was optimized for isolating the human DNA with reduced PCR inhibitors present in stool. Fecal samples were collected from 10 colonoscopy-negative adult volunteers and 10 patients with CRC. Stool (1 g) was extracted using phenol/chloroform based protocol. The amplification of P53 exon 9 was examined to evaluate the extraction efficiency for human genomic targets and also compared its efficiency with Machiels et al. and Ito et al. protocols. The amplification of exon 9 of P53 from isolated fecal DNA was possible in most cases in 35 rounds of PCR using no additional purification procedure for elimination of the remaining inhibitors.inhibitors. A useful, rapid and easy protocol for routine extraction of DNA from stool was introduced and compared with two previous protocols.

  4. Simple and Rapid Molecular Techniques for Identification of Amylose Levels in Rice Varieties

    Science.gov (United States)

    Cheng, Acga; Ismail, Ismanizan; Osman, Mohamad; Hashim, Habibuddin

    2012-01-01

    The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)10, (CT)11, (CT)16, (CT)17, and (CT)18 were identified. Of these, (CT)11 and (CT)17 were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)10 and (CT)11, while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5′-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE. PMID:22754356

  5. Rapid and reliable determination of p-nitroaniline in wastewater by molecularly imprinted fluorescent polymeric ionic liquid microspheres.

    Science.gov (United States)

    Lu, Xing; Yang, Yiwen; Zeng, Yanbo; Li, Lei; Wu, Xiaohua

    2018-01-15

    Rapid and efficient detecting trace amount of environmental p-nitroaniline (p-NA) is in urgent need for security concerns and pollution supervision. In this work we report the use of molecularly imprinted polymeric ionic liquid (MIPIL) microspheres to construct recognizable surfaces for detection of p-NA through fluorescence quenching. The p-NA imprinted microspheres are synthesized by precipitation polymerization upon co-polymerization of 3-(anthracen-9-ylmethyl)-1-vinyl-1H-imidazol-3-ium chloride (Fluorescent IL monomer) with ethyleneglycol dimethacrylate (EGDMA). The electron-rich group alkenyl imidazole in IL functional monomer can dramatically improve the emission of anthracene fluorophore and the π-π stacking, electronic, and hydrogen bond between p-NA and MIPIL can efficiently enhance the selective recognition force. The as-synthesized MIPIL microspheres present spherical shape, high fluorescence emission intensity and specific recognition, which showed rapid detection rate (1min), stable reusable property (at least 4 time recycles), wonderful selectivity over several structural analogs, wide linear range (10nM to 10M) with a correlation coefficient of 0.992, and excellent sensitivity (LOD, 9nM). As synthesis and surface functionalization of MIPIL microspheres are well established, the methods reported in this work are facile, rapid and efficient for monitoring p-NA in environmental wastewater. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

    Directory of Open Access Journals (Sweden)

    Ida eSteinberger-Levy

    2016-05-01

    Full Text Available Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test.

  7. A novel molecular toolkit for rapid detection of the pathogen and primary vector of thousand cankers disease.

    Directory of Open Access Journals (Sweden)

    Emel Oren

    Full Text Available Thousand Cankers Disease (TCD of Juglans and Pterocarya (Juglandaceae involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA, but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location: California-J. hindsii (heavy disease incidence; Tennessee-J. nigra (mild disease incidence; and outside the known TCD zone (Missouri-J. nigra, no record of the disease. California samples had the highest incidence of the TCD organisms (85%, 34/40. Tennessee had intermediate incidence (42.5%, 17/40, whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD.

  8. Molecular typing of Salmonella from Sergipe, Northeastern Brazil, showing the epidemiological relationship between poultry and human infection.

    Science.gov (United States)

    Góis, P B P; Carneiro, M R P; Jain, S; Santos, M I S; Batista, M V A; Cândido, A L

    2015-09-25

    Randomly amplified polymorphic DNA (RAPD) has been widely used for epidemiological and phylogenetic purposes ow-ing to its rapidity and efficiency. The aim of this study was to perform genome typing of Salmonella samples isolated from different sources by RAPD profiling. Thirty-three Salmonella samples from the bacterial collection of the Laboratório de Virologia Comparada, Departamento de Morfologia, Universidade Federal de Sergipe, Brazil, and two standard samples were used. RAPD profiling was conducted using six primers of the Ready-To-Go RAPD system. The amplified products were electro-phoresed on 5% polyacrylamide gel and silver-stained. RAPD analysis resulted in reproducible and stable banding patterns and showed high genetic diversity among the isolated strains. The Primer P1-generated dendrogram showed an epidemiologic relationship between the human and poultry isolated samples, highlighting the usefulness of RAPD for molecular typing and epidemiological studies.

  9. Relative abundance of Mycobacterium bovis molecular types in cattle: a simulation study of potential epidemiological drivers.

    Science.gov (United States)

    Trewby, Hannah; Wright, David M; Skuce, Robin A; McCormick, Carl; Mallon, Thomas R; Presho, Eleanor L; Kao, Rowland R; Haydon, Daniel T; Biek, Roman

    2017-08-22

    The patterns of relative species abundance are commonly studied in ecology and epidemiology to provide insights into underlying dynamical processes. Molecular types (MVLA-types) of Mycobacterium bovis, the causal agent of bovine tuberculosis, are now routinely recorded in culture-confirmed bovine tuberculosis cases in Northern Ireland. In this study, we use ecological approaches and simulation modelling to investigate the distribution of relative abundances of MVLA-types and its potential drivers. We explore four biologically plausible hypotheses regarding the processes driving molecular type relative abundances: sampling and speciation; structuring of the pathogen population; historical changes in population size; and transmission heterogeneity (superspreading). Northern Irish herd-level MVLA-type surveillance shows a right-skewed distribution of MVLA-types, with a small number of types present at very high frequencies and the majority of types very rare. We demonstrate that this skew is too extreme to be accounted for by simple neutral ecological processes. Simulation results indicate that the process of MVLA-type speciation and the manner in which the MVLA-typing loci were chosen in Northern Ireland cannot account for the observed skew. Similarly, we find that pathogen population structure, assuming for example a reservoir of infection in a separate host, would drive the relative abundance distribution in the opposite direction to that observed, generating more even abundances of molecular types. However, we find that historical increases in bovine tuberculosis prevalence and/or transmission heterogeneity (superspreading) are both capable of generating the skewed MVLA-type distribution, consistent with findings of previous work examining the distribution of molecular types in human tuberculosis. Although the distribution of MVLA-type abundances does not fit classical neutral predictions, our simulations show that increases in pathogen population size and

  10. Selective and rapid uptake of adeno-associated virus type 2 in brain.

    Science.gov (United States)

    Bartlett, J S; Samulski, R J; McCown, T J

    1998-05-20

    Recombinant adeno-associated virus (AAV) vectors effectively transfer and express foreign genes in the brain. The transferred genes, however, are selectively expressed in neurons, and the cause of this specificity is not understood. To address this question, wild-type AAV-2 capsids were covalently labeled with the fluorophore, Cy3, and infused into the inferior colliculus or the hippocampus. Using antibodies to identify neurons (NeuN), astrocytes (GFAP), or oligodendrocytes (OX-42), clear neuron-specific uptake of the virus was observed as early as 6 min after the start of the infusion. By 30 min postinfusion, AAV particles were present in the nucleus of neurons, yet in both the inferior colliculus and hippocampus, a subset of neurons did not take up the virus particles. No AAV particles were found in astrocytes 1.5 min or 24 hr after virus infusion. Interestingly, 1 hr postinfusion, no AAV particles were found in microglia, yet by 24 hr postinfusion, a punctate pattern of AAV particles was found in microglia. To test whether virus uptake correlated with vector-transduced cells, an rAAV-CMV-GFP virus was infused. By 3 days postinfusion, GFP was localized to neuronal populations with no expression in astrocytes or microglia, similar to that of fluorescent virus uptake. These findings demonstrate that in brain, AAV particles rapidly bind and enter primarily neurons with a pattern similar to that of in vivo vector transduction. In addition, these studies indicate that viral binding and uptake, independent of promoter tropism, can explain the specificity of AAV brain transduction. Thus, this first description of AAV kinetic disposition in vivo should facilitate targeted application of this vector for human brain gene therapy.

  11. Rapid and molecular selective electrochemical sensing of phthalates in aqueous solution

    KAUST Repository

    Zia, Asif I.

    2015-05-01

    Reported research work presents real time non-invasive detection of phthalates in spiked aqueous samples by employing electrochemical impedance spectroscopy (EIS) technique incorporating a novel interdigital capacitive sensor with multiple sensing thin film gold micro-electrodes fabricated on native silicon dioxide layer grown on semiconducting single crystal silicon wafer. The sensing surface was functionalized by a self-assembled monolayer of 3-aminopropyltrietoxysilane (APTES) with embedded molecular imprinted polymer (MIP) to introduce selectivity for the di(2-ethylhexyl) phthalate (DEHP) molecule. Various concentrations (1-100. ppm) of DEHP in deionized MilliQ water were tested using the functionalized sensing surface to capture the analyte. Frequency response analyzer (FRA) algorithm was used to obtain impedance spectra so as to determine sample conductance and capacitance for evaluation of phthalate concentration in the sample solution. Spectrum analysis algorithm interpreted the experimentally obtained impedance spectra by applying complex nonlinear least square (CNLS) curve fitting in order to obtain electrochemical equivalent circuit and corresponding circuit parameters describing the kinetics of the electrochemical cell. Principal component analysis was applied to deduce the effects of surface immobilized molecular imprinted polymer layer on the evaluated circuit parameters and its electrical response. The results obtained by the testing system were validated using commercially available high performance liquid chromatography diode array detector system.

  12. Neurofibromatosis type 2 (NF2: A clinical and molecular review

    Directory of Open Access Journals (Sweden)

    Evans D Gareth R

    2009-06-01

    Full Text Available Abstract Neurofibromatosis type 2 (NF2 is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000 is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular schwannomas may also cause dizziness or imbalance as a first symptom. Nausea, vomiting or true vertigo are rare symptoms, except in late-stage disease. The other main tumours are schwannomas of the other cranial, spinal and peripheral nerves; meningiomas both intracranial (including optic nerve meningiomas and intraspinal, and some low-grade central nervous system malignancies (ependymomas. Ophthalmic features are also prominent and include reduced visual acuity and cataract. About 70% of NF2 patients have skin tumours (intracutaneous plaque-like lesions or more deep-seated subcutaneous nodular tumours. Neurofibromatosis type 2 is a dominantly inherited tumour predisposition syndrome caused by mutations in the NF2 gene on chromosome 22. More than 50% of patients represent new mutations and as many as one-third are mosaic for the underlying disease-causing mutation. Although truncating mutations (nonsense and frameshifts are the most frequent germline event and cause the most severe disease, single and multiple exon deletions are common. A strategy for detection of the latter is vital for a sensitive analysis. Diagnosis is based on clinical and neuroimaging studies. Presymptomatic genetic testing is an integral part of the management of NF2 families. Prenatal diagnosis and pre-implantation genetic diagnosis is possible. The main differential diagnosis of NF2 is schwannomatosis. NF2 represents a difficult management problem with most patients facing substantial

  13. Neurofibromatosis type 2 (NF2): a clinical and molecular review.

    Science.gov (United States)

    Evans, D Gareth R

    2009-06-19

    Neurofibromatosis type 2 (NF2) is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000) is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular schwannomas may also cause dizziness or imbalance as a first symptom. Nausea, vomiting or true vertigo are rare symptoms, except in late-stage disease. The other main tumours are schwannomas of the other cranial, spinal and peripheral nerves; meningiomas both intracranial (including optic nerve meningiomas) and intraspinal, and some low-grade central nervous system malignancies (ependymomas). Ophthalmic features are also prominent and include reduced visual acuity and cataract. About 70% of NF2 patients have skin tumours (intracutaneous plaque-like lesions or more deep-seated subcutaneous nodular tumours). Neurofibromatosis type 2 is a dominantly inherited tumour predisposition syndrome caused by mutations in the NF2 gene on chromosome 22. More than 50% of patients represent new mutations and as many as one-third are mosaic for the underlying disease-causing mutation. Although truncating mutations (nonsense and frameshifts) are the most frequent germline event and cause the most severe disease, single and multiple exon deletions are common. A strategy for detection of the latter is vital for a sensitive analysis. Diagnosis is based on clinical and neuroimaging studies. Presymptomatic genetic testing is an integral part of the management of NF2 families. Prenatal diagnosis and pre-implantation genetic diagnosis is possible. The main differential diagnosis of NF2 is schwannomatosis. NF2 represents a difficult management problem with most patients facing substantial morbidity and reduced

  14. Molecular typing of Treponema pallidum: a systematic review and meta-analysis.

    Science.gov (United States)

    Peng, Rui-Rui; Wang, Alberta L; Li, Jing; Tucker, Joseph D; Yin, Yue-Ping; Chen, Xiang-Sheng

    2011-11-01

    Syphilis is resurgent in many regions of the world. Molecular typing is a robust tool for investigating strain diversity and epidemiology. This study aimed to review original research on molecular typing of Treponema pallidum (T. pallidum) with three objectives: (1) to determine specimen types most suitable for molecular typing; (2) to determine T. pallidum subtype distribution across geographic areas; and (3) to summarize available information on subtypes associated with neurosyphilis and macrolide resistance. Two researchers independently searched five databases from 1998 through 2010, assessed for eligibility and study quality, and extracted data. Search terms included "Treponema pallidum," or "syphilis," combined with the subject headings "molecular," "subtyping," "typing," "genotype," and "epidemiology." Sixteen eligible studies were included. Publication bias was not statistically significant by the Begg rank correlation test. Medians, inter-quartile ranges, and 95% confidence intervals were determined for DNA extraction and full typing efficiency. A random-effects model was used to perform subgroup analyses to reduce obvious between-study heterogeneity. Primary and secondary lesions and ear lobe blood specimens had an average higher yield of T. pallidum DNA (83.0% vs. 28.2%, χ(2) = 247.6, ppallidum DNA than blood. There was wide geographic variation in T. pallidum subtypes. More research is needed on the relationship between clinical presentation and subtype, and further validation of ear lobe blood for obtaining T. pallidum DNA would be useful for future molecular studies of syphilis.

  15. Molecular genetic analysis of Type II diabetes associated m.3243A ...

    African Journals Online (AJOL)

    Saidul Abrar

    Molecular genetic analysis of Type II diabetes associated m.3243A>G ... Article history: Received 6 October 2016. Accepted 8 December 2016. Available online 18 January 2017. Keywords: Type II diabetes. Mitochondrial DNA. PCR ... Production and hosting by Elsevier B.V. This is an open access article under.

  16. Trichorhinophalangeal syndrome type I - Clinical, microscopic, and molecular features

    Directory of Open Access Journals (Sweden)

    Jiehyun Jeon

    2014-01-01

    Full Text Available Trichorhinophalangeal syndrome type I (TRPS I is an autosomal dominant malformation syndrome characterized by a triad of hair alteration, craniofacial and skeletal abnormalities. TRPS1 gene was first identified in 2000 and mapped on chromosome 8q23.3. A 39-year-old female patient with short stature (149 cm visited for fine sparse and slow-growing hair with receded medio-occipital hairline of roughly triangular shape since infancy. A typical pear-shaped nose and elongated philtrum were noticeable. In addition, she reported deviation of middle phalanges, bilateral coxa varus in both hips and brachydactyly on bilateral fourth digits. Mutation analysis identified a transition of cytosine to thymine at position 1630 (exon 4, which results in amino acid change R544X and a premature stop of translation. There is no established treatment. But through careful evaluation of suspicious cases to identify potential mutation carriers, the patient can receive information about the disease and genetic counseling.

  17. Plasmids in Gram negatives: molecular typing of resistance plasmids.

    Science.gov (United States)

    Carattoli, Alessandra

    2011-12-01

    A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread. Copyright © 2011 Elsevier GmbH. All rights reserved.

  18. A molecular census of arcuate hypothalamus and median eminence cell types

    DEFF Research Database (Denmark)

    Campbell, John N; Macosko, Evan Z; Fenselau, Henning

    2017-01-01

    The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult m......-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred....

  19. Pyrosequencing for rapid molecular identification of Schistosoma japonicum and S. mekongi eggs and cercariae.

    Science.gov (United States)

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M; Sri-Aroon, Pusadee; Limpanont, Yanin; Lulitanond, Viraphong; Janwan, Penchom; Sanpool, Oranuch; Tourtip, Somjintana; Maleewong, Wanchai

    2013-09-01

    Schistosomiasis, which is caused by Schistosoma japonicum and S. mekongi, is a chronic and dangerous widespread disease affecting several countries in Asia. Differentiation between S. japonicum and S. mekongi eggs and/or cercariae via microscopic examination is difficult due to morphological similarities. It is important to identify these etiological agents isolated from animals and humans at the species or genotype level. In this study, a pyrosequencing assay designed to detect S. japonicum and S. mekongi DNA in fecal samples and infected snails was developed and evaluated as an alternative tool to diagnose schistosomiasis. New primers targeting the 18S ribosomal RNA gene were designated for specific amplification. S. japonicum and S. mekongi were identified using a 43-nucleotide pattern of the 18S ribosomal RNA gene and were differentiated using 7 nucleotides within this region. S. japonicum and S. mekongi-infected snails and fecal samples derived from infected mice and rats were differentially detected within a short period of time. The analytical sensitivity of the method enabled the identification of as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and 2 eggs inoculated in 100mg of non-infected fecal sample. To evaluate the comparative efficacy of the assay, identical samples were also analyzed via microscopy and Sanger sequencing. The pyrosequencing technique was found to be superior to the microscopy method and more rapid than the Sanger sequencing method. These results suggest that the pyrosequencing assay is rapid, simple, sensitive and accurate in identifying S. japonicum and S. mekongi in intermediate hosts and fecal samples of the final host. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Assessing health systems for type 1 diabetes in sub-Saharan Africa: developing a 'Rapid Assessment Protocol for Insulin Access'

    DEFF Research Database (Denmark)

    Beran, David; Yudkin, John S; de Courten, Maximilian

    2006-01-01

    In order to improve the health of people with Type 1 diabetes in developing countries, a clear analysis of the constraints to insulin access and diabetes care is needed. We developed a Rapid Assessment Protocol for Insulin Access, comprising a series of questionnaires as well as a protocol for th...

  1. Development of an ELISA for Rapid Detection of Anti-Type VII Collagen Autoantibodies in Epidermolysis Bullosa Acquisita

    National Research Council Canada - National Science Library

    Chen, M; Chan, L S; Cai, X; O'Toole, E A; Sample, J C; Woodley, D T

    1997-01-01

    .... In this study, we developed a rapid, quantitative enzyme-linked immunosorbent assay (ELISA) to detect autoantibody activity against the complete NC1 domain of type VII collagen with the use of an eukaryotic-expressed, recombinant human NC1 antigen...

  2. The catalase reaction of Shigella species and its use in rapid screening for epidemic Shigella dysenteriae type 1.

    Science.gov (United States)

    Karas, J A; Pillay, D G; Sturm, A W

    2007-01-01

    As epidemic dysentery caused by Shigella dysenteriae type 1 is associated with high mortality, early identification of outbreaks is important. Since S. dysenteriae type 1 differs from most of the Enterobacteriaceae in that it does not produce catalase, a test for catalase may provide a useful screening method. The ability of a catalase test to provide rapid identification of S. dysenteriae type 1 has now been assessed, using isolates of this pathogen from five continents, Shigella of other species, and entero-invasive (EIEC) and Shiga-toxin-producing Escherichia coli (STEC). All of the isolates of S. dysenteriae type 1, as well as S. dysenteriae of types 3, 4, 6, 9, 11 and 12 and S. boydii of type 12, were found catalase-negative. All the other bacteria tested were positive for catalase. In an epidemic setting in South Africa, 406 xylose-negative and lysine-decarboxylase-negative isolates, collected from xylose-lysine-deoxycholate (XLD) agar, were tested for catalase. All 356 of the catalase-negative isolates were confirmed to be of S. dysenteriae type 1. None of the catalase-positive isolates were of S. dysenteriae type 1. The catalase test is useful in the rapid, presumptive identification of S. dysenteriae type 1, from appropriate culture media, because of its high predictive value, simplicity and speed. It would be particularly useful during dysentery outbreaks, when other Shigella would be uncommon. There was no association between the absence of catalase activity and the production of Shiga toxin.

  3. Molecular investigation of genetic assimilation during the rapid adaptive radiations of East African cichlid fishes.

    Science.gov (United States)

    Gunter, Helen M; Schneider, Ralf F; Karner, Immanuel; Sturmbauer, Christian; Meyer, Axel

    2017-12-01

    Adaptive radiations are characterized by adaptive diversification intertwined with rapid speciation within a lineage resulting in many ecologically specialized, phenotypically diverse species. It has been proposed that adaptive radiations can originate from ancestral lineages with pronounced phenotypic plasticity in adaptive traits, facilitating ecologically driven phenotypic diversification that is ultimately fixed through genetic assimilation of gene regulatory regions. This study aimed to investigate how phenotypic plasticity is reflected in gene expression patterns in the trophic apparatus of several lineages of East African cichlid fishes, and whether the observed patterns support genetic assimilation. This investigation used a split brood experimental design to compare adaptive plasticity in species from within and outside of adaptive radiations. The plastic response was induced in the crushing pharyngeal jaws through feeding individuals either a hard or soft diet. We find that nonradiating, basal lineages show higher levels of adaptive morphological plasticity than the derived, radiated lineages, suggesting that these differences have become partially genetically fixed during the formation of the adaptive radiations. Two candidate genes that may have undergone genetic assimilation, gif and alas1, were identified, in addition to alterations in the wiring of LPJ patterning networks. Taken together, our results suggest that genetic assimilation may have dampened the inducibility of plasticity related genes during the adaptive radiations of East African cichlids, flattening the reaction norms and canalizing their feeding phenotypes, driving adaptation to progressively more narrow ecological niches. © 2017 John Wiley & Sons Ltd.

  4. Rapid and sensitive detection of rotavirus molecular signatures using surface enhanced Raman spectroscopy.

    Directory of Open Access Journals (Sweden)

    Jeremy D Driskell

    Full Text Available Human enteric virus infections range from gastroenteritis to life threatening diseases such as myocarditis and aseptic meningitis. Rotavirus is one of the most common enteric agents and mortality associated with infection can be very significant in developing countries. Most enteric viruses produce diseases that are not distinct from other pathogens, and current diagnostics is limited in breadth and sensitivity required to advance virus detection schemes for disease intervention strategies. A spectroscopic assay based on surface enhanced Raman scattering (SERS has been developed for rapid and sensitive detection of rotavirus. The SERS method relies on the fabrication of silver nanorod array substrates that are extremely SERS-active allowing for direct structural characterization of viruses. SERS spectra for eight rotavirus strains were analyzed to qualitatively identify rotaviruses and to classify each according to G and P genotype and strain with >96% accuracy, and a quantitative model based on partial least squares regression analysis was evaluated. This novel SERS-based virus detection method shows that SERS can be used to identify spectral fingerprints of human rotaviruses, and suggests that this detection method can be used for pathogen detection central to human health care.

  5. Molecular typing of Treponema pallidum: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Rui-Rui Peng

    2011-11-01

    Full Text Available Syphilis is resurgent in many regions of the world. Molecular typing is a robust tool for investigating strain diversity and epidemiology. This study aimed to review original research on molecular typing of Treponema pallidum (T. pallidum with three objectives: (1 to determine specimen types most suitable for molecular typing; (2 to determine T. pallidum subtype distribution across geographic areas; and (3 to summarize available information on subtypes associated with neurosyphilis and macrolide resistance.Two researchers independently searched five databases from 1998 through 2010, assessed for eligibility and study quality, and extracted data. Search terms included "Treponema pallidum," or "syphilis," combined with the subject headings "molecular," "subtyping," "typing," "genotype," and "epidemiology." Sixteen eligible studies were included. Publication bias was not statistically significant by the Begg rank correlation test. Medians, inter-quartile ranges, and 95% confidence intervals were determined for DNA extraction and full typing efficiency. A random-effects model was used to perform subgroup analyses to reduce obvious between-study heterogeneity. Primary and secondary lesions and ear lobe blood specimens had an average higher yield of T. pallidum DNA (83.0% vs. 28.2%, χ(2 = 247.6, p<0.001 and an average higher efficiency of full molecular typing (80.9% vs. 43.1%, χ(2 = 102.3, p<0.001 compared to plasma, whole blood, and cerebrospinal fluid. A pooled analysis of subtype distribution based on country location showed that 14d was the most common subtype, and subtype distribution varied across geographic areas. Subtype data associated with macrolide resistance and neurosyphilis were limited.Primary lesion was a better specimen for obtaining T. pallidum DNA than blood. There was wide geographic variation in T. pallidum subtypes. More research is needed on the relationship between clinical presentation and subtype, and further

  6. Accessible surfaces of beta proteins increase with increasing protein molecular mass more rapidly than those of other proteins.

    Directory of Open Access Journals (Sweden)

    Anna V Glyakina

    Full Text Available Here we present a systematic analysis of accessible surface areas and hydrogen bonds of 2554 globular proteins from four structural classes (all-α, all-β, α/β and α+β proteins that is aimed to learn in which structural class the accessible surface area increases with increasing protein molecular mass more rapidly than in other classes, and what structural peculiarities are responsible for this effect. The beta structural class of proteins was found to be the leader, with the following possible explanations of this fact. First, in beta structural proteins, the fraction of residues not included in the regular secondary structure is the largest, and second, the accessible surface area of packaged elements of the beta-structure increases more rapidly with increasing molecular mass in comparison with the alpha-structure. Moreover, in the beta structure, the probability of formation of backbone hydrogen bonds is higher than that in the alpha helix for all residues of α+β proteins (the average probability is 0.73±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without proline and α/β proteins, except for asparagine, aspartic acid, glycine, threonine, and serine (0.70±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without the proline residue. There is a linear relationship between the number of hydrogen bonds and the number of amino acid residues in the protein (Number of hydrogen bonds=0.678·number of residues-3.350.

  7. Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid.

    Science.gov (United States)

    Liu, Xin; Li, Ying; Liang, Jing; Zhu, Wenyue; Xu, Jingyue; Su, Ruifang; Yuan, Lei; Sun, Chunyan

    2016-11-01

    In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3' and 5'-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the "open" configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Molecular epidemiology of wild poliovirus type 1 circulation in West and Central Africa, from 1997 to 1999, using genotyping with a restriction fragment length polymorphism assay.

    Science.gov (United States)

    Gouandjika-Vasilache, Ionela; Burns, Cara C; Gumede, Nicksy; Guillot, Sophie; Ménard, Didier; Dosseh, Annick; Akoua-Koffi, Chantal; Pallansch, Mark A; Kew, Olen M; Delpeyroux, Francis

    2008-01-01

    Virological surveillance is an important element in the Polio Eradication Initiative to provide information rapidly about circulating wild polioviruses. Molecular tools have been developed to identify the serotype of the poliovirus strains and whether they are of vaccine or wild origin (intratypic differentiation) and to perform the molecular epidemiology of wild strains. The main objective of this study was to show that restriction fragment length polymorphism (RFLP) is a tool that can be used for molecular epidemiology of wild polioviruses. This is retrospective study of poliovirus type 1 strains received at the Institut Pasteur of Bangui (IPB), a WHO Regional Reference Laboratory for Africa, since 1994. We describe our experience with isolates from Western and Central Africa and show a positive correlation between the genotypes as determined by sequencing the gene for the VP1 capsid protein and the RFLP patterns. Although genomic sequencing is the gold standard method for detailed molecular epidemiology analysis of poliovirus isolates, these results show that RFLP is a potentially valuable tool for molecular epidemiological analysis of poliovirus type 1 strains: it could be used by many laboratories as a rapid method for ITD and genotype screening where sequencing capacity is not readily available.

  9. Galleria mellonella model identifies highly virulent strains among all major molecular types of Cryptococcus gattii.

    Directory of Open Access Journals (Sweden)

    Carolina Firacative

    Full Text Available Cryptococcosis is mainly caused by Cryptococcus neoformans. However, the number of cases due to C. gattii is increasing, affecting mainly immunocompetent hosts. C. gattii is divided into four major molecular types, VGI to VGIV, which differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. Besides studies on the Vancouver Island outbreak strains, which showed that the subtype VGIIa is highly virulent compared to the subtype VGIIb, little is known about the virulence of the other major molecular types. To elucidate the virulence potential of the major molecular types of C. gattii, Galleria mellonella larvae were inoculated with ten globally selected strains per molecular type. Survival rates were recorded and known virulence factors were studied. One VGII, one VGIII and one VGIV strain were more virulent (p 0.05, 21 (five VGI, five VGII, four VGIII and seven VGIV were less virulent (p <0.05 while one strain of each molecular type were avirulent. Cell and capsule size of all strains increased markedly during larvae infection (p <0.001. No differences in growth rate at 37°C were observed. Melanin synthesis was directly related with the level of virulence: more virulent strains produced more melanin than less virulent strains (p <0.05. The results indicate that all C. gattii major molecular types exhibit a range of virulence, with some strains having the potential to be more virulent. The study highlights the necessity to further investigate the genetic background of more and less virulent strains in order to recognize critical features, other than the known virulence factors (capsule, melanin and growth at mammalian body temperature, that maybe crucial for the development and progression of cryptococcosis.

  10. Molecular typing of uropathogenic Escherichia coli isolated from Korean children with urinary tract infection.

    Science.gov (United States)

    Yun, Ki Wook; Kim, Do Soo; Kim, Wonyong; Lim, In Seok

    2015-01-01

    We investigated the molecular types of uropathogenic Escherichia coli (UPEC) by using conventional phylogrouping, multilocus sequence typing (MLST), and fimH genotyping. Samples of patients younger than 18 years of age were collected from the Chung-Ang University Hospital over 2 years. Conventional phylogenetic grouping for UPEC strains was performed by polymerase chain reaction (PCR). Bacterial strain sequence types (STs) were classified on the basis of the results of partial sequencing of seven housekeeping genes. In addition, we analyzed nucleotide variations in a 424-base pair fragment of fimH, a major virulence factor in UPEC. Sixty-four UPEC isolates were analyzed in this study. Phylogenetic grouping revealed that group B2 was the most common type (n=54, 84%). We identified 16 distinctive STs using MLST. The most common STs were ST95 (35.9%), ST73 (15.6%), ST131 (12.5%), ST69 (7.8%), and ST14 (6.3%). Fourteen fimH allele types were identified, of which 11 had been previously reported, and the remaining three were identified in this study. f1 (n=28, 45.2%) was found to be the most common allele type, followed by f6 and f9 (n=7, 11.3% each). Comparative analysis of the results from the three different molecular typing techniques revealed that both MLST and fimH typing generated more discriminatory UPEC types than did PCR-based phylogrouping. We characterized UPEC molecular types isolated from Korean children by MLST and fimH genotyping. fimH genotyping might serve as a useful molecular test for large epidemiologic studies of UPEC isolates.

  11. Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

    Science.gov (United States)

    Sulakvelidze, Alexander; Kekelidze, Merab; Gomelauri, Tsaro; Deng, Yingkang; Khetsuriani, Nino; Kobaidze, Ketino; De Zoysa, Aruni; Efstratiou, Androulla; Morris, J. Glenn; Imnadze, Paata

    1999-01-01

    Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates. PMID:10488190

  12. Apta-nanosensor preparation and in vitro assay for rapid Diazinon detection using a computational molecular approach.

    Science.gov (United States)

    Jokar, Mahmoud; Safaralizadeh, Mohammad Hassan; Hadizadeh, Farzin; Rahmani, Fatemeh; Kalani, Mohamad Reza

    2017-02-01

    Aptamers (ss-DNA or ss-RNA), also known as artificial antibodies, have been selected in vitro median to bind target molecules with high affinity and selectivity. Diazinon is one of the most widely used organophosphorus insecticides in developing and underdeveloped countries as insecticide and acaricide. Diazinon is readily absorbed from the gastrointestinal system and rapidly distributed throughout the body. Thus, the design of clinical and laboratory diagnostics using nanobiosensors is necessary. A computational approach allows us to screen or rank receptor structure and predict interaction outcomes with a deeper understanding, and it is much more cost effective than laboratory attempts. In this research, the best sequence (high affinity bind Diazinon-ssDNA) was ranked among 12 aptamers isolated from SELEX experimentation. Docking results, as the first virtual screening stage and static technique, selected frequent conformation of each aptamer. Then, the quantity and quality of aptamer-Diazinon interaction were simulated using molecular dynamics as a mobility technique. RMSD, RMSF, radius of gyration, and the number of hydrogen bonds formed between Diazinon-aptamer were monitored to assess the quantity and quality of interactions. G-quadruplex DNA aptamer (DF20) showed to be a reliable candidate for Diazinon biosensing. The apta-nanosensor designed using simulation results allowed with linearity detection in the range of .141-.65 nM and a LOD of 17.903 nM, and it was validated using a computational molecular approach.

  13. Preparation of molecularly imprinted polymer with double templates for rapid simultaneous determination of melamine and dicyandiamide in dairy products.

    Science.gov (United States)

    Liu, Jiang; Song, Han; Liu, Jie; Liu, Yuan; Li, Le; Tang, Hui; Li, Yingchun

    2015-03-01

    In this study, a rapid and accurate determination strategy was established for simultaneous measurement of melamine (MLM) and dicyandiamide (DCD) directly in powdered milk by coupling molecularly imprinted solid-phase extraction (MISPE) with high performance liquid chromatography (HPLC). A novel double-template technique was adopted for preparing SPE packing agent and the obtained double-templated (MLM and DCD) molecularly imprinted polymers (MD-MIPs) was characterized by Fourier-transform infrared spectroscopy and scanning electron microscope (SEM). The molecular recognition ability and the binding capability of the as-prepared polymers towards MLM and DCD were evaluated via static and dynamic binding tests, and it was found that the MD-MIPs showed better affinity and selectivity for both templates compared with single-templated MIPs and non-imprinted polymers (NIPs). An approach based on MISPE and HPLC was then developed and optimized to detect MLM and DCD in powdered milk. The detection limit of the method (S/N=3) were 0.13 μg/g for MLM and 0.07 μg/g for DCD, and the relative standard deviation (RSD) of intra-day and inter-day determination for MLM was 3.3% and 4.7%, and 3.5% and 5.9% for DCD. The recoveries in MLM and DCD analysis at three spiked levels were 93.1-100.1% and 75.7-82.5%, respectively, with all RSD less than 5.2%. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Molecular cloning and characterization of a novel Cys 2 /His 2 -type ...

    African Journals Online (AJOL)

    A novel member of the Cys2/His2-type zinc finger protein gene family, designated DgZFP3, was isolated from chrysanthemum by rapid amplification of cDNA ends (RACE). The DgZFP3 encodes a protein of 248 amino acids, including two conserved Cys2/His2-type zinc finger motifs with a plant-specific QALGGH motif in ...

  15. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    Energy Technology Data Exchange (ETDEWEB)

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  16. Response to “Rapid tests for HIV type discrimination in West Africa may perform differently”

    National Research Council Canada - National Science Library

    Tchounga, Boris K; Ekouevi, Didier K; Eholie, Serge P

    2015-01-01

    ... of each test used for the initial discrimination of HIV‐positive patients. Our team previously conducted in 2004 a field evaluation of rapid HIV serologic tests in Côte d'Ivoire and highlighted the lower accuracy of Genie II for differentiating between HIV‐1, HIV‐2 and dually reactive patients [ 4 ]. In our most recent study, the initial HIV diag...

  17. Response to "Rapid tests for HIV type discrimination in West Africa may perform differently"

    National Research Council Canada - National Science Library

    Tchounga, Boris K; Ekouevi, Didier K; Eholie, Serge P

    2015-01-01

    ... test used for the initial discrimination of HIV-positive patients. Our team previously conducted in 2004 a field evaluation of rapid HIV serologic tests in Cote d'Ivoire and highlighted the lower accuracy of Genie II for differentiating between HIV-1, HIV-2 and dually reactive patients [4]. In our most recent study, the initial HIV diagnostic te...

  18. Combined Campylobacter jejuni and Campylobacter coli Rapid Testing and Molecular Epidemiology in Conventional Broiler Flocks.

    Science.gov (United States)

    Schallegger, G; Muri-Klinger, S; Brugger, K; Lindhardt, C; John, L; Glatzl, M; Wagner, M; Stessl, B

    2016-12-01

    Campylobacter spp. are important causes of bacterial zoonosis, most often transmitted by contaminated poultry meat. From an epidemiological and risk assessment perspective, further knowledge should be obtained on Campylobacter prevalence and genotype distribution in primary production. Consequently, 15 Austrian broiler flocks were surveyed in summer for their thermophilic Campylobacter spp. contamination status. Chicken droppings, dust and drinking water samples were collected from each flock at three separate sampling periods. Isolates were confirmed by PCR and subtyped. We also compared three alternative methods (culture-based enrichment in Bolton broth, culture-independent real-time PCR and a lateral-flow test) for their applicability in chicken droppings. Twelve flocks were found to be positive for thermophilic Campylobacter spp. during the entire sampling period. Seven flocks (46.6%) were contaminated with both, C. jejuni and C. coli, five flocks harboured solely one species. We observed to a majority flock-specific C. jejuni and C. coli genotypes, which dominated the respective flock. Flocks within a distance jejuni genotypes indicating a cross-contamination event via the environment or personnel vectors. Multilocus sequence typing (MLST) of C. jejuni revealed that the majority of isolates were assigned to globally distributed clonal complexes or had a strong link to the human interface (CC ST-446 and ST4373). The combination of techniques poses an advantage over risk assessment studies based on cultures alone, as, in the case of Campylobacter, occurrence of a high variety of genotypes might be present among a broiler flock. We suggest applying the lateral-flow test under field conditions to identify 'high-shedding' broiler flocks at the farm level. Consequently, poultry farmers and veterinarians could improve hygiene measurements and direct sanitation activities, especially during the thinning period. Ultimately, real-time PCR could be applied to quantify

  19. Molecular Typing of Treponema pallidum in Denmark: A Nationwide Study of Syphilis.

    Science.gov (United States)

    Salado-Rasmussen, Kirsten; Cowan, Susan; Gerstoft, Jan; Larsen, Helle Kiellberg; Hoffmann, Steen; Knudsen, Troels Bygum; Katzenstein, Terese Lea; Jensen, Jørgen Skov

    2016-02-01

    The aim of this nationwide study is to determine the strain type diversity among patients diagnosed with syphilis by PCR during a 4-year period in Denmark. Epidemiological data, including HIV status, for all patients were obtained from the Danish national syphilis registration system. Molecular strain typing was based on characterization of 3 variable treponemal genes, arp, tpr and tp0548. A total of 278 specimens from 269 patients were included. Among the fully typeable specimens (n = 197), 22 strain types were identified, with 1 type, 14d/g, accounting for 54%. The majority (93%) of the patients reported acquiring syphilis in Denmark. Among patients with concurrent HIV, 9 full strain types were identified and no difference in strain type was found by HIV status (p = 0.197). In conclusion, the majority of patients were infected in Denmark and the HIV-infected syphilis patients were diagnosed with a wide spectrum of different strain types of Treponema pallidum.

  20. Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

    Science.gov (United States)

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A.; Brennan, Patrick J.

    2012-01-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations. PMID:22170923

  1. The Risk and Clinical/Molecular Characteristics of Breast Cancer in Women with Neurofibromatosis Type 1

    Science.gov (United States)

    2015-09-01

    risk of breast cancer in women with NF1. Plexiform neurofibroma may be a predictor for MPNST. Learning disability and European ancestry may be a...1 Award Number: W81XWH-11-1-0671 TITLE: The Risk and Clinical/Molecular Characteristics of Breast Cancer in Women with Neurofibromatosis Type 1...Characteristics of Breast Cancer in Women with Neurofibromatosis Type 1” 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER

  2. Phenological Metrics Extraction for Agricultural Land-use Types Using RapidEye and MODIS

    Science.gov (United States)

    Xu, Xingmei; Doktor, Daniel; Conrad, Christopher

    2016-04-01

    Crop phenology involves the various agricultural events, such as planting, emergence, flowering, development of fruit and harvest. These phenological stages of a crop contain essential information for practical agricultural management, crop productivity estimation, investigations of crop-weather relationships, and also play an important role in improving agricultural land-use classification. In this study, we used MODIS and RapidEye images to extract phenological metrics in central Germany between 2010 and 2014. The Best Index Slope Extraction algorithm was used to remove undesirable data noise from Normalized Difference Vegetation Index (NDVI) time series of both satellite data before fast Fourier transformation was applied. Metrics optimization for phenology of major crops in the study area (winter wheat, winter barley, winter oilseed rape and sugar beet) and validation were performed with intensive ground observations from the German Weather Service (2010-2014) and our own measurements of BBCH code (Biologische Bundesanstalt für Land- und Forstwirtschaft, Bundessortenamt und CHemische Industrie) (in 2014). We found that the dates with maximum NDVI have a close link to the heading stage of cereals (RMSE = 9.48 days for MODIS and RMSE = 13.55 days for RapidEye), and the dates of local half maximum during senescence period of winter crops was strongly related to ripeness stage (BBCH: 87) (RMSE = 8.87 days for MODIS and RMSE = 9.62 days for RapidEye). The root-mean-square errors (RMSE) of derived green up dates for both winter and summer crops were larger than 2 weeks, which was caused by limited number of good quality images during the winter season. Comparison between RapidEye and homogeneous MODIS pixels indicated that phenological metrics derived from both satellites were similar to the crop calendar in this region. We also investigated the influence of spatial aggregation of RapidEye-scale phenology to MODIS scale as well as the effect of decreasing the

  3. C-type lectin interactions with Schistosoma mansoni SEA : Molecular basis and function

    NARCIS (Netherlands)

    Liempt, van P.A.G.

    2007-01-01

    Outline of this thesis The studies described in this thesis have been performed to gain more insight in the recognition of Schistosoma mansoni glycans by C-type lectins and the consequences for dendritic cell mediated immune responses. As a first approach to understand the molecular interactions

  4. Molecular misreading: a new type of transcript mutation expressed during aging

    NARCIS (Netherlands)

    van Leeuwen, F. W.; Fischer, D. F.; Kamel, D.; Sluijs, J. A.; Sonnemans, M. A.; Benne, R.; Swaab, D. F.; Salehi, A.; Hol, E. M.

    2000-01-01

    Dinucleotide deletions (e.g. DeltaGA, DeltaGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs. As a result, the reading frame shifts to the +1 frame, and so-called "+1 proteins" are subsequently synthesized. +1 Proteins have a wild-type N-terminus, but an

  5. VIRO-TypeNed, systematic molecular surveillance of enteroviruses in the Netherlands between 2010 and 2014

    NARCIS (Netherlands)

    K. Benschop (Kimberley); J. Rahamat-Langendoen (Janette); H.G.A.M. van der Avoort (Harrie); E.C.J. Claas (Eric); S.D. Pas (Suzan); R. Schuurman (Rob); J.J. Verweij (Jaco); K.C. Wolthers (Katja); H.G.M. Niesters (Bert); M.P.G. Koopmans D.V.M. (Marion)

    2016-01-01

    textabstractVIRO-TypeNed is a collaborative molecular surveillance platform facilitated through a web-based database. Genetic data in combination with epidemiological, clinical and patient data are shared between clinical and public health laboratories, as part of the surveillance underpinning

  6. VIRO-TypeNed, systematic molecular surveillance of enteroviruses in the Netherlands between 2010 and 2014

    NARCIS (Netherlands)

    Benschop, K. S. M.; Rahamat-Langendoen, J. C.; van der Avoort, H. G. A. M.; Claas, E. C. J.; Pas, S. D.; Schuurman, R.; Verweij, J. J.; Wolthers, K. C.; Niesters, H. G. M.; Koopmans, M. P. G.

    VIRO-TypeNed is a collaborative molecular surveillance platform facilitated through a web-based database. Genetic data in combination with epidemiological, clinical and patient data are shared between clinical and public health laboratories, as part of the surveillance underpinning poliovirus

  7. Molecular genetic analysis of Type II diabetes associated m.3243A ...

    African Journals Online (AJOL)

    Saidul Abrar

    Molecular genetic analysis of Type II diabetes associated m.3243A>G mitochondrial DNA mutation in a Pakistani family. Saidul Abrar a, Khushi Muhammad b, Hasnain Zaman c, Suleman Khan b, Faisal Nouroz a,d, Nousheen Bibi a,* a Department of Bioinformatics, Hazara University Mansehra, Pakistan b Department of ...

  8. Beneficial autoimmunity at body surfaces– immune surveillance and rapid type 2 immunity regulate tissue homeostasis and cancer

    Directory of Open Access Journals (Sweden)

    Tim eDalessandri

    2014-07-01

    Full Text Available Epithelial cells line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation or toxins cause activation of epithelial cells with release of cytokines and chemokines as well as alterations in the expression of cell surface ligands. Such display of epithelial stress is rapidly sensed by tissue resident immunocytes, which can directly interact with self-moieties on epithelial cells and initiate both local and systemic immune responses. Epithelial cells are thus key drivers of immune surveillance at body surface tissues. However, epithelial cells have a propensity to drive type 2 immunity (rather than type 1 upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis.

  9. A refined method for molecular typing reveals that co-occurrence of PrPSc types in Creutzfeldt-Jakob disease is not the rule

    NARCIS (Netherlands)

    Notari, S.; Capellari, S.; Langeveld, J.P.M.; Giese, A.; Strammiello, R.; Gambetti, P.; Kretzschmar, H.A.; Parchi, P.

    2007-01-01

    Molecular typing in Creutzfeldt¿Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrPSc) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrPSc types

  10. Reinterpreting a community outbreak of Salmonella enterica serotype Enteritidis in the light of molecular typing

    Directory of Open Access Journals (Sweden)

    Nastasi Antonino

    2007-09-01

    Full Text Available Abstract Background In November 2005, a large outbreak due to Salmonella enterica serotype Enteritidis (S. Enteritidis was observed within children who had eaten their meals at 53 school cafeterias in Florence and the surrounding area. A total of 154 isolates of S. Enteritidis were recovered from human cases between November 2005 and January 2006. All strains were assigned phage type 8 (PT8 and a common XbaI pulsotype. This paper reports the findings of a molecular epidemiological investigation performed on 124 strains of S. Enteritidis isolated in the years 2005 and 2006 in Florence and the surrounding area, including the epidemic isolates. Methods One hundred twenty-four human isolates of S. Enteritidis identified in the period January 2005 – December 2006 were submitted to molecular typing by single enzyme – amplified fragment length polymorphism (SE-AFLP. Results Molecular subtyping by SE-AFLP yielded five different profiles. In the pre-epidemic phase, type A included 78.4% of isolates, whereas only three (8.1% belonged to type C. All isolates, but one, of the epidemic phase were indistinguishable and attributed to type C. In the post-epidemic period, a polymorphic pattern of SE-AFLP types was again recognized but type C accounted for 73.3% of the isolates during the first six months of 2006, whereas during the remaining six months type A regained the first place, including 52.0% of the isolates. Conclusion The epidemic event was attributed to the emergence and clonal expansion of a strain of S. Enteritidis PT8-SE-AFLP type C. Circulation of the epidemic clone was much more extensive than the surveillance and traditional laboratory data demonstrated.

  11. Next-Generation Sequencing-Aided Rapid Molecular Diagnosis of Occult Macular Dystrophy in a Chinese Family

    Directory of Open Access Journals (Sweden)

    Yu-He Qi

    2017-08-01

    Full Text Available Purpose: To show early, rapid and accurate molecular diagnosis of occult macular dystrophy (OMD in a four-generation Chinese family with inherited macular dystrophy.Methods: In the current study, we comprehensively screened 130 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the proband of a four-generation Chinese family that has suffered from maculopathy without a definitive diagnosis for over 10 years. Variants were filtered and analyzed to identify possible disease-causing variants before validation by Sanger sequencing.Results: Two heterozygous mutations—RP1L1 c.133 C > T (p.Arg45Trp, which is a hot spot for OMD, and ABCA4 c.6119 G > A (p.Arg2040Gln, which was identified in Stargardt’s disease were found in three patients, but neither of the mutations was found in the unaffected individuals in the same family, who are phenotypically normal or in the normal control volunteers.Conclusion: These results cannot only confirm the diagnosis of OMD in the proband, but also provide presymptomatic diagnosis of the proband’s children before the onset of visual acuity impairment and guidance regarding the prognosis and management of these patients. Heterozygous mutations of RP1L1 c.133 C > T (p.Arg45Trp and ABCA4 c.6119 G > A (p.Arg2040Gln are likely responsible for OMD. Our results further extend our current understanding of the genetic basis of OMD, and emphasize the importance of molecular diagnosis and genetic counseling for OMD.

  12. Next-Generation Sequencing-Aided Rapid Molecular Diagnosis of Occult Macular Dystrophy in a Chinese Family.

    Science.gov (United States)

    Qi, Yu-He; Gao, Feng-Juan; Hu, Fang-Yuan; Zhang, Sheng-Hai; Chen, Jun-Yi; Huang, Wan-Jing; Tian, Guo-Hong; Wang, Min; Gan, De-Kang; Wu, Ji-Hong; Xu, Ge-Zhi

    2017-01-01

    Purpose: To show early, rapid and accurate molecular diagnosis of occult macular dystrophy (OMD) in a four-generation Chinese family with inherited macular dystrophy. Methods: In the current study, we comprehensively screened 130 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the proband of a four-generation Chinese family that has suffered from maculopathy without a definitive diagnosis for over 10 years. Variants were filtered and analyzed to identify possible disease-causing variants before validation by Sanger sequencing. Results: Two heterozygous mutations-RP1L1 c.133 C > T (p.Arg45Trp), which is a hot spot for OMD, and ABCA4 c.6119 G > A (p.Arg2040Gln), which was identified in Stargardt's disease were found in three patients, but neither of the mutations was found in the unaffected individuals in the same family, who are phenotypically normal or in the normal control volunteers. Conclusion: These results cannot only confirm the diagnosis of OMD in the proband, but also provide presymptomatic diagnosis of the proband's children before the onset of visual acuity impairment and guidance regarding the prognosis and management of these patients. Heterozygous mutations of RP1L1 c.133 C > T (p.Arg45Trp) and ABCA4 c.6119 G > A (p.Arg2040Gln) are likely responsible for OMD. Our results further extend our current understanding of the genetic basis of OMD, and emphasize the importance of molecular diagnosis and genetic counseling for OMD.

  13. MDMS: Molecular dynamics meta-simulator for evaluating exchange type sampling methods

    Science.gov (United States)

    Smith, Daniel B.; Okur, Asim; Brooks, Bernard R.

    2012-08-01

    Replica exchange methods have become popular tools to explore conformational space for small proteins. For larger biological systems, even with enhanced sampling methods, exploring the free energy landscape remains computationally challenging. This problem has led to the development of many improved replica exchange methods. Unfortunately, testing these methods remains expensive. We propose a molecular dynamics meta-simulator (MDMS) based on transition state theory to simulate a replica exchange simulation, eliminating the need to run explicit dynamics between exchange attempts. MDMS simulations allow for rapid testing of new replica exchange based methods, greatly reducing the amount of time needed for new method development.

  14. Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

    Science.gov (United States)

    Benitez, Alvaro J; Winchell, Jonas M

    2016-04-01

    We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

  15. Osteogenesis imperfecta type I: Molecular heterogeneity for COL1A1 null alleles of type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Willing, M.C.; Deschenes, S.P.; Pitts, S.H.; Arikat, H.; Roberts, E.J.; Scott, D.A.; Slayton, R.L. [Univ. of Iowa, Iowa City, IA (United States); Byers, P.H. [Univ. of Washington, Seattle, WA (United States)

    1994-10-01

    Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 {open_quotes}null{close_quotes} allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5{prime} donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype. 58 refs., 4 figs., 1 tab.

  16. A molecular dynamics study on thin film liquid boiling characteristics under rapid linear boundary heating: Effect of liquid film thickness

    Science.gov (United States)

    Rabbi, Kazi Fazle; Tamim, Saiful Islam; Faisal, A. H. M.; Mukut, K. M.; Hasan, Mohammad Nasim

    2017-06-01

    This study is a molecular dynamics investigation of phase change phenomena i.e. boiling of thin liquid films subjected to rapid linear heating at the boundary. The purpose of this study is to understand the phase change heat transfer phenomena at nano scale level. In the simulation, a thin film of liquid argon over a platinum surface has been considered. The simulation domain herein is a three-phase system consisting of liquid and vapor argon atoms placed over a platinum wall. Initially the whole system is brought to an equilibrium state at 90 K and then the temperature of the bottom wall is increased to a higher temperature (250K) within a finite time interval. Four different liquid argon film thicknesses have been considered (3 nm, 4 nm, 5 nm and 6 nm) in this study. The boundary heating rate (40×109 K/s) is kept constant in all these cases. Variation in system temperature, pressure, net evaporation number, spatial number density of the argon region with time for different film thickness have been demonstrated and analyzed. The present study indicates that the pattern of phase transition may be significantly different (i.e. evaporation or explosive boiling) depending on the liquid film thickness. Among the four cases considered in the present study, explosive boiling has been observed only for the liquid films of 5nm and 6nm thickness, while for the other cases, evaporation take place.

  17. Optimization of Molecularly Imprinted Polymer Method for Rapid Screening of 17β-Estradiol in Water by Fluorescence Quenching

    Directory of Open Access Journals (Sweden)

    Yu Yang

    2011-01-01

    Full Text Available A new method was optimized for rapid screening of 17β-estradiol (E2 in water under 10 min. Molecularly imprinted polymer (MIP particles (325 ± 25 nm were added in a water sample at pH 5.5 and 20∘C to form a suspension. Fluorescence emission from E2 nonspecifically bound onto the MIP particles was first quenched by large gold nanoparticles (43 ± 5 nm. The Stern-Volmer plot was linear, with dynamic quenching constants (Ksv of 2.9 ×104 M-1. Fluorescence emission from E2 specifically bound inside the MIP particles was next quenched by small nitrite anions that easily penetrated the imprinted cavities. The Stern-Volmer plot became nonlinear, with Ksv = 2.1 × 102 M-1 and static quenching constant (V below 1.0 M-1. The difference between these two emission intensities varied as the initial E2 concentration in water, generating a Scatchard calibration curve with R2>0.97 from 0.1 to 10 ppb.

  18. Use of Molecular Methods for the Rapid Mass Detection of Schistosoma mansoni (Platyhelminthes: Trematoda) in Biomphalaria spp. (Gastropoda: Planorbidae).

    Science.gov (United States)

    Caldeira, Roberta Lima; Jannotti-Passos, Liana Konovaloffi; Dos Santos Carvalho, Omar

    2017-01-01

    The low stringency-polymerase chain reaction (LS-PCR) and loop-mediated isothermal amplification (LAMP) assays were used to detect the presence of S. mansoni DNA in (1) Brazilian intermediate hosts (Biomphalaria glabrata, B. straminea, and B. tenagophila) with patent S. mansoni infections, (2) B. glabrata snails with prepatent S. mansoni infections, (3) various mixtures of infected and noninfected snails; and (4) snails infected with other trematode species. The assays showed high sensitivity and specificity and could detect S. mansoni DNA when one positive snail was included in a pool of 1,000 negative specimens of Biomphalaria. These molecular approaches can provide a low-cost, effective, and rapid method for detecting the presence of S. mansoni in pooled samples of field-collected Biomphalaria. These assays should aid mapping of transmission sites in endemic areas, especially in low prevalence regions and improve schistosomiasis surveillance. It will be a useful tool to monitor low infection rates of snails in areas where control interventions are leading towards the elimination of schistosomiasis.

  19. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  20. Quiescent-phase evolution of a surge-type glacier: Black Rapids Glacier, Alaska, U.S.A.

    Science.gov (United States)

    Heinrichs, T.A.; Mayo, L.R.; Echelmeyer, K.A.; Harrison, W.D.

    1996-01-01

    Black Rapids Glacier, a surge-type glacier in the Alaska Range, most recently surged in 1936-37 and is currently in its quiescent phase. Mass balance, ice velocity and thickness change have been measured at three to ten sites from 1972 to 1994. The annual speed has undergone cyclical fluctuations of as much as 45% about the mean speed. Ice thickness and surface slope did not change enough to cause the speed fluctuations through changes in ice deformation, which indicates that they are being driven by changes in basal motion. The behavior of Black Rapids Glacier during this quiescent phase is significantly different from that of Variegated Glacier, another well-studied surge-type glacier in Alaska. The present medial-moraine configuration of Black Rapids Glacier indicates that a surge could occur at any time. However, ice velocity data indicate that the next surge may not be imminent. We believe that there is little chance that the next surge will cross and dam the Delta River.

  1. Rapid nickel diffusion in cold-worked type 316 austenitic steel at 360-500 C

    Energy Technology Data Exchange (ETDEWEB)

    Arioka, Koji [Institute of Nuclear Safety Systems, Inc., Mihama (Japan); Iijima, Yoshiaki [Tohoku Univ., Sendai (Japan). Dept. of Materials Science; Miyamoto, Tomoki [Kobe Material Testing Laboratory Co. Ltd., Harima (Japan)

    2017-10-15

    The diffusion coefficient of nickel in cold-worked Type 316 austenitic steel was determined by the diffusion couple method in the temperature range between 360 and 500 C. A diffusion couple was prepared by electroless nickel plating on the surface of a 20 % cold-worked Type 316 austenitic steel specimen. The growth in width of the interdiffusion zone was proportional to the square root of diffusion time until 14 055 h. The diffusion coefficient of nickel (D{sub Ni}) in cold-worked Type 316 austenitic steel was determined by extrapolating the concentration-dependent interdiffusion coefficient to 11 at.% of nickel. The value of D{sub Ni} at 360 C was about 5 000 times higher than the lattice diffusion coefficient of nickel in Type 316 austenitic steel. The determined activation energy 117 kJ mol{sup -1} was 46.6 % of the activation energy 251 kJ mol{sup -1} for the lattice diffusion of nickel in Type 316 austenitic steel.

  2. Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

    Science.gov (United States)

    Zhang, Yonghua; Ai, Junjie; Gu, Qiaorong; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2017-03-01

    Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

  3. Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods.

    Science.gov (United States)

    Ei, Phyu Win; Aung, Wah Wah; Lee, Jong Seok; Choi, Go Eun; Chang, Chulhun L

    2016-11-01

    Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units-variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

  4. Short communication: HIV type 1 escapes inactivation by saliva via rapid escape into oral epithelial cells.

    Science.gov (United States)

    Dietrich, Elizabeth A; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Kappes, John C; Ross, Karen F; Herzberg, Mark C

    2012-12-01

    Saliva contains anti-HIV-1 factors, which show unclear efficacy in thwarting mucosal infection. When incubated in fresh, unfractionated whole saliva, infectious HIV-1 IIIb and BaL (X4- and R5-tropic, respectively) persisted from 4 to at least 30 min in a saliva concentration-dependent manner. In salivary supernatant for up to 6 h, both infectious HIV-1 strains "escaped" into immortalized oral epithelial cells; infectious BaL showed selectively enhanced escape in the presence of saliva. Fluorescently labeled HIV-1 virus-like particles entered oral epithelial cells within minutes of exposure. Using a previously unrecognized mechanism, therefore, strains of HIV-1 escape inactivation by saliva via rapid uptake into oral epithelial cells.

  5. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  6. Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

    Science.gov (United States)

    Goller, K V; Dill, V; Madi, M; Martin, P; Van der Stede, Y; Vandenberge, V; Haas, B; Van Borm, S; Koenen, F; Kasanga, C J; Ndusilo, N; Beer, M; Liu, L; Mioulet, V; Armson, B; King, D P; Fowler, V L

    2017-11-09

    Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world. © 2017 The Authors. Transboundary and Emerging Diseases

  7. KRAS mutation testing in borderline ovarian tumors and low-grade ovarian carcinomas with a rapid, fully integrated molecular diagnostic system.

    Science.gov (United States)

    Sadlecki, Pawel; Antosik, Paulina; Grzanka, Dariusz; Grabiec, Marek; Walentowicz-Sadlecka, Malgorzata

    2017-10-01

    Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors. Implementation of targeted therapeutic strategies requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is fully integrated, real-time polymerase chain reaction-based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded tumor samples. The primary aim of this study was to verify whether fully integrated real-time polymerase chain reaction-based Idylla system may be useful in determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas. The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as borderline ovarian tumors. Seven patients examined with Idylla KRAS Mutation Test tested positive for KRAS mutation. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors. Mean survival of the study subjects was 48.51 months (range 3-60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue.

  8. Molecular characterization of spa type t127, sequence type 1 methicillin-resistant Staphylococcus aureus from pigs.

    Science.gov (United States)

    Franco, Alessia; Hasman, Henrik; Iurescia, Manuela; Lorenzetti, Raniero; Stegger, Marc; Pantosti, Annalisa; Feltrin, Fabiola; Ianzano, Angela; Porrero, Maria Concepción; Liapi, Maria; Battisti, Antonio

    2011-06-01

    The aim of this study was to provide molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) spa type t127, sequence type (ST) 1 isolates, detected in a European baseline survey in holdings of breeding pigs, to determine phenotypic and genotypic drug resistance and to compare the results with those obtained from a collection of t127, ST1 MRSA and methicillin-susceptible S. aureus (MSSA) clinical isolates. Twenty-four t127, ST1 MRSA from dust sampled in different breeding holdings in Italy, Spain and Cyprus were studied, along with 2 t127, ST1 MRSA from fattening pigs and 11 human t127, ST1 MRSA and MSSA. Genotyping was performed using multilocus sequence typing (MLST), spa typing and PFGE. SCCmec elements were characterized by multiplex-PCR and resistance and pathogenicity genes by PCR and microarray. PFGE patterns separated a porcine cluster (PC) from a human cluster (HC), with 75% similarity. The PC carried SCCmec cassette type V, while all isolates of the HC carried SCCmec cassette type IVa. Kanamycin resistance mediated by aadD, fluoroquinolone and erm(A)-mediated macrolide resistance and the absence of the sakA gene were features of the PC only. All isolates of both clusters were positive for LukE-LukD and LuF-LukS-HlgA leukotoxin genes and one human MSSA harboured Panton-Valentine leucocidin genes. Despite differences in the host-specific genetic features, the possibility of PC transmission to humans cannot be excluded. MRSA spa type t127, ST1 from pigs possesses several virulence and resistance genes towards major classes of antimicrobials and may represent a serious therapeutic challenge in case of invasive infections in humans.

  9. Rapid calibration of a projection-type holographic light-field display using hierarchically upconverted binary sinusoidal patterns.

    Science.gov (United States)

    Nakamura, Tomoya; Yamaguchi, Masahiro

    2017-12-01

    A projection-type holographic light-field (LF) display is a full-parallax, full-color, and glass-free three-dimensional (3D) display with a holographic optical element and a projector. The display has unique characteristics, including transparency; however, a rapid calibration method has not yet been established. In this paper, we propose a rapid calibration method for a holographic LF display without sacrificing its accuracy. The proposed method performs calibration via the projection of binary sinusoidal patterns whose frequencies are iteratively and hierarchically upconverted. Compared to the conventional method, in the proposed method, the required number of projections is reduced from linear to logarithmic with the projector's resolution. We confirm the successful reconstruction of the 3D image using the proposed method.

  10. Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2017-01-01

    In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

  11. Best practice guidelines for the molecular genetic diagnosis of Type 1 (HFE-related hereditary haemochromatosis

    Directory of Open Access Journals (Sweden)

    Barton David E

    2006-11-01

    Full Text Available Abstract Background Hereditary haemochromatosis (HH is a recessively-inherited disorder of iron over-absorption prevalent in Caucasian populations. Affected individuals for Type 1 HH are usually either homozygous for a cysteine to tyrosine amino acid substitution at position 282 (C282Y of the HFE gene, or compound heterozygotes for C282Y and for a histidine to aspartic acid change at position 63 (H63D. Molecular genetic testing for these two mutations has become widespread in recent years. With diverse testing methods and reporting practices in use, there was a clear need for agreed guidelines for haemochromatosis genetic testing. The UK Clinical Molecular Genetics Society has elaborated a consensus process for the development of disease-specific best practice guidelines for genetic testing. Methods A survey of current practice in the molecular diagnosis of haemochromatosis was conducted. Based on the results of this survey, draft guidelines were prepared using the template developed by UK Clinical Molecular Genetics Society. A workshop was held to develop the draft into a consensus document. The consensus document was then posted on the Clinical Molecular Genetics Society website for broader consultation and amendment. Results Consensus or near-consensus was achieved on all points in the draft guidelines. The consensus and consultation processes worked well, and outstanding issues were documented in an appendix to the guidelines. Conclusion An agreed set of best practice guidelines were developed for diagnostic, predictive and carrier testing for hereditary haemochromatosis and for reporting the results of such testing.

  12. Molecular Typing and Epidemiology of Human Listeriosis Cases, Denmark, 2002–20121

    Science.gov (United States)

    Björkman, Jonas T.; Ethelberg, Steen; Kiil, Kristoffer; Kemp, Michael; Nielsen, Eva Møller

    2016-01-01

    Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002–2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005–2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting the need for more refined discriminatory typing methods to identify outbreaks within frequently occurring L. monocytogenes PFGE types. PMID:26982714

  13. Molecular and Phylogenetic Analysis of Bovine Papillomavirus Type 1: First Report in Iraqi Cattle

    OpenAIRE

    Mohammed A Hamad; Al-Shammari, Ahmed M; Odisho, Shoni M.; Yaseen, Nahi Y.

    2016-01-01

    This study aimed to provide the first molecular characterization of bovine papillomavirus type 1 (BPV-1) in Iraq. BPV is a widely spread oncogenic virus in Iraqi cattle and is associated with the formation of both benign and malignant lesions, resulting in notable economic losses in dairy and beef cattle. In the current study, 140 cutaneous papilloma specimens were collected from cattle in central Iraq. These samples were submitted to histopathological examination, PCR, and sequencing analysi...

  14. Energy-efficient hydrogen separation by AB-type ladder-polymer molecular sieves.

    Science.gov (United States)

    Ghanem, Bader S; Swaidan, Raja; Ma, Xiaohua; Litwiller, Eric; Pinnau, Ingo

    2014-10-22

    Increases in hydrogen selectivity of more than 100% compared with the most selective ladder polymer of intrinsic microporosity (PIM) reported to date are achieved with self-polymerized A-B-type ladder monomers comprising rigid and three-dimensional 9,10-dialkyl-substituted triptycene moieties. The selectivities match those of materials commercially employed in hydrogen separation, but the gas permeabilities are 150-fold higher. This new polymer molecular sieve is also the most selective PIM for air separation.

  15. Peranan Terapi Awal dan Terapi Pemeliharaan pada Rapidly Progressive Periodontitis Type 1

    Directory of Open Access Journals (Sweden)

    Anita H. Joedo

    2015-08-01

    Full Text Available Rapidly Progressive Periodontitis (RPP is a severe form of a periodontal disease which starts since a puberty age. The disease if a generalized periodontal destruction without a specific distribution mode: it develops more progressively but does not in accordance to local factors. The first step to the RPP treatment is initial therapy: i.e. DHE, scaling and root planing, and eliminating predisposing local factors and continued with a maintence therapy which will support the success of a surgery later. A study case: a 21-year old RPP woman showed hyperaemia, an abscess, a 10 mm mesial pocket depth, a 5 mm distal pocker depth, a 5 mm buccal pocket depth, a 2nd degree tooth mobility and a 3 mm buccal recession on 25. In the initial therapy she was given an amoxicillin, a metronidazole for killing a supra and subgingival baterial, vitamins B and C, and also a chlorhexidine 0.2% mouth wash for a week. After a week the abscess and the inflammation decreased, but the mobility was still in the same condition and the DHE was still evaluated because of the patient's social factor, the FO was delayed. The next visit was done every 2 monts for a year for maintenance care. The clinical result showed the gingival inflammation and the tooth mobility disappeared. Radiographically, the alveolar bone showed more radiopaque and the lamina dura was seen. In conclusio, the initial and the maintenance therapy was seen to heal the RPP.

  16. Novel automated point collection software facilitates rapid, high-density electroanatomical mapping with multiple catheter types.

    Science.gov (United States)

    Ptaszek, Leon M; Moon, Boyce; Rozen, Guy; Mahapatra, Srijoy; Mansour, Moussa

    2018-01-01

    Manual, point-by-point electroanatomical mapping requires the operator to directly evaluate each point during map construction. Consequently, point collection can be a slow process. An automated 3D mapping system was developed with the goal of improving key mapping metrics, including map completion time and point density. Automated 3D mapping software that includes morphology and cycle length discrimination functions for surface and intracardiac electrograms was developed. In five swine, electroanatomical maps (EAMs) of all four cardiac chambers were generated in sinus rhythm. Four catheters were used: two different four-pole ablation catheters, a 20-pole circular catheter, and a 64-pole basket catheter. Automated and manual 3D mapping were compared for 12 different catheter-chamber combinations (paired sets of 10 maps for most combinations, for a total of 156 maps). Automated 3D mapping produced more than twofold increase in the number of points per map, as compared with manual 3D mapping (P ≤0.007 for all catheter-chamber combinations tested). Automated 3D mapping also reduced map completion time by an average of 29% (P software described in this study is significantly faster than manual, point-by-point 3D mapping. This resulted in shorter mapping time and higher point density. The morphology discrimination functions effectively excluded ectopic beats during mapping in sinus rhythm and allowed for rapid mapping of intermittent ventricular ectopic beats. © 2017 Wiley Periodicals, Inc.

  17. Quantum Interference Effects in Molecular Y- and Rhomb-Type Systems

    Science.gov (United States)

    Xia, Hui-Rong; Ye, Cen-Yun; Xu, Jian-We; Ding, Liang-En

    1996-01-01

    In this paper we report the first observation of molecular population trapping in four level systems. Constructive and destructive quantum interferences between two sum-frequncy two-photon transitions in Y- and rhomb-type four-level systems, respectively, im sodium molecules have been experimentally achieved by using only one laser source. Their energy level schemes are featured by the extremely near-resonant enhancement of the equal-frequency two-photon transitions, sharing both the initial and the intermediate levels for the Y-type, and sharing both the initial and the final levels for the rhomb-type systems. Their novel spectral effects are to show seriously restrained Doppler-free UV peak at the nominal location of the induced two-photon transition with visible fluorescence in rhomb-type schems, and to show a strong extra UV peak but null visible fluorescence in the middle between the two dipole allowed two-photon transitions.

  18. Rapid Assay for Simultaneous Detection and Differentiation of Immunoglobulin G Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2

    OpenAIRE

    Vallari, Ana S.; Hickman, Robert K.; Hackett, John R.; Brennan, Catherine A.; Varitek, Vincent A.; Devare, Sushil G.

    1998-01-01

    A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western b...

  19. Rapid changes in plasma androgens during insulin withdrawal in male type 1 (insulin-dependent) diabetics

    DEFF Research Database (Denmark)

    Madsbad, S; Gluud, C; Bennett, Patrick

    1986-01-01

    Plasma concentrations of testosterone, androstenedione and dihydrotestosterone were measured in 15 Type 1 (insulin-dependent) diabetics with (n = 8) and without (n = 7) B-cell function during 12 h of insulin withdrawal and compared with those of 8 normal subjects. Before insulin withdrawal...... the patients without B-cell function were more metabolically decompensated from after 4 h of insulin withdrawal compared with patients with B-cell function, no significant differences were found in androgen concentrations between the two groups although a tendency to lower concentrations were seen in the group...... without B-cell function....

  20. Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification.

    Science.gov (United States)

    Wang, Jianchang; Liu, Libing; Li, Ruiwen; Wang, Jinfeng; Fu, Qi; Yuan, Wanzhe

    2016-04-01

    A novel recombinase polymerase amplification (RPA)-based method for detection of canine parvovirus type 2 (CPV-2) was developed. Sensitivity analysis showed that the detection limit of RPA was 10 copies of CPV-2 genomic DNA. RPA amplified both CPV-2a and -2b DNA but did not amplify the template of other important dog viruses (CCoV, PRV or CDV), demonstrating high specificity. The method was further validated with 57 canine fecal samples. An outstanding advantage of RPA is that it is an isothermal reaction and can be performed in a water bath, making RPA a potential alternative method for CPV-2 detection in resource-limited settings.

  1. A molecular epidemiological perspective of rhinovirus types circulating in Amsterdam from 2007 to 2012.

    Science.gov (United States)

    van der Linden, L; Bruning, A H L; Thomas, X V; Minnaar, R P; Rebers, S P H; Schinkel, J; de Jong, M D; Pajkrt, D; Wolthers, K C

    2016-12-01

    Rhinoviruses (RVs) are frequently detected respiratory viruses that cause mild common cold symptoms, but may also lead to more severe respiratory tract infections. The large number of RV types, classified into species A, B and C, hampers clear insights into the epidemiology and clinical significance of each RV type. The aim of this study was to map the circulation of RV types in the Amsterdam area. RV-positive nasopharyngeal and oropharyngeal samples, collected from 2007 to 2012 in the Academic Medical Centre (Amsterdam, the Netherlands), were typed based on the sequence of the region coding for capsid proteins VP4 and VP2. RV-A, RV-B and RV-C were found in proportions of of 52.4% (334/637), 11.3% (72/637), and 36.2% (231/637), respectively. We detected 129 of the 167 currently classified types. RVs circulated throughout the entire year with a peak in the autumn and a decline in the summer. Some RV types were observed throughout the entire sampling period and others had a more seasonal pattern. Nine RV-A and four RV-B novel provisionally assigned types were identified. This study provides an insight into the molecular epidemiology of RVs in the Amsterdam area. The RVs circulating are diverse and include several provisionally new types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  2. Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World.

    Science.gov (United States)

    Fraga, Jorge; Veland, Nicolas; Montalvo, Ana M; Praet, Nicolas; Boggild, Andrea K; Valencia, Braulio M; Arévalo, Jorge; Llanos-Cuentas, Alejandro; Dujardin, Jean-Claude; Van der Auwera, Gert

    2012-10-01

    The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Rapidly rising incidence of childhood type 1 diabetes in Chinese population: epidemiology in Shanghai during 1997-2011.

    Science.gov (United States)

    Zhao, Zhuhui; Sun, Chengjun; Wang, Chunfang; Li, Pin; Wang, Wei; Ye, Jun; Gu, Xuefan; Wang, Xiaodong; Shen, Shuixian; Zhi, Dijing; Lu, Zhong; Ye, Rong; Cheng, Ruoqian; Xi, Li; Li, Xiaojing; Zheng, Zhangqian; Zhang, Miaoying; Luo, Feihong

    2014-12-01

    The aim of this study was to investigate incidence trend of childhood type 1 diabetes in Shanghai, a megalopolis in east China. We established a population-based retrospective registry for the disease in the city's registered population during 1997-2011 and collected 622 incident type 1 diabetes in children aged 0-14 years. Standardized incidence rates and 95 % CI were estimated by applying the capture-recapture method and assuming Poisson distribution. Incidence trend was analyzed using the Poisson regression model. The mean annual incidence of childhood type 1 diabetes was 3.1 per 100,000 person-years. We did not observe significant difference in incidence between boys and girls. The incidence is unstable and had a mean annual increase 14.2 % per year during the studied period. A faster annual increase was observed in boys, warmer seasons, and in the outer regions of the city. If present trends continue, the number of new type 1 diabetes cases will double from 2016 to 2020, and prevalent cases will sextuple by 2025. Our results showed the incidence of childhood type 1 diabetes was rising rapidly in Shanghai. More studies are needed to analyze incidence changes in other regions of China for appropriate allocation of healthcare resources.

  4. The use of high-resolution melting analysis for rapid spa typing on methicillin-resistant Staphylococcus aureus clinical isolates.

    Science.gov (United States)

    Chen, Jonathan Hon-Kwan; Cheng, Vincent Chi-Chung; Chan, Jasper Fuk-Woo; She, Kevin Kin-Kwan; Yan, Mei-Kum; Yau, Miranda Chong-Yee; Kwan, Grace See-Wai; Yam, Wing-Cheong; Yuen, Kwok-Yung

    2013-02-15

    Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hong Kong for three decades. This study evaluated the practical use of high-resolution melting (HRM) real-time PCR analysis on MRSA staphylococcal Protein A (spa) typing on local MRSA isolates. Among 55 clinical MRSA isolates collected in 2011, 12 different spa types were observed by the conventional PCR-sequencing method including the locally predominant spa type t1081 and two locally predominant community acquired MRSA spa types t019 and t437. By using the HRM method, it could differentiate all 12 spa genotypes by distinct melting curves and HRM difference plot analysis. These two methods demonstrated 100% concordance whereas the HRM method required only 3h of turnaround time and one-fifth of reagent cost compared to the conventional method. Our study confirmed that the cost effective and rapid HRM typing approach is practically useful for MRSA community transmission monitoring and nosocomial outbreak control in Hong Kong. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum▿

    Science.gov (United States)

    Muniz, Mauro de Medeiros; Morais e Silva Tavares, Patrícia; Meyer, Wieland; Nosanchuk, Joshua Daniel; Zancope-Oliveira, Rosely Maria

    2010-01-01

    Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited. PMID:20453140

  6. Linking rapid magma reservoir assembly and eruption trigger mechanisms at evolved Yellowstone-type supervolcanoes

    Science.gov (United States)

    Wotzlaw, J.F.; Bindeman, I.N.; Watts, Kathryn E.; Schmitt, A.K.; Caricchi, L.; Schaltegger, U.

    2014-01-01

    The geological record contains evidence of volcanic eruptions that were as much as two orders of magnitude larger than the most voluminous eruption experienced by modern civilizations, the A.D. 1815 Tambora (Indonesia) eruption. Perhaps nowhere on Earth are deposits of such supereruptions more prominent than in the Snake River Plain–Yellowstone Plateau (SRP-YP) volcanic province (northwest United States). While magmatic activity at Yellowstone is still ongoing, the Heise volcanic field in eastern Idaho represents the youngest complete caldera cycle in the SRP-YP, and thus is particularly instructive for current and future volcanic activity at Yellowstone. The Heise caldera cycle culminated 4.5 Ma ago in the eruption of the ∼1800 km3 Kilgore Tuff. Accessory zircons in the Kilgore Tuff display significant intercrystalline and intracrystalline oxygen isotopic heterogeneity, and the vast majority are 18O depleted. This suggests that zircons crystallized from isotopically distinct magma batches that were generated by remelting of subcaldera silicic rocks previously altered by low-δ18O meteoric-hydrothermal fluids. Prior to eruption these magma batches were assembled and homogenized into a single voluminous reservoir. U-Pb geochronology of isotopically diverse zircons using chemical abrasion–isotope dilution–thermal ionization mass spectrometry yielded indistinguishable crystallization ages with a weighted mean 206Pb/238U date of 4.4876 ± 0.0023 Ma (MSWD = 1.5; n = 24). These zircon crystallization ages are also indistinguishable from the sanidine 40Ar/39Ar dates, and thus zircons crystallized close to eruption. This requires that shallow crustal melting, assembly of isolated batches into a supervolcanic magma reservoir, homogenization, and eruption occurred extremely rapidly, within the resolution of our geochronology (103–104 yr). The crystal-scale image of the reservoir configuration, with several isolated magma batches, is very similar to the

  7. Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.

    Science.gov (United States)

    Speletas, Matthaios; Kioumi, Anna; Loules, Gedeon; Hytiroglou, Prodromos; Tsitouridis, John; Christakis, John; Germenis, Anastasios E

    2008-01-01

    Mutations in the SLC40A1 gene result in a dominant genetic disorder [ferroportin disease; hereditary hemochromatosis type (HH) IV], characterized by iron overload with two different clinical manifestations, normal transferrin saturation with macrophage iron accumulation (the most prevalent type) or high transferrin saturation with hepatocyte iron accumulation (classical hemochromatosis phenotype). In previous studies, the mutational analysis of SLC40A1 gene has been performed at the genomic DNA level by PCR amplification and direct sequencing of all coding regions and flanking intron-exon boundaries (usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA (non-isotopic RNase cleavage assay). This protocol turned out to be rapid, sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations in two patients with hyperferritinemia, normal transferrin saturation and iron accumulation predominantly in macrophages and Kupffer cells. The first one displayed the well-described alteration V162 Delta and the second a novel mutation (R178G) that was further detected in two relatives in a pedigree analysis. The proposed procedure would facilitate the wide-range molecular analysis of the SLC40A1 gene, contributing to better understanding the pathogenesis of the ferroportin disease.

  8. A combination of four cell types for rapid detection of enteroviruses in clinical specimens.

    Science.gov (United States)

    Dagan, R; Menegus, M A

    1986-07-01

    Isolation in cell culture is currently the most sensitive and reliable way to demonstrate enterovirus (EV) in clinical specimens. During July-October 1982 and 1983, we studied the impact of adding two new cell lines, Buffalo green monkey kidney (BGM) and human rhabdomyosarcoma (RD), to the more traditional cell combination used for EV isolation, human embryonic lung (HEL) and primary cynomolgus monkey kidney (CMK) cells; 2,558 specimens were studied: 632 fecal, 677 respiratory, 537 CSF, and 712 blood. An EV was isolated from 417 (16%); of these, 77 (18%) were positive only in BGM or RD; 35% (146/417) of the specimens were positive in BGM, RD, or both, at least one day earlier than in the traditional cells. BGM cells were helpful in isolation of group B coxsackieviruses (CB): 99% of 121 positive specimens were detected in BGM vs 73% in CMK and 23% in HEL; 72% of the CB isolates were detected by day 2 in BGM vs 48% in CMK and 0% in HEL. RD cells were helpful in the isolation of echoviruses: 59% of the 189 positive specimens were detected in RD vs 67% in HEL and 58% in CMK. RD was the only positive cell type in 28/189 (15%) positive specimens; 31% of the echovirus isolates were detected by day 2 in RD, vs 20% in HEL and 19% in CMK. Using the cell types described, we provided the clinician with results in 42% of the EV-positive specimens by day 2 after inoculation and in 61% by day 4.

  9. Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita.

    Science.gov (United States)

    Chen, M; Chan, L S; Cai, X; O'Toole, E A; Sample, J C; Woodley, D T

    1997-01-01

    Epidermolysis bullosa acquisita (EBA) is an acquired blistering skin disease characterized by the presence of IgG autoantibodies to type VII collagen. EBA autoantibodies recognize four major immunodominant epitopes localized within the amino-terminal, noncollagenous (NC1) domain. In this study, we developed a rapid, quantitative enzyme-linked immunosorbent assay (ELISA) to detect autoantibody activity against the complete NC1 domain of type VII collagen with the use of an eukaryotic-expressed, recombinant human NC1 antigen. With the ELISA, we tested serum from patients with EBA (n = 24), bullous systemic lupus erythematosus (BSLE) (n = 3), bullous pemphigoid (n = 16), pemphigus (n = 11), and normal controls (n = 12). All EBA and BSLE serum, including four sera that were negative by indirect immunofluorescence, demonstrated reactivity with immobilized NC1 in the ELISA. In contrast, none of the sera from healthy control subjects or patients with unrelated blistering skin diseases reacted with NC1. The EBA sera also reacted with recombinant NC1 by immunoblot analysis but with less sensitivity. Thus, the newly developed ELISA using recombinant NC1 is a sensitive, specific assay and a useful tool for rapidly screening EBA and BSLE serum.

  10. Successful Rehabilitation of Partial Edentulous Maxilla and Mandible with New Type of Implants: Molecular Precision Implants

    Directory of Open Access Journals (Sweden)

    Matteo Danza

    2014-01-01

    Full Text Available The extraction of teeth results in rapid bone resorption both vertically and horizontally in the first month. The loss of alveolar ridge reduces the chance of implant rehabilitation. Atraumatic extraction, implant placement in extraction socket, and an immediate prosthesis have been proposed as alternative therapies to maintain the volume and contours tissue and reduce time and cost of treatment. The immediate load of implants is a universally practiced procedure; nevertheless a successful procedure requires expertise in both the clinical and the reconstructive stages using a solid implant system. Excellent primary stability and high bone-implant contact are only minimal requirements for any type of implant procedure. In this paper we present a case report using a new type of implants. The new type of implants, due to its sophisticated control system of production, provides to the implantologist a safe and reliable implant, with a macromorphology designed to ensure a close contact with the surrounding bone.

  11. Successful rehabilitation of partial edentulous maxilla and mandible with new type of implants: molecular precision implants.

    Science.gov (United States)

    Danza, Matteo; Lauritano, Dorina; Carinci, Francesco

    2014-01-01

    The extraction of teeth results in rapid bone resorption both vertically and horizontally in the first month. The loss of alveolar ridge reduces the chance of implant rehabilitation. Atraumatic extraction, implant placement in extraction socket, and an immediate prosthesis have been proposed as alternative therapies to maintain the volume and contours tissue and reduce time and cost of treatment. The immediate load of implants is a universally practiced procedure; nevertheless a successful procedure requires expertise in both the clinical and the reconstructive stages using a solid implant system. Excellent primary stability and high bone-implant contact are only minimal requirements for any type of implant procedure. In this paper we present a case report using a new type of implants. The new type of implants, due to its sophisticated control system of production, provides to the implantologist a safe and reliable implant, with a macromorphology designed to ensure a close contact with the surrounding bone.

  12. Current Methods in the Molecular Typing of Mycobacterium tuberculosis and Other Mycobacteria

    Science.gov (United States)

    van Ingen, Jakko; Dziadek, Jarosław; Mazur, Paweł K.; Bielecki, Jacek

    2014-01-01

    In the epidemiology of tuberculosis (TB) and nontuberculous mycobacterial (NTM) diseases, as in all infectious diseases, the key issue is to define the source of infection and to disclose its routes of transmission and dissemination in the environment. For this to be accomplished, the ability of discerning and tracking individual Mycobacterium strains is of critical importance. Molecular typing methods have greatly improved our understanding of the biology of mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. The utility of various typing methods depends on the Mycobacterium species under investigation as well as on the research question. For tuberculosis, different methods have different roles in phylogenetic analyses and person-to-person transmission studies. In NTM diseases, most investigations involve the search for environmental sources or phylogenetic relationships. Here, too, the type of setting determines which methodology is most suitable. Within this review, we summarize currently available molecular methods for strain typing of M. tuberculosis and some NTM species, most commonly associated with human disease. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed. PMID:24527454

  13. Molecular prediction for atherogenic risks across different cell types of leukocytes

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    Cheng Feng

    2012-01-01

    Full Text Available Abstract Background Diagnosing subclinical atherosclerosis is often difficult since patients are asymptomatic. In order to alleviate this limitation, we have developed a molecular prediction technique for predicting patients with atherogenic risks using multi-gene expression biomarkers on leukocytes. Methods We first discovered 356 expression biomarkers which showed significant differential expression between genome-wide microarray data of monocytes from patients with familial hyperlipidemia and increased risk of atherosclerosis compared to normal controls. These biomarkers were further triaged with 56 biomarkers known to be directly related to atherogenic risks. We also applied a COXEN algorithm to identify concordantly expressed biomarkers between monocytes and each of three different cell types of leukocytes. We then developed a multi-gene predictor using all or three subsets of these 56 biomarkers on the monocyte patient data. These predictors were then applied to multiple independent patient sets from three cell types of leukocytes (macrophages, circulating T cells, or whole white blood cells to predict patients with atherogenic risks. Results When the 56 predictor was applied to the three patient sets from different cell types of leukocytes, all significantly stratified patients with atherogenic risks from healthy people in these independent cohorts. Concordantly expressed biomarkers identified by the COXEN algorithm provided slightly better prediction results. Conclusion These results demonstrated the potential of molecular prediction of atherogenic risks across different cell types of leukocytes.

  14. Two sympatric types of Plasmodium ovale and discrimination by molecular methods

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    Myo Thura Zaw

    2017-10-01

    Full Text Available Plasmodium ovale is widely distributed in tropical countries, whereas it has not been reported in the Americas. It is not a problem globally because it is rarely detected by microscopy owing to low parasite density, which is a feature of clinical ovale malaria. P.o. curtisi and P.o. wallikeri are widespread in both Africa and Asia, and were known to be sympatric in many African countries and in southeast Asian countries. Small subunit ribosomal RNA (SSUrRNA gene, cytochrome b (cytb gene, and merozoite surface protein-1 (msp-1 gene were initially studied for molecular discrimination of P.o. curtisi and P.o. wallikeri using polymerase chain reaction (PCR and DNA sequencing. DNA sequences of other genes from P. ovale in Southeast Asia and the southwestern Pacific regions were also targeted to differentiate the two sympatric types. In terms of clinical manifestations, P.o. wallikeri tended to produce higher parasitemia levels and more severe symptoms. To date, there have been a few studies that used the quantitative PCR method for discrimination of the two distinct P. ovale types. Conventional PCR with consequent DNA sequencing is the common method used to differentiate these two types. It is necessary to identify these two types because relapse periodicity, drug susceptibility, and mosquito species preference need to be studied to reduce ovale malaria. In this article, an easier method of molecular-level discrimination of P.o. curtisi and P.o. wallikeri is proposed.

  15. A Molecular Census of Arcuate Hypothalamus and Median Eminence Cell Types

    Science.gov (United States)

    Campbell, John N.; Macosko, Evan Z.; Fenselau, Henning; Pers, Tune H.; Lyubetskaya, Anna; Tenen, Danielle; Goldman, Melissa; Verstegen, Anne M.J.; Resch, Jon M.; McCarroll, Steven A.; Rosen, Evan D.; Lowell, Bradford B.; Tsai, Linus

    2017-01-01

    The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility, and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a novel leptin-sensing neuronal population, multiple AgRP and POMC subtypes, and an orexigenic somatostatin neuronal population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinctly responsive subtypes of AgRP and POMC neurons. Finally, integrating our data with human GWAS data implicates two previously unknown neuronal subtypes in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred. PMID:28166221

  16. Noninvasive buccal swab antigen sample and molecular testing provides extended antigen typing for patients with hemoglobinopathies.

    Science.gov (United States)

    Rampersad, Angeli; Hampton, Kisha; Duncan, Natalie; Roberson, Chris; Slayten, Jayanna; Davisson, Suzanne; Aronowitz, Jessica; Shapiro, Amy

    2014-11-01

    To demonstrate the feasibility of performing a noninvasive, molecular-based red blood cell (RBC) antigen test on infants and very young children with sickle cell disease as part of a statewide newborn screening follow-up program. A prospective pilot project was conducted using a noninvasive buccal swab and test kit to perform DNA-based, extended RBC phenotyping in 92 children participating in a newborn hemoglobinopathy screening follow-up program. Reported data include the extended panel of antigens detected by molecular analysis compared with unaffected population estimates. Molecular-based RBC antigen testing was successful, with extended RBC typing generated for all subjects. Molecular testing detected several rare negative or rare positive phenotypes, demonstrating the utility of obtaining an extended antigen panel. This study demonstrates the feasibility of performing antigen testing on buccal swab specimens from children with sickle cell disease as part of a newborn screening follow-up program with the aim of allowing specific unit matching to prevent alloimmunization with RBC transfusions. The general applicability of testing may be limited by a lack of uniform insurance coverage for buccal swab testing, however. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. The Search for Molecular Prognostic Markers of Diabetic Nephropathy in Patients with Type 2 Diabetes Mellitus

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    V. M. Ibragimov

    2016-03-01

    Full Text Available The purpose of this study was to search for molecular prognostic markers of diabetic nephropathy (DN in patients with type 2 diabetes mellitus (T2DM. The study included 205 patients with T2DM and DN (stages 1 to 4. All patients were stratified by the MDRD equation. The control group included 30 healthy individuals. All T2DM patients were divided into 4 groups depending on the DN stages. Group 1 included 42 patients with DN-Stage 1 (prenephropathy, Group 2 included 48 patients with DN-Stage 2 (incipient nephropathy; Group 3 included 65 patients with DN-Stage 3 (overt nephropathy, and Group 4 included 50 patients with DN-Stage 4 (kidney failure. Molecular phenotyping of urine was processed with methods of proteomics: the prefractionation, the separation of proteins with standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, «Bruker», USA, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, «Bruker», USA. The data of the molecular interactions and functional features of proteins were received with STRING 10.0 database. Potentially new molecular markers of DN development were identified. The research into signaling pathways and the molecules that are involved in ECM formation may help in developing strategies to prevent DN.

  18. Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of Campylobacter jejuni

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    Nash John HE

    2008-08-01

    Full Text Available Abstract Background Multi-Locus Sequence Typing (MLST has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci. Results Our results indicate that, in general, there is significant concordance between strain relationships established by MLST and those based on shared gene content as established by CGH. While MLST has significant predictive power with respect to overall genome similarity of isolates, we also found evidence for significant differences in genomic content among strains that would otherwise appear to be highly related based on their MLST profiles. Conclusion The extensive genomic mosaicism between closely related strains has important implications in the context of establishing strain to strain relationships because it suggests that the exact gene content of strains, and by extension their phenotype, is less likely to be "predicted" based on a small number of typing loci. This in turn suggests that a greater emphasis should be placed on analyzing genes of clinical interest as we forge ahead with the next generation of molecular typing methods.

  19. Optimization of a pulsed-field gel electrophoresis for molecular typing of Proteus mirabilis

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    Alper Karagöz

    2013-09-01

    Full Text Available Objective: For the detection of outbreaks caused byProteus mirabilis, strains clonal relations are determinedmethods as “pulsed-field gel electrophoresis (PFGE”.The aim of this study was optimization of a pulsed-fieldgel electrophoresis for molecular typing of P. mirabilis.Methods: In this study, PFGE’ protocol is optimized foruse in molecular typing of P. mirabilis. Phylogenetic analyzesof strains were evaluated with Bionumerics softwaresystem (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium.Results: This protocol compared with Gram-negativebacteria PFGE protocols, NotI enzyme is suitable for thisbacterium. Electrophoresis conditions should be revealedas; - block 1: initial pulse duration 1 sec, ending pulseduration 30 sec, striking angle 120°, the current 6 V/cm2,temperature 14°C, time 8 hours; - block 2: initial pulseduration 30 sec, ending pulse duration 70 sec, strikingangle 120°, the current 6 V/cm2, temperature 14°C, time16 hours; - TBE, pH=8.4.Conclusion: P. mirabilis strains were typed by PFGE andBionumerics analysis program were determined clonal relationships.The procedure was simple, reproducible andsuitable for these bacteria. Also it was evaluated, becauseof reducing time, the solution volumes and enzymes canbe economically. Outbreaks of nosocomial infections dueto bacteria studied assessment and the potential to provideuseful information about the degree of prevalence.This optimized protocol is allowed different centers’ PFGEresults to compare with other laboratories results. J ClinExp Invest 2013; 4 (3: 306-312Key words: Proteus mirabilis, molecular typing, pulsedfieldgel electrophoresis.

  20. Antimicrobial Resistance and Molecular Typing of Pseudomonas Aeruginosa Isolated from Surgical Wounds in Lagos, Nigeria

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    Kehinde Akinsinde

    2012-06-01

    Full Text Available The aim of the study was to determine the resistance patterns of Pseudomonas aeruginosa isolates recovered from patients with surgical wounds in hospitals and also to investigate their epidemiological relatedness using molecular typing techniques. Twenty Pseudomonas sp. isolated from surgical wounds were subjected to antibiotic susceptibility testing by disk diffusion, plasmid profile, SDS-PAGE and PCR using the parC, gyr A gene and RAPD using the 1254 primer. The isolates showed resistance to 12 different antibiotics with six being 100% resistant. Plasmids were detected in 16 (80% of the isolates. The RAPD-PCR using the primer 1254, SDS-PAGE classified the 20 Pseudomonas spp. into 5 and 6 types respectively. Pseudomona aeruginosa strains isolated from surgical wounds were generally resistant to a broad range of antibiotics and this is rather worrisome. The typing techniques classified the 20 isolates into 5 and 6 groups.

  1. Antimicrobial resistance and molecular typing of pseudomonas aeruginosa isolated from surgical wounds in Lagos, Nigeria.

    Science.gov (United States)

    Smith, Stella; Ganiyu, Olaniyi; John, Rachael; Fowora, Muinah; Akinsinde, Kehinde; Odeigah, Peter

    2012-01-01

    The aim of the study was to determine the resistance patterns of Pseudomonas aeruginosa isolates recovered from patients with surgical wounds in hospitals and also to investigate their epidemiological relatedness using molecular typing techniques. Twenty Pseudomonas sp. isolated from surgical wounds were subjected to antibiotic susceptibility testing by disk diffusion, plasmid profile, SDS-PAGE and PCR using the parC, gyr A gene and RAPD using the 1254 primer. The isolates showed resistance to 12 different antibiotics with six being 100% resistant. Plasmids were detected in 16 (80%) of the isolates. The RAPD-PCR using the primer 1254, SDS-PAGE classified the 20 Pseudomonas spp. into 5 and 6 types respectively. Pseudomona aeruginosa strains isolated from surgical wounds were generally resistant to a broad range of antibiotics and this is rather worrisome. The typing techniques classified the 20 isolates into 5 and 6 groups.

  2. Rapid dissemination of colistin and carbapenem resistant Acinetobacter baumannii in Central Greece: mechanisms of resistance, molecular identification and epidemiological data.

    Science.gov (United States)

    Oikonomou, O; Sarrou, S; Papagiannitsis, C C; Georgiadou, S; Mantzarlis, K; Zakynthinos, E; Dalekos, G N; Petinaki, E

    2015-12-09

    Colistin-resistant/carbapenem-resistant Acinetobacter baumannii is a significant challenge for antibiotic treatment and infection control policies. Since 2012, in Central Greece an increase of colistin/pan- resistant A. baumannii has occurred, indicating the need for further analysis. A total of 86 colistin-resistant/carbapenem-resistant out of 1228 A. baumannii clinical isolates, consecutively collected between 2012 and 2014 in a tertiary Greek hospital of Central Greece, as well as one environmental isolate from surveillance cultures were studied. Molecular typing and mechanisms of resistance to colistin and to carbapenems were assessed, whereas, epidemiological and clinical data of the patients were reviewed. During the study period, the rate of colistin resistance gradually increased and reached 21.1 % in 2014. All colistin-resistant/carbapenem-resistant A. baumannii belonged to 3LST ST101 clone that corresponds to the international clonal lineage II. Carbapenem resistance was associated with the presence of bla oxa-23-like, while resistance to colistin probably correlated with G54E and R109H amino acid substitutions in PmrA and PmrC, respectively. Epidemiological data of the patients indicated that the first detection of colistin-resistant/carbapenem-resistant ST101 clone in the University Hospital of Larissa (UHL) was associated with a patient who previously had received colistin, while, the movement of the infected patients into the hospital probably resulted to its spread.

  3. Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

    Science.gov (United States)

    Bockstael, Olivier; Melas, Catherine; Pythoud, Catherine; Levivier, Marc; McCarty, Douglas; Samulski, R Jude; De Witte, Olivier; Tenenbaum, Liliane

    2012-07-01

    The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.

  4. Photoinduced Pedalo-Type Motion in an Azodicarboxamide-Based Molecular Switch.

    Science.gov (United States)

    Amirjalayer, Saeed; Martinez-Cuezva, Alberto; Berna, Jose; Woutersen, Sander; Buma, Wybren Jan

    2017-11-15

    Well-defined structural changes of molecular units that can be triggered by light are crucial for the development of photoactive functional materials. Herein, we report on a novel switch that has azodicarboxamide as its photo-triggerable element. Time-resolved UV-pump/IR probe spectroscopy in combination with quantum-chemical calculations shows that the azodicarboxamide functionality, in contrast to other azo-based chromophores, does not undergo trans-cis photoisomerization. Instead, a photoinduced pedalo-type motion occurs, which because of its volume-conserving properties enables the design of functional molecular systems with controllable motion in a confined space. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  5. Assessing health systems for type 1 diabetes in sub-Saharan Africa: developing a 'Rapid Assessment Protocol for Insulin Access'

    Directory of Open Access Journals (Sweden)

    de Courten Maximilian

    2006-02-01

    Full Text Available Abstract Background In order to improve the health of people with Type 1 diabetes in developing countries, a clear analysis of the constraints to insulin access and diabetes care is needed. We developed a Rapid Assessment Protocol for Insulin Access, comprising a series of questionnaires as well as a protocol for the gathering of other data through site visits, discussions, and document reviews. Methods The Rapid Assessment Protocol for Insulin Access draws on the principles of Rapid Assessment Protocols which have been developed and implemented in several different areas. This protocol was adapted through a thorough literature review on diabetes, chronic condition management and medicine supply in developing countries. A visit to three countries in sub-Saharan Africa and meetings with different experts in the field of diabetes helped refine the questionnaires. Following the development of the questionnaires these were tested with various people familiar with diabetes and/or healthcare in developing countries. The Protocol was piloted in Mozambique then refined and had two further iterations in Zambia and Mali. Translations of questionnaires were made into local languages when necessary, with back translation to ensure precision. Results In each country the protocol was implemented in 3 areas – the capital city, a large urban centre and a predominantly rural area and their respective surroundings. Interviews were carried out by local teams trained on how to use the tool. Data was then collected and entered into a database for analysis. Conclusion The Rapid Assessment Protocol for Insulin Access was developed to provide a situational analysis of Type 1 diabetes, in order to make recommendations to the national Ministries of Health and Diabetes Associations. It provided valuable information on patients' access to insulin, syringes, monitoring and care. It was thus able to sketch a picture of the health care system with regards to its ability to

  6. Normal Morning Melanin-Concentrating Hormone Levels and No Association with Rapid Eye Movement or Non-Rapid Eye Movement Sleep Parameters in Narcolepsy Type 1 and Type 2.

    Science.gov (United States)

    Schrölkamp, Maren; Jennum, Poul J; Gammeltoft, Steen; Holm, Anja; Kornum, Birgitte R; Knudsen, Stine

    2017-02-15

    Other than hypocretin-1 (HCRT-1) deficiency in narcolepsy type 1 (NT1), the neurochemical imbalance of NT1 and narcolepsy type 2 (NT2) with normal HCRT-1 levels is largely unknown. The neuropeptide melanin-concentrating hormone (MCH) is mainly secreted during sleep and is involved in rapid eye movement (REM) and non-rapid eye movement (NREM) sleep regulation. Hypocretin neurons reciprocally interact with MCH neurons. We hypothesized that altered MCH secretion contributes to the symptoms and sleep abnormalities of narcolepsy and that this is reflected in morning cerebrospinal fluid (CSF) MCH levels, in contrast to previously reported normal evening/afternoon levels. Lumbar CSF and plasma were collected from 07:00 to 10:00 from 57 patients with narcolepsy (subtypes: 47 NT1; 10 NT2) diagnosed according to International Classification of Sleep Disorders, Third Edition (ICSD-3) and 20 healthy controls. HCRT-1 and MCH levels were quantified by radioimmunoassay and correlated with clinical symptoms, polysomnography (PSG), and Multiple Sleep Latency Test (MSLT) parameters. CSF and plasma MCH levels were not significantly different between narcolepsy patients regardless of ICSD-3 subtype, HCRT-1 levels, or compared to controls. CSF MCH and HCRT-1 levels were not significantly correlated. Multivariate regression models of CSF MCH levels, age, sex, and body mass index predicting clinical, PSG, and MSLT parameters did not reveal any significant associations to CSF MCH levels. Our study shows that MCH levels in CSF collected in the morning are normal in narcolepsy and not associated with the clinical symptoms, REM sleep abnormalities, nor number of muscle movements during REM or NREM sleep of the patients. We conclude that morning lumbar CSF MCH measurement is not an informative diagnostic marker for narcolepsy.

  7. Molecular identification of 7 human papillomavirus types in recurrent respiratory papillomatosis.

    Science.gov (United States)

    Peñaloza-Plascencia, M; Montoya-Fuentes, H; Flores-Martínez, S E; Fierro-Velasco, F J; Peñaloza-González, J M; Sánchez-Corona, J

    2000-09-01

    Recurrent respiratory papillomatosis (RRP) is the most frequent benign neoplasm in childhood; it originates as a mild dysphonia and results in asphyxia. The RRP has been associated with an infection caused by human papillomavirus (HPV), mainly types 6 and 11, the latter being associated with more severe RRP. To analyze the frequency of the association of RRP with the HPV types in our juvenile population and to classify it according to severity. Observational descriptive trial. Forty-seven samples of paraffin-embedded papillomas, from 26 female and 21 male children (age range, 2 weeks to 17 years) were analyzed. DNA was isolated and a 188-base pair fragment was amplified from a consensus sequence in the E1 open reading frame of several HPVs by polymerase chain reaction. The corresponding band was recovered and reamplified. The fragment was digested with the restriction enzyme RsaI. The digestion products were compared with patterns of molecular weight markers for viral type identification. The patients' clinical records were reviewed, and RRP was classified as mild or aggressive. The presence of HPV types 6, 11, 16, 31, 33, 35, or 39 was confirmed in all the cases with different combinations. The chi(2) test showed no significant differences in clinical aggressiveness among the viral types. A logistic regression analysis demonstrated no association between clinical aggressiveness and any viral type or viral combination. These results show that RRP is caused by infection with HPV types 6 and 11 in addition to many other types, with no relationship between HPV type and clinical severity.

  8. Molecular and structural studies of Japanese patients with sialidosis type 1.

    Science.gov (United States)

    Naganawa, Y; Itoh, K; Shimmoto, M; Takiguchi, K; Doi, H; Nishizawa, Y; Kobayashi, T; Kamei, S; Lukong, K E; Pshezhetsky, A V; Sakuraba, H

    2000-01-01

    To gain insight into the pathogenesis of sialidosis type 1, we performed molecular investigations of two unrelated Japanese patients. Both of them are compound heterozygotes for base substitutions of 649G-to-A and 727G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosomal neuraminidase was constructed and the structural changes caused by these missense mutations were deduced. The predicted change due to V217M was smaller than that caused by G243R, the latter resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed neuraminidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although a G243R mutant was retained in the endoplasmic reticulum/Golgi area and had completely lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis type 1 with a late onset and moderate clinical course.

  9. The Changing Face of the Epidemiology of Tuberculosis due to Molecular Strain Typing: A Review

    Directory of Open Access Journals (Sweden)

    Philip N Suffys

    1997-05-01

    Full Text Available About one third of the world population is infected with tubercle bacilli, causing eight million new cases of tuberculosis (TB and three million deaths each year. After years of lack of interest in the disease, World Health Organization recently declared TB a global emergency and it is clear that there is need for more efficient national TB programs and newly defined research priorities. A more complete epidemiology of tuberculosis will lead to a better identification of index cases and to a more efficient treatment of the disease. Recently, new molecular tools became available for the identification of strains of Mycobacterium tuberculosis (M. tuberculosis, allowing a better recognition of transmission routes of defined strains. Both a standardized restriction-fragment-length-polymorphism-based methodology for epidemiological studies on a large scale and deoxyribonucleic acids (DNA amplification-based methods that allow rapid detection of outbreaks with multidrug-resistant (MDR strains, often characterized by high mortality rates, have been developed. This review comments on the existing methods of DNA-based recognition of M. tuberculosis strains and their peculiarities. It also summarizes literature data on the application of molecular fingerprinting for detection of outbreaks of M. tuberculosis, for identification of index cases, for study of interaction between TB and infection with the human immunodeficiency virus, for analysis of the behavior of MDR strains, for a better understanding of risk factors for transmission of TB within communities and for population-based studies of TB transmission within and between countries

  10. Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil.

    Science.gov (United States)

    Alves, Gleica Soyan Barbosa; Freire, Ana Karla Lima; Bentes, Amaury Dos Santos; Pinheiro, José Felipe de Souza; de Souza, João Vicente Braga; Wanke, Bodo; Matsuura, Takeshi; Jackisch-Matsuura, Ani Beatriz

    2016-08-01

    Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark-brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine-glycine-bromothymol blue agar. Molecular typing was done by PCR-RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest(®) strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus. © 2016 Blackwell Verlag GmbH.

  11. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  12. Wine yeast molecular typing using a simplified method for simultaneously extracting mtDNA, nuclear DNA and virus dsRNA.

    Science.gov (United States)

    Maqueda, Matilde; Zamora, Emiliano; Rodríguez-Cousiño, Nieves; Ramírez, Manuel

    2010-04-01

    Quick and accurate methods are required for the identification of industrial, environmental, and clinical yeast strains. We propose a rapid method for the simultaneous extraction of yeast mtDNA, nuclear DNA, and virus dsRNA. It is simpler, cheaper, and faster than the previously reported methods. It allows one to choose among a broad range of molecular analysis approaches for yeast typing, avoiding the need to use of several different methods for the separate extraction of each nucleic acid type. The application of this method followed by the combined analysis of mtDNA and dsRNA (ScV-M and W) is a highly attractive option for fast and efficient wine yeast typing.

  13. Banting Lecture 2009: An Unfinished Journey: Molecular Pathogenesis to Prevention of Type 1A Diabetes

    Science.gov (United States)

    Eisenbarth, George S.

    2010-01-01

    The Banting Medal for Scientific Achievement Award is the American Diabetes Association's highest scientific award and honors an individual who has made significant, long-term contributions to the understanding of diabetes, its treatment, and/or prevention. The award is named after Nobel Prize winner Sir Frederick Banting, who codiscovered insulin treatment for diabetes. Dr. Eisenbarth received the American Diabetes Association's Banting Medal for Scientific Achievement at the Association's 69th Scientific Sessions, June 5–9, 2009, in New Orleans, Louisiana. He presented the Banting Lecture, An Unfinished Journey—Type 1 Diabetes—Molecular Pathogenesis to Prevention, on Sunday, June 7, 2009. PMID:20350969

  14. High-current operation of vertical-type organic transistor with preferentially oriented molecular film

    Science.gov (United States)

    Fukagawa, Hirohiko; Watanabe, Yasuyuki; Kudo, Kazuhiro; Nishida, Jun-ichi; Yamashita, Yoshiro; Fujikake, Hideo; Tokito, Shizuo; Yamamoto, Toshihiro

    2016-04-01

    A high-performance vertical-type organic transistor has been fabricated using bis(l,2,5-thiadiazolo)-p-quinobis(l,3-dithiole) (BTQBT) for the channel layer. The BTQBT molecules are oriented horizontally, with the molecular plane of each monolayer parallel to the substrate. The π-π stacking direction of the BTQBT molecules is aligned with the carrier transport direction in this vertical transistor. The modulated drain current density exceeded 1 A cm-2 upon the application of a gate voltage of less than 5 V. In addition, the device exhibits a high on/off current ratio of over 105.

  15. Physiological and molecular characterization of an IRK-type inward rectifier K+ channel in a tumour mast cell line.

    Science.gov (United States)

    Wischmeyer, E; Lentes, K U; Karschin, A

    1995-04-01

    The basophilic leucaemia cell line RBL-2H3 exhibits a robust inwardly rectifying potassium current, IKIR, which is likely to be modulated by G proteins. We examined the physiological and molecular properties of this KIR conductance to define the nature of the underlying channel species. The macroscopic conductance revealed characteristics typical of classical K+ inward rectifiers of the IRK type. Channel gating was rapid, first order (tau approximately 1 ms at -100 mV) and steeply voltage dependent. Both activation potential and slope conductance were dependent on extracellular K+ concentration ([K+]o) and inward rectification persisted in the absence of internal Mg2+. The current was susceptible to a concentration- and voltage-dependent block by extracellular Na+, Cs+ and Ba2+. Initial IKIR whole-cell amplitudes as well as current rundown were dependent on the presence of 1 mM internal ATP. Perfusion of intracellular guanosine 5'-Q-(3-thiotriphosphate) (GTP[gamma S]) suppressed IKIR with an average half-time of decline of approximately 400 s. It was demonstrated that the dominant IRK-type 25 pS conductance channel was indeed suppressed by 100 microM preloaded GTP[gamma S]. Reverse transcriptase-polymerase chain reactions (RT-PCR) with RBL cell poly(A)+ RNA identified a full length K+ inward rectifier with 94% base pair homology to the recently cloned mouse IRK1 channel. It is concluded that RBL cells express a classical voltage-dependent IRK-type K+ inward rectifier RBL-IRK1 which is negatively controlled by G proteins.

  16. Rapid development of thymic neuroendocrine carcinoma despite transcervical thymectomy in a patient with multiple endocrine neoplasia type 1

    Directory of Open Access Journals (Sweden)

    Dhalapathy Sadacharan

    2013-01-01

    Full Text Available Thymic neuroendocrine (NE tumors are a rare manifestation of multiple endocrine neoplasia syndrome type 1 (MEN-1. They are malignant and aggressive tumors and form a major cause of mortality in MEN-1. Transcervical thymectomy (TCT at the time of parathyroid surgery for primary hyperparathyroidism (PHPT in MEN-1 usually prevents thymic NE tumors. We report a 56-year-old nonsmoker male with sporadic MEN-1 who presented with thymic NE carcinoma developing rapidly within a span of 8 months after subtotal parathyroidectomy and TCT for PHPT. We present a brief review of literature on this rare NE malignancy, focusing on its occurrence despite TCT. This case highlights the fact that thymic NE carcinoma may develop even after TCT in MEN-1. Regular surveillance for these aggressive thymic NE tumors is mandatory even after TCT in MEN-1 setting.

  17. Rapid Access to β-Trifluoromethyl-Substituted Ketones: Harnessing Inductive Effects in Wacker-Type Oxidations of Internal Alkenes

    KAUST Repository

    Lerch, Michael M.

    2014-07-18

    We present a practical trifluoromethyl-directed Wacker-type oxidation of internal alkenes that enables rapid access to β-trifluoromethyl-substituted ketones. Allylic trifluoromethyl-substituted alkenes bearing a wide range of functional groups can be oxidized in high yield and regioselectivity. The distance dependence of the regioselectivity was established by systematic variation of the number of methylene units between the double bond and the trifluoromethyl group. The regioselectivity enforced by traditional directing groups could even be reversed by introduction of a competing trifluoromethyl group. Besides being a new powerful synthetic method to prepare fluorinated molecules, this work directly probes the role of inductive effects on nucleopalladation events. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Molecular organization of the 5S rDNA gene type II in elasmobranchs.

    Science.gov (United States)

    Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

    2016-01-01

    The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

  19. An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need.

    Science.gov (United States)

    Wilkes, Rebecca P; Lee, Pei-Yu A; Tsai, Yun-Long; Tsai, Chuan-Fu; Chang, Hsiu-Hui; Chang, Hsiao-Fen G; Wang, Hwa-Tang T

    2015-08-01

    Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Synthesis of molecular imprinted dye-silica nanocomposites with high selectivity and sensitivity: Fluorescent imprinted sensor for rapid and efficient detection of τ-fluvalinate in vodka.

    Science.gov (United States)

    Wang, Yunyun; Wang, Jixiang; Cheng, Rujia; Sun, Lin; Dai, Xiaohui; Yan, Yongsheng

    2018-02-01

    An imprinted fluorescent sensor was fabricated based on SiO2 nanoparticles encapsulated with molecular imprinted polymer containing allyl fluorescein. High fluorine cypermethirin as template molecules, methyl methacrylate as functional monomer, and allyl fluorescein as optical materials synthesized a core-shell fluorescent molecular imprinted sensor, which showed a high and rapid sensitivity and selectivity for the detection of τ-fluvalinate. The sensor presented appreciable sensitivity with a limit of 13.251 nM, rapid detection that reached to equilibrium within 3 min, great linear relationship in the relevant concentration range from 0 to 150 nM and excellent selectivity over structural analogues. In addition, the fluorescent sensor demonstrated desirable regeneration ability (eight cycling operation). The molecular imprinted polymers ensured specificity, while the fluorescent dyes provided the stabile sensitivity. Finally, an effective application of the sensor was implemented by the detection of τ-fluvalinate in real samples from vodka. The molecular imprinted fluorescent sensor showed a promising potential in environmental monitoring and food safety. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. Correlation among phenotypical and molecular techniques in comparing ascomycetous yeast type strains.

    Science.gov (United States)

    De Nicola, Raffaele; Corte, Laura; Lattanzi, Monia; Martini, Alessandro; Fatichenti, Fabrizio; Cardinali, Gianluigi

    2005-01-01

    Different phenotypical or molecular techniques can be used to describe and classify microorganisms for taxonomic, phylogenetic or genetic purposes. In yeast taxonomy the official hierarchic classification, based on morphological and physiological characters, is used together with more convenient molecular techniques such as the DNA sequencing. The question on whether these procedures produce coherent classifications is critical both to interpret taxonomic data consistently and to outline species correctly. In this paper, a set of type strains from the major genera of the budding hemiascomycetes yeast is examined with a series of physiological and molecular techniques, widely employed in taxonomy, in order to compare the among-strains correlations obtained with different methods. Results showed that the level of correlation among different techniques is relatively low, showing that different classifications and species organization could be obtained with diverse approaches. This is particularly interesting, considering that the official description of the yeast species is based on characters different from those becoming increasingly popular in the routine identification.

  2. Discretization of Gene Expression Data Unmasks Molecular Subgroups Recurring in Different Human Cancer Types.

    Directory of Open Access Journals (Sweden)

    Manfred Beleut

    Full Text Available Despite the individually different molecular alterations in tumors, the malignancy associated biological traits are strikingly similar. Results of a previous study using renal cell carcinoma (RCC as a model pointed towards cancer-related features, which could be visualized as three groups by microarray based gene expression analysis. In this study, we used a mathematic model to verify the presence of these groups in RCC as well as in other cancer types. We developed an algorithm for gene-expression deviation profiling for analyzing gene expression data of a total of 8397 patients with 13 different cancer types and normal tissues. We revealed three common Cancer Transcriptomic Profiles (CTPs which recurred in all investigated tumors. Additionally, CTPs remained robust regardless of the functions or numbers of genes analyzed. CTPs may represent common genetic fingerprints, which potentially reflect the closely related biological traits of human cancers.

  3. Hoveyda–Grubbs type metathesis catalyst immobilized on mesoporous molecular sieves MCM-41 and SBA-15

    Directory of Open Access Journals (Sweden)

    Zdeněk Bastl

    2011-01-01

    Full Text Available A commercially available Hoveyda–Grubbs type catalyst (RC303 Zhannan Pharma was immobilized on mesoporous molecular sieves MCM-41 and on SBA-15 by direct interaction with the sieve wall surface. The immobilized catalysts exhibited high activity and nearly 100% selectivity in several types of alkene metathesis reactions. Ru leaching was found to depend on the substrate and solvent used (the lowest leaching was found for ring-closing metathesis of 1,7-octadiene in cyclohexane – 0.04% of catalyst Ru content. Results of XPS, UV–vis and NMR spectroscopy showed that at least 76% of the Ru content was bound to the support surface non-covalently and could be removed from the catalyst by washing with THF.

  4. Molecular Strain Typing of Clinical Isolates, Trichophyton rubrum using Non Transcribed Spacer (NTS) Region as a Molecular Marker.

    Science.gov (United States)

    Ramaraj, Vijayakumar; Vijayaraman, Rajyoganandh S; Elavarashi, Elangovan; Rangarajan, Sudha; Kindo, Anupma Jyoti

    2017-05-01

    Dermatophytes are a group of fungi which infect keratinized tissues and causes superficial mycoses in humans and animals. The group comprises of three major genera, Trichophyton, Microsporum and Epidermophyton. Among them Trichophyton rubrum is a predominant anthropophilic fungi which causes chronic infections. Although, the infection is superficial and treatable, reinfection/coinfection causes inflation in the treatment cost. Identifying the source and mode of transmission is essential to prevent its transmission. Accurate discrimination is required to understand the clinical (relapse or reinfection) and epidemiological implications of the genetic heterogeneity of this species. Polymorphism in the Non Transcribed Spacer (NTS) region of ribosomal DNA (rDNA) clusters renders an effective way to discriminate strains among T. rubrum. To carry out the strain typing of the clinical isolates, Trichophyton rubrum using NTS as a molecular marker. Seventy T.rubrum clinical isolates obtained from April-2011-March 2013, from Sri Ramachandra Medical Centre, Chennai, Tamil Nadu, India, were identified by conventional phenotypic methods and included in this prospective study. The isolates were then subjected to Polymerase Chain Reaction (PCR) targeting two subrepeat elements (SREs), TRS-1 and TRS-2 of the NTS region. Strain-specific polymorphism was observed in both subrepeat loci. Total, nine different strains were obtained on combining both TRS-1 and TRS-2, SREs. The outcome has given a strong representation for using NTS region amplification in discriminating the T. rubrum clinical isolates. The method can be adapted as a tool for conducting epidemiology and population based study in T. rubrum infections. This will help in future exploration of the epidemiology of T. rubrum.

  5. Molecular cloning, sequencing, and gene expression analysis of tributyltin-binding protein type 1 in Japanese medaka fish, Oryzias latipes.

    Science.gov (United States)

    Nassef, Mohamed; Kato-Unoki, Yoko; Furuta, Tomohisa; Nakayama, Kei; Satone, Hina; Shimasaki, Yohei; Honjo, Tsuneo; Oshima, Yuji

    2011-04-01

    The full-length cDNA sequence of tributyltin-binding protein type 1 in Japanese medaka (Oryzias latipes) (Olat.TBT-bp1) was determined by means of rapid amplification of cDNA ends (RACE) of liver tissue. Analysis of the structure of the gene encoding Olat.TBT-bp1 revealed that the exonintron organization of this gene corresponds to that of the genes encoding lipocalin superfamily proteins, suggesting that Olat.TBT-bp1 can be categorized as a member of the lipocalin superfamily, which may play an important role in transportation, detoxification, and excretion of xenobiotic compounds. Reverse transcription - PCR revealed that Olat.TBT-bp1 was expressed mainly in the liver, and upregulation of its expression was detected 1, 2, and 4 weeks post hatching. Relative expression of the Olat.TBT-bp1 gene was significantly downregulated, compared with that in the solvent control, by exposure to tributyltin at 0.01 mg/l or triclosan at 1.7 mg/l. Further studies on Olat.TBT-bp1 expression in conjunction with other biochemical and physiological toxicities in response to chemical exposures are needed to increase our understanding and information of TBT-bps mechanisms and as molecular biomarkers of chemical exposures. The role of Olat.TBT-bp1 in xenobiotic detoxification and/or excretion needs more investigations.

  6. Toxicology and drug delivery by cucurbit[n]uril type molecular containers.

    Science.gov (United States)

    Hettiarachchi, Gaya; Nguyen, Duc; Wu, Jing; Lucas, Derick; Ma, Da; Isaacs, Lyle; Briken, Volker

    2010-05-06

    Many drug delivery systems are based on the ability of certain macrocyclic compounds - such as cyclodextrins (CDs) - to act as molecular containers for pharmaceutical agents in water. Indeed beta-CD and its derivatives have been widely used in the formulation of hydrophobic pharmaceuticals despite their poor abilities to act as a molecular container (e.g., weak binding (K(a)containers that bind to a variety of cationic and neutral species with high affinity (K(a)>10(4) M(-1)) and therefore show great promise as a drug delivery system. In this study we investigated the toxicology, uptake, and bioactivity of two cucurbit[n]urils (CB[5] and CB[7]) and three CB[n]-type containers (Pentamer 1, methyl hexamer 2, and phenyl hexamer 3). All five containers demonstrated high cell tolerance at concentrations of up to 1 mM in cell lines originating from kidney, liver or blood tissue using assays for metabolic activity and cytotoxicity. Furthermore, the CB[7] molecular container was efficiently internalized by macrophages indicating their potential for the intracellular delivery of drugs. Bioactivity assays showed that the first-line tuberculosis drug, ethambutol, was as efficient in treating mycobacteria infected macrophages when loaded into CB[7] as when given in the unbound form. This result suggests that CB[7]-bound drug molecules can be released from the container to find their intracellular target. Our study reveals very low toxicity of five members of the cucurbit[n]uril family of nanocontainers. It demonstrates the uptake of containers by cells and intracellular release of container-loaded drugs. These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system.

  7. Metallization of solid molecular hydrogen in two dimensions: Mott-Hubbard-type transition

    Science.gov (United States)

    Biborski, Andrzej; Kådzielawa, Andrzej P.; Spałek, Józef

    2017-08-01

    We analyze the pressure-induced metal-insulator transition in a two-dimensional vertical stack of H2 molecules in (x-y) plane, and show that it represents a striking example of the Mott-Hubbard-type transition. Our combined exact diagonalization approach, formulated and solved in the second quantization formalism, includes also simultaneous ab initio readjustment of the single-particle wave functions, contained in the model microscopic parameters. The system is studied as a function of applied side force (generalized pressure), both in the H2-molecular and H-quasiatomic states. Extended Hubbard model is taken at the start, together with longer-range electron-electron interactions incorporated into the scheme. The stacked molecular plane transforms discontinuously into a (quasi)atomic state under the applied force via a two-step transition: the first between molecular insulating phases and the second from the molecular to the quasiatomic metallic phase. No quasiatomic insulating phase occurs. All the transitions are accompanied by abrupt changes of the bond length and the intermolecular distance (lattice parameter), as well as by discontinuous changes of the principal electronic properties, which are characteristic of the Mott-Hubbard transition here associated with the jumps of the predetermined equilibrium lattice parameter and the effective bond length. The phase transition can be interpreted in terms of the solid hydrogen metallization under pressure exerted by, e.g., the substrate covered with a monomolecular H2 film of the vertically stacked molecules. Both the Mott and Hubbard criteria at the insulator to metal transition are discussed.

  8. Molecular epidemiology of rabies in Botswana: a comparison between antibody typing and nucleotide sequence phylogeny.

    Science.gov (United States)

    Johnson, N; Letshwenyo, M; Baipoledi, E K; Thobokwe, G; Fooks, A R

    2004-06-10

    A panel of rabies virus isolates (RABV) endemic within Botswana between 1988 and 1992 have been typed by anti-nucleocapsid monoclonal antibodies (MAb) into two dominant groups. The first associated with the domestic dog (Canis familiaris) and the second associated with a range of wildlife species. Using nucleoprotein coding sequence data, we have applied molecular phylogenetic techniques to the same panel of 35 well-characterised rabies virus isolates from throughout Botswana in an attempt to compare both techniques and to further investigate the virus/host species relationships within this African country. The results confirm that there are indeed two major groups and that these are related primarily to biotype. The wildlife-associated biotype appeared more phylogenetically diverse and was more commonly isolated in the southeast of the country, with the canine-associated group dominating the north of the country. In addition, molecular phylogeny identified further groupings within both biotypes and a small number of isolates, which were not classified by MAb typing, could be assigned to a group. During the study period (4 years) there appeared to be little sequence variation within groups suggesting that distinct lineages persisted throughout the study and that there appears to be little evolutionary pressure on the nucleoprotein coding region of the viral genome. Copyright 2004 Elsiever B.V.

  9. In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

    Science.gov (United States)

    Hu, Bo; Lara-Tejero, Maria; Kong, Qingke; Galán, Jorge E; Liu, Jun

    2017-03-09

    Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2.

    Science.gov (United States)

    Kamsteeg, Erik-Jan; Kress, Wolfram; Catalli, Claudio; Hertz, Jens M; Witsch-Baumgartner, Martina; Buckley, Michael F; van Engelen, Baziel G M; Schwartz, Marianne; Scheffer, Hans

    2012-12-01

    Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and congenital forms, which frequently result in death because of respiratory deficiency, through to late-onset baldness and cataract. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age and have a more severe course (anticipation). In myotonic dystrophy type 2 (DM2), no anticipation is described, but cardiac conduction abnormalities as in DM1 are observed and patients with DM2 additionally have muscle pain and stiffness. Both DM1 and DM2 are caused by unstable DNA repeats in untranslated regions of different genes: A (CTG)n repeat in the 3'-UTR of the DMPK gene and a (CCTG)n repeat in intron 1 of the CNBP (formerly ZNF9) gene, respectively. The length of the (CTG)n repeat expansion in DM1 correlates with disease severity and age of onset. Nevertheless, these repeat sizes have limited predictive values on individual bases. Because of the disease characteristics in DM1 and DM2, appropriate molecular testing and reporting is very important for the optimal counseling in myotonic dystrophy. Here, we describe best practice guidelines for clinical molecular genetic analysis and reporting in DM1 and DM2, including presymptomatic and prenatal testing.

  11. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2

    Science.gov (United States)

    Kamsteeg, Erik-Jan; Kress, Wolfram; Catalli, Claudio; Hertz, Jens M; Witsch-Baumgartner, Martina; Buckley, Michael F; van Engelen, Baziel G M; Schwartz, Marianne; Scheffer, Hans

    2012-01-01

    Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and congenital forms, which frequently result in death because of respiratory deficiency, through to late-onset baldness and cataract. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age and have a more severe course (anticipation). In myotonic dystrophy type 2 (DM2), no anticipation is described, but cardiac conduction abnormalities as in DM1 are observed and patients with DM2 additionally have muscle pain and stiffness. Both DM1 and DM2 are caused by unstable DNA repeats in untranslated regions of different genes: A (CTG)n repeat in the 3'-UTR of the DMPK gene and a (CCTG)n repeat in intron 1 of the CNBP (formerly ZNF9) gene, respectively. The length of the (CTG)n repeat expansion in DM1 correlates with disease severity and age of onset. Nevertheless, these repeat sizes have limited predictive values on individual bases. Because of the disease characteristics in DM1 and DM2, appropriate molecular testing and reporting is very important for the optimal counseling in myotonic dystrophy. Here, we describe best practice guidelines for clinical molecular genetic analysis and reporting in DM1 and DM2, including presymptomatic and prenatal testing. PMID:22643181

  12. Molecular typing of uropathogenic E. coli strains by the ERIC-PCR method.

    Science.gov (United States)

    Ardakani, Maryam Afkhami; Ranjbar, Reza

    2016-04-01

    Escherichia coli (E. coli) is the most common cause of urinary infections in hospitals. The aim of this study was to evaluate the ERIC-PCR method for molecular typing of uropathogenic E. coli strains isolated from hospitalized patients. In a cross sectional study, 98 E. coli samples were collected from urine samples taken from patients admitted to Baqiyatallah Hospital from June 2014 to January 2015. The disk agar diffusion method was used to determine antibiotic sensitivity. DNA proliferation based on repetitive intergenic consensus was used to classify the E. coli strains. The products of proliferation were electrophoresed on 1.5% agarose gel, and their dendrograms were drawn. The data were analyzed by online Insillico software. The method used in this research proliferated numerous bands (4-17 bands), ranging from 100 to 3000 base pairs. The detected strains were classified into six clusters (E1-E6) with 70% similarity between them. In this study, uropathogenic E. coli strains belonged to different genotypic clusters. It was found that ERIC-PCR had good differentiation power for molecular typing of uropathogenic E. coli strains isolated from the patients in the study.

  13. Molecular identification of 13 new enterovirus types, EV79-88, EV97, and EV100-101, members of the species Human Enterovirus B.

    Science.gov (United States)

    Oberste, M Steven; Maher, Kaija; Nix, William A; Michele, Suzanne M; Uddin, Moyez; Schnurr, David; al-Busaidy, Suleiman; Akoua-Koffi, Chantal; Pallansch, Mark A

    2007-09-01

    Molecular methods have enabled the rapid identification of new enterovirus (EV) serotypes that are untypeable using existing neutralizing antisera. As a result, sequencing of the VP1 capsid gene has been developed as a surrogate for antigenic typing to distinguish enterovirus types. In this study, 17 enterovirus isolates from four countries were identified as members of 13 new types within the species Human Enterovirus B (HEV-B) by complete genome sequencing. Members of each of these new types are at least 75% identical to one another (91% amino acid identity) in VP1, but members of different types differ from one another and from other enteroviruses by at least 27% in nucleotide sequence (26% amino acid sequence difference). The complete P1 (capsid) sequences of the new types are at least 17% different from those of all other enterovirus serotypes (14.5% amino acid sequence difference), but they are highly conserved within a type (93% amino acid identity), confirming that the 17 new strains belong to HEV-B. We propose that these 17 isolates be classified as members of 13 new human enterovirus types, enteroviruses 79-88, 97, and 100-101.

  14. Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR

    Directory of Open Access Journals (Sweden)

    Zhang Chunling

    2010-12-01

    Full Text Available Abstract Porcine circovirus type 2 (PCV2 and the associated disease postweaning multisystemic wasting syndrome (PMWS have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.

  15. Rapid PCR-Based Diagnosis of Septic Arthritis by Early Gram-Type Classification and Pathogen Identification▿

    Science.gov (United States)

    Yang, Samuel; Ramachandran, Padmini; Hardick, Andrew; Hsieh, Yu-Hsiang; Quianzon, Celeste; Kuroki, Marcos; Hardick, Justin; Kecojevic, Aleksandar; Abeygunawardena, Avanthi; Zenilman, Jonathan; Melendez, Johan; Doshi, Vishal; Gaydos, Charlotte; Rothman, Richard E.

    2008-01-01

    Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 101 CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions. PMID:18305128

  16. Rapid degradation of Congo red by molecularly imprinted polypyrrole-coated magnetic TiO{sub 2} nanoparticles in dark at ambient conditions

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Shoutai; Hu, Xiaolei; Liu, Hualong; Wang, Qiang; He, Chiyang, E-mail: chiyanghe@hotmail.com

    2015-08-30

    Highlights: • Molecularly imprinted polypyrrole-coated magnetic TiO{sub 2} catalyst was prepared. • The catalyst degraded Congo red rapidly in dark at ambient conditions. • Degradation mechanism was proposed according to LC–MS analysis. • The catalyst can be easily recycled by a magnet. - Abstract: A novel molecularly imprinted polymer (MIP)-coated magnetic TiO{sub 2} nanocomposite was prepared, using methyl orange (MO) as the dummy template and pyrrole as functional monomer, for degradation of Congo red (CR). The nanocomposite was characterized by Fourier transform infrared spectroscopy, thermo-gravimetric analysis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer. The imprinting efficiency of the imprinted nanoparticles was investigated by static binding test, and their degradation ability toward CR was also studied. Moreover, the effects of pH, temperature, dissolved oxygen and oscillation rate on degradation rate of CR were investigated. Results showed that the imprinted nanocomposite had higher adsorption ability for MO compared with the non-imprinted one. Moreover, it could degrade CR rapidly in dark at room temperature and atmospheric pressure and could be recycled easily by a magnet with a good reusability. A degradation mechanism was proposed according to LC–MS analysis of degradation products of CR. The new imprinted nanoparticles showed high catalytic activity at ambient conditions without light illumination and additional chemicals, and therefore, it can be potentially applied to the rapid, “green” and low-cost degradation of CR in industrial printing and dyeing wastewater.

  17. Moving toward rapid and low-cost point-of-care molecular diagnostics with a repurposed 3D printer and RPA.

    Science.gov (United States)

    Chan, Kamfai; Wong, Pui-Yan; Parikh, Chaitanya; Wong, Season

    2018-01-12

    Traditionally, the majority of nucleic acid amplification-based molecular diagnostic tests are done in centralized settings. In recent years, point-of-care tests have been developed for use in low-resource settings away from central laboratories. While most experts agree that point-of-care molecular tests are greatly needed, their availability as cost-effective and easy-to-operate tests remains an unmet goal. In this article, we discuss our efforts to develop a recombinase polymerase amplification reaction-based test that will meet these criteria. First, we describe our efforts in repurposing a low-cost 3D printer as a platform that can carry out medium-throughput, rapid, and high-performing nucleic acid extraction. Next, we address how these purified templates can be rapidly amplified and analyzed using the 3D printer's heated bed or the deconstructed, low-cost thermal cycler we have developed. In both approaches, real-time isothermal amplification and detection of template DNA or RNA can be accomplished using a low-cost portable detector or smartphone camera. Last, we demonstrate the capability of our technologies using foodborne pathogens and the Zika virus. Our low-cost approach does not employ complicated and high-cost components, making it suitable for resource-limited settings. When integrated and commercialized, it will offer simple sample-to-answer molecular diagnostics. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Molecular Gas in Debris Disks around Young A-type Stars

    Science.gov (United States)

    Moór, Attila; Curé, Michel; Kóspál, Ágnes; Ábrahám, Péter; Csengeri, Timea; Eiroa, Carlos; Gunawan, Diah; Henning, Thomas; Hughes, A. Meredith; Juhász, Attila; Pawellek, Nicole; Wyatt, Mark

    2017-11-01

    According to the current paradigm of circumstellar disk evolution, gas-rich primordial disks evolve into gas-poor debris disks that are composed of second-generation dust. To explore the transition between these phases, we searched for 12CO, 13CO, and C18O emission in seven dust-rich debris disks around young A-type stars, using the Atacama Large Millimeter/submillimeter Array (ALMA) in Band 6. We discovered molecular gas in three debris disks. In all of these disks, the 12CO line was optically thick, highlighting the importance of less abundant molecules in reliable mass estimates. By supplementing our target list with literature data, we compiled a volume-limited sample of dust-rich debris disks around young A-type stars within 150 pc. We obtained a CO detection rate of 11/16 above a 12CO J = 2–1 line luminosity threshold of ∼1.4 × 104 Jy km s‑1pc2 in the sample. This high incidence implies that the presence of CO gas in the bright debris disks around young A-type stars is more likely the rule than the exception. Interestingly, dust-rich debris disks around young FG-type stars exhibit, with the same detectability threshold as A-type stars, a significantly lower gas incidence. While the transition from the protoplanetary phase to the debris phase is associated with a drop in the dust content, our results exhibit a large spread in the CO mass in our debris sample, with peak values that are comparable to those in the protoplanetary Herbig Ae disks. In the particularly CO-rich debris systems, the gas may have a primordial origin, which is a characteristic of a hybrid disk.

  19. Preparation of magnetic molecularly imprinted polymers by atom transfer radical polymerization for the rapid extraction of avermectin from fish samples.

    Science.gov (United States)

    You, Xiaoxiao; Gao, Lei; Qin, Dongli; Chen, Ligang

    2017-01-01

    A novel and highly efficient approach to obtain magnetic molecularly imprinted polymers is described to detect avermectin in fish samples. The magnetic molecularly imprinted polymers were synthesized by surface imprinting polymerization using magnetic multiwalled carbon nanotubes as the support materials, atom transfer radical polymerization as the polymerization method, avermectin as template, acrylamide as functional monomer, and ethylene glycol dimethacrylate as crosslinker. The characteristics of the magnetic molecularly imprinted polymers were assessed by using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, vibrating sample magnetometry, X-ray diffraction, and thermogravimetric analysis. The binding characteristics of magnetic molecularly imprinted polymers were researched through isothermal adsorption experiment, kinetics adsorption experiment, and the selectivity experiment. Coupled with ultra high performance liquid chromatography and tandem mass spectrometry, the extraction conditions of the magnetic molecularly imprinted polymers as adsorbents for avermectin were investigated in detail. The recovery of avermectin was 84.2-97.0%, and the limit of detection was 0.075 μg/kg. Relative standard deviations of intra- and inter-day precisions were in the range of 1.7-2.9% and 3.4-5.6%, respectively. The results demonstrated that the extraction method not only has high selectivity and accuracy, but also is convenient for the determination of avermectin in fish samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Molecular typing of Staphylococcus aureus isolate responsible for staphylococcal poisoning incident in homemade food

    Directory of Open Access Journals (Sweden)

    Guerrino Macori

    2016-06-01

    Full Text Available In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA and D (SED, respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

  1. Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food

    Science.gov (United States)

    Bellio, Alberto; Bianchi, Daniela Manila; Gallina, Silvia; Adriano, Daniela; Zuccon, Fabio; Chiesa, Francesco; Acutis, Pier Luigi; Casalinuovo, Francesco; Decastelli, Lucia

    2016-01-01

    In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves. PMID:27800449

  2. Rapid tumor progression in a patient with HPV type 16 associated anal squamous cell carcinoma suffering from long-standing Crohn's disease: A case report

    Directory of Open Access Journals (Sweden)

    Fischer AK

    2016-10-01

    Full Text Available Background and aim: Squamous cell carcinoma (SCC is the most common cancer of the anal region, typically associated with high-risk (hr HPV infection. Furthermore, there is evidence that Crohn's disease predisposes to adenocarcinoma in patients with perianal disease. Materials and methods: A 57-year old patient presenting with long history of Crohn's disease since the age of mid-twenties, went through several surgeries including ileocolectomy and anal fistula resection, combined with immunosuppressive therapy additionally periodically since 2008. One year before death (in 2015 a painful fistula was diagnosed with extensive high grade anal intraepithelial neoplasia (AIN-HG and evidence of invasive growth as non-keratinizing SCC. Tissue samples from several previous and current resection specimens were re-evaluated and extensively investigated for Crohn´s type inflammation, dysplasia and HPV both by immunohistochemistry (p16/Ki67 and molecular subtyping of HPV. Results: AIN-HG and invasive anal squamous cell carcinoma turned out to be strongly positive for p16/Ki67 staining and molecular analysis disclosed a HPV-16 subtype. In contrast, HPV-analysis was negative in all available previous tissue samples including one anal fistula resected five years before (in 2009 which was lined by non-keratinized squamous epithelium without any evidence of dysplasia. Thus, the patient was diagnosed as Crohn's disease with hr-HPV infection that rapidly (< 5ys progressed to AIN-HG and anal SCC. Finally, osseous metastases occurred and the patient died shortly after. Conclusions: This case of a patient diagnosed with SCC of the anal canal in combination with Crohn's disease as well as HPV Type 16 infection, points to the pathomechanism leading to dysplasia and finally cancer. We assume that immunosuppressive therapy in Crohn's disease may predispose to both persistent HPV infection and HPV related invasive anal carcinoma. The accelerated progression of HPV

  3. Circumnuclear Molecular Disks in Early-type Galaxies: Physical Properties and Precision Black Hole Mass Measurements

    Science.gov (United States)

    Boizelle, Benjamin

    2018-01-01

    ALMA is now capable of providing the most precise determinations of the masses of supermassive black holes in early-type galaxies (ETGs). In ALMA Cycle 2 we began a program to map the molecular gas kinematics in nearby ETGs that host central dust disks as seen in Hubble Space Telescope imaging. These initial observations targeted CO(2-1) emission at ~0.3" resolution, corresponding roughly to the projected radii of influence of the central black holes. In all cases we detect significant (~108 M⊙) molecular gas reservoirs that are in dynamically cold rotation, providing the most sensitive probes of the inner gravitational potentials of luminous ETGs. Using these gas kinematics, we verify that these molecular disks are formally stable against gravitational fragmentation and collapse. In several galaxies we detect central high-velocity gas rotation that provides direct kinematic evidence for a black hole. For two of these targets, NGC 1332 and NGC 3258, we have obtained higher-resolution observations (0.044" and 0.09") in Cycles 3 and 4 that more fully map out the gas rotation within the gravitational sphere of influence. We present dynamical modeling results for these targets, demonstrating that ALMA observations can enable black hole mass measurements at a precision of 10% or better, with minimal susceptibility to the systematic uncertainties that affect other methods of black hole mass measurement in ETGs. We discuss the impact of future high-resolution ALMA observations on black hole demographics and their potential to refine the high-mass end of the black hole-host galaxy scaling relationships.

  4. Single-molecule toroics in Ising-type lanthanide molecular clusters.

    Science.gov (United States)

    Ungur, Liviu; Lin, Shuang-Yan; Tang, Jinkui; Chibotaru, Liviu F

    2014-01-01

    Single-molecule toroics (SMTs) are defined, by analogy with single-molecule magnets, as bistable molecules with a toroidal magnetic state, and seem to be most promising for future applications in quantum computing and information storage and use as multiferroic materials with magnetoelectric effect. As an interdisciplinary research area that spans chemistry, physics and material sciences, synthetic chemists have produced systems suitable for detailed study by physicists and materials scientists, while ab initio calculations have been playing a major role in the detection of toroidal magnetization and the advancement of this field. In this tutorial review, we demonstrate the research developed in the fascinating and challenging field of molecular-based SMTs with particular focus on how recent studies tend to address the issue of toroidal arrangement of the magnetic moment in these systems. Herein, nine typical SMTs are summarized, showing that the assembly of wheel-shaped complexes with the high symmetry of the molecule unit and strong intra-molecular dipolar interactions using strong anisotropy metal ions represents the most promising route toward the design of a toroidal moment. Furthermore, the linkage of such robust toroidal moment units with ferromagnetic type through appropriate bridging ligands enhances the toroidal magnetic moment per unit cell.

  5. Molecular Analysis of the Coprinus Cinereus Mating Type a Factor Demonstrates an Unexpectedly Complex Structure

    Science.gov (United States)

    May, G.; Chevanton, L. L.; Pukkila, P. J.

    1991-01-01

    We report here the molecular cloning of the A43 mating type factor from Coprinus cinereus, a basidiomycetous fungus. Our molecular analyses revealed an unexpected source of variation in the A factor. Though genetic studies have demonstrated that A has two subunits, α and β, we located three nonoverlapping fragments in the A43 region that have A factor function following DNA-mediated transformation. The three fragments demonstrate no similarity to one another as judged by restriction enzyme maps and by hybridization on Southern blots. We conclude that the A43 factor is composed of at least three subunits. When strains carrying different A factors are examined by hybridization to the cloned subunits, extensive polymorphism is seen. Both intensity of hybridization and restriction fragment lengths vary between strains. Some strains fail to show any hybridization to a probe. In contrast, other strains from widely separated geographic locations apparently share very similar subunits. From comparative restriction enzyme mapping of A43 and a mutated A43 factor, we inferred that a 12-kb deletion in the A factor was responsible for the constitutive, dominant phenotype of the mutated A factor. The results of transformation experiments support an activator model for the activity of the A factor in regulating the A pathway. PMID:1678725

  6. Influence of Extractant and Soil Type on Molecular Characteristics of Humic Substances From Two Brazilian Soils

    Directory of Open Access Journals (Sweden)

    Dick Deborah Pinheiro

    1999-01-01

    Full Text Available In a previous study it was observed that humic substances (HS extracted with NaOH solution and with Na4P2O7 solution presented different molecular weights, and also that the extracted HS yield by each method varied between an Oxisol and a Mollisol from South Brazil. In the present study, we further investigated the organic matter in these soils by characterizing HS extracted with 0.5 mol L-1 NaOH and with neutral 0.15 mol L-1 Na4P2O7 solutions from the above mentioned samples, using elemental analysis and nuclear magnetic ressonance spectroscopy (liquid state ¹H- and 13C-NMR, and by relating the molecular differences to the extraction method and soil type. HS extracted with pyrophosphate were more humified, showing a higher aromaticity and higher carboxylic content. The NaOH-extracted HS were more aliphatic and contained a higher O-alkyl proportion, which is indicative of a less humified nature than the pyrophosphate-extracted HS.

  7. Molecular Evaluation of Genetic Diversity in Wild-Type Mastic Tree (Pistacia lentiscus L.).

    Science.gov (United States)

    Abuduli, Alimu; Aydin, Yıldız; Sakiroglu, Muhammet; Onay, Ahmet; Ercisli, Sezai; Uncuoglu, Ahu Altinkut

    2016-10-01

    In this study, the patterns of genetic variation and phylogenetic relationships of mastic tree (Pistacia lentiscus L.) genotypes including 12 males and 12 females were evaluated using SSR, RAPD, ISSR, and ITS markers yielding 40, 703, 929 alleles, and 260-292 base pairs for ITS1 region, respectively. The average number of alleles produced from SSR, RAPD, and ISSR primers were 5.7, 14, and 18, respectively. The grouping pattern obtained from Bayesian clustering method based on each marker dataset was produced. Principal component analyses (PCA) of molecular data was investigated and neighbor joining dendrograms were subsequently created. Overall, the results indicated that ISSR and RAPD markers were the most powerful to differentiate the genotypes in comparison with other types of molecular markers used in this study. The ISSR results indicated that male and female genotypes were distinctly separated from each other. In this frame, M9 (Alaçatı) and M10 (Mesta Sakız Adası-Chios) were the closest genotypes and while F11 (Seferihisar) and F12 (Bornova/Gökdere) genotypes fall into same cluster and showing closer genetic relation. The RAPD pattern indicated that M8 (Urla) and M10 (Mesta Sakız Adası-Chios), and F10 (Mesta Sakız Adası-Chios) and F11 (Seferihisar) genotypes were the closest male and female genotypes, respectively.

  8. Molecular characterization of a Korean bovine parainfluenza virus type 3 isolate.

    Science.gov (United States)

    Oem, Jae-Ku; Lee, Eun-Yong; Lee, Kyoung-Ki; Kim, Seong-Hee; Lee, Myoung-Heon; Hyun, Bang-Hun

    2013-02-22

    Bovine parainfluenza virus type 3 (BPIV-3) was isolated from Korean native cattle that presented clinical signs of mild pneumonia. The complete genome of a representative isolate (12Q061) was sequenced. The newly identified strain, which was found to be distinct from the previously reported genotypes A (BPIV-3a) and B (BPIV-3b) and closely related to the Chinese strain SD0835, was tentatively classified as genotype C (BPIV-3c). Our results suggest a relationship between BPIV-3 genetic variation and the geographic location of its isolation. Identification of these new BPIV-3 genotypes may facilitate the development of improved diagnostic methods and vaccines. This is to our knowledge the first report of the identification and molecular characterization of BPIV-3 in Korea. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Predicting hydration free energies of amphetamine-type stimulants with a customized molecular model

    Science.gov (United States)

    Li, Jipeng; Fu, Jia; Huang, Xing; Lu, Diannan; Wu, Jianzhong

    2016-09-01

    Amphetamine-type stimulants (ATS) are a group of incitation and psychedelic drugs affecting the central nervous system. Physicochemical data for these compounds are essential for understanding the stimulating mechanism, for assessing their environmental impacts, and for developing new drug detection methods. However, experimental data are scarce due to tight regulation of such illicit drugs, yet conventional methods to estimate their properties are often unreliable. Here we introduce a tailor-made multiscale procedure for predicting the hydration free energies and the solvation structures of ATS molecules by a combination of first principles calculations and the classical density functional theory. We demonstrate that the multiscale procedure performs well for a training set with similar molecular characteristics and yields good agreement with a testing set not used in the training. The theoretical predictions serve as a benchmark for the missing experimental data and, importantly, provide microscopic insights into manipulating the hydrophobicity of ATS compounds by chemical modifications.

  10. Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers.

    Science.gov (United States)

    Masneuf-Pomarède, I; Le Jeune, C; Durrens, P; Lollier, M; Aigle, M; Dubourdieu, D

    2007-01-01

    The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.

  11. Rapid Formation of Molecular Bromine from Deliquesced NaBr Aerosol in the Presence of Ozone and UV Light

    Science.gov (United States)

    The formation of gas-phase bromine from aqueous sodium bromide aerosols is investigated through a combination of chamber experiments and chemical kinetics modeling. Experiments show that Br2(g) is produced rapidly from deliquesced NaBr aerosols in the presence of OH radicals prod...

  12. Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm

    NARCIS (Netherlands)

    Simons, S.O.; Laan, T. van der; Mulder, A.; Ingen, J. van; Rigouts, L.; Dekhuijzen, P.N.R.; Boeree, M.J.; Soolingen, D. van

    2014-01-01

    There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with

  13. Antibiotic Susceptibility and Molecular Typing by REP-PCR among Acinetobacter baumannii Isolates

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    Delsuz Rezaee

    2014-04-01

    Full Text Available Background & Objectives : Acinetobacter baumannii is an opportunistic Gram-negative pathogen with increasing relevance in a variety of hospital-acquired infections especially among intensive care unit patients. A. baumannii is mostly a cause of septicemia, pneumonia and urinary tract infection following hospitalization of patients. In this study antibiotic susceptibility pattern of A.baumannii isolates and molecular typing among isolates resistant by REP-PCR were determined.   Methods : During study, the A. baumannii, were isolated from hospitals in Tehran. The isolates were identified using standard biochemical tests and antibiotic susceptibility was determined by the disk diffusion method. Extraction of DNA and molecular typing of isolates performed using CTAB method and REP-PCR, respectively.   Results : In this study 75 A. baumannii isolates separated from patients with an average age of 51 ± 18.45 years . The highest resistance rate was against azteronam (97%, ceftazidim (93%, cefepime (93%, piperacillin-tazobactam (93%, ciprofloxacin (93% and ticarcillin (93% while the lowest resistance rate was against tigecycline (n= 51, 68%, followed by tobramycin (n=24, 32%, ampicillin-sulbactam (n=21, 28%, amikacin (n=16, 21%, and carbapenems (n=11, 15%. The REP-PCR in resistant of A. baumannii isolates showed that the genotypes of A, B and C are the predominant genotypes in the resistant antibiotics.   Conclusion: This study showed a high percentage of resistance to antimicrobial agents among genotypes A, B, and C of the A. baumannii isolates therefore strategies to control the spread of A. baumannii isolates must be designed and evaluated.

  14. Dummy-surface molecularly imprinted polymers on magnetic graphene oxide for rapid and selective quantification of acrylamide in heat-processed (including fried) foods.

    Science.gov (United States)

    Ning, Fangjian; Qiu, Tingting; Wang, Qi; Peng, Hailong; Li, Yanbin; Wu, Xiaqing; Zhang, Zhong; Chen, Linxin; Xiong, Hua

    2017-04-15

    Novel nano-sized dummy-surface molecularly imprinted polymers (DSMIPs) on a magnetic graphene oxide (GO-Fe3O4) surface were developed as substrates, using propionamide as a dummy template molecule for the selective recognition and rapid pre-concentration and removal of acrylamide (AM) from food samples. These products showed rapid kinetics, high binding capacity (adsorption at 3.68mg·g-1), and selectivity (imprinting factor α 2.83); the adsorption processes followed the Langmuir-Freundlich isotherm and pseudo-second-order kinetic models. Excellent recognition selectivity toward acrylamide was achieved compared to structural analogs, such as propionic and acrylic acids (selectivity factor β 2.33, and 2.20, respectively). Moreover, DSMIPs-GO-Fe3O4 was used to quantify acrylamide in food samples, yielding satisfactory recovery (86.7-94.3%) and low relative standard deviation (acrylamide from food samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Evaluation of Xpert MTB/RIF assay for rapid molecular diagnosis of tuberculosis in a two-year period in Croatia

    Directory of Open Access Journals (Sweden)

    Ljiljana Zmak

    2013-01-01

    Full Text Available Mycobacterium tuberculosis remains a major global health problem and is currently killing 1.5 million people every year. One of the most important steps in tuberculosis control is the rapid and accurate laboratory diagnosis. The Xpert MTB/RIF assay is a novel molecular, easy-to-use assay, which can lead to tuberculosis identification in less than 2 h. In this study, the Xpert MTB/RIF assay performance for rapid diagnosis of tuberculosis was evaluated in comparison with conventional culture methods; 361 pulmonary and extrapulmonary patient samples were collected between October 2010 and October 2012 and were analyzed at the National Reference laboratory for Mycobacteria, Zagreb, Croatia. For pulmonary samples the sensitivity and specificity were 86% and 100%, while for extrapulmonary samples the sensitivity and specificity were 75% and 99%, respectively. It was concluded that Xpert MTB/RIF assay has high sensitivity and specificity for both pulmonary and extrapulmonary specimens.

  16. Extranodal NK/T-cell lymphoma, nasal type, manifesting as rapidly progressive dementia without any mass or enhancing brain lesion.

    Science.gov (United States)

    Shimatani, Yoshimitsu; Nakano, Yuta; Tsuyama, Naoko; Murayama, Shigeo; Oki, Ryosuke; Miyamoto, Ryosuke; Murakami, Nagahisa; Fujita, Koji; Watanabe, Syunsuke; Uehara, Hisanori; Abe, Takashi; Nodera, Hiroyuki; Kawarai, Toshitaka; Izumi, Yuishin; Kaji, Ryuji

    2016-10-01

    Among the many potential etiologies for rapidly progressive dementia (RPD), primary central nervous system extranodal NK/T-cell lymphoma, nasal-type (ENKL) is a rare entity. We present the first reported case of autopsy-proven RPD due to ENKL without any mass or enhancing lesion of the brain. A 54-year-old immunocompetent man presented with RPD, myoclonus and ataxia. The mini-mental state examination (MMSE) score was 22/30. His brain MRI revealed progressive brain atrophy without gadolinium enhancement or mass lesion. Five months after the initial evaluation, cognitive impairment further worsened with an MMSE score of 3/30. At the advanced stage, lumbar MRI showed swollen cauda equina with gadolinium enhancement. The number of Epstein-Barr virus (EBV) DNA in cerebrospinal fluid had gradually increased. Twelve months after onset, the patient died of respiratory failure. Pathological findings revealed that lymphoma cells had diffusely invaded the meninges, parenchyma of the brain, spinal cord and cauda equina. Cells were positive for CD3, CD56 and EBV-encoded small RNAs and negative for CD20. No evidence of malignancy was identified in the visceral organs. This report indicates that ENKL should be recognized as one of the rare causes of RPD. Early testing for EBV-DNA in cerebrospinal fluid and imaging of cauda equina would be useful diagnostic tools. © 2016 Japanese Society of Neuropathology.

  17. Low-cost, rapidly-developed, 3D printed in vitro corpus callosum model for mucopolysaccharidosis type I.

    Science.gov (United States)

    Tabet, Anthony; Gardner, Matthew; Swanson, Sebastian; Crump, Sydney; McMeekin, Austin; Gong, Diana; Tabet, Rebecca; Hacker, Benjamin; Nestrasil, Igor

    2016-01-01

    The rising prevalence of high throughput screening and the general inability of (1) two dimensional (2D) cell culture and (2) in vitro release studies to predict in vivo neurobiological and pharmacokinetic responses in humans has led to greater interest in more realistic three dimensional (3D) benchtop platforms. Advantages of 3D human cell culture over its 2D analogue, or even animal models, include taking the effects of microgeometry and long-range topological features into consideration. In the era of personalized medicine, it has become increasingly valuable to screen candidate molecules and synergistic therapeutics at a patient-specific level, in particular for diseases that manifest in highly variable ways. The lack of established standards and the relatively arbitrary choice of probing conditions has limited in vitro drug release to a largely qualitative assessment as opposed to a predictive, quantitative measure of pharmacokinetics and pharmacodynamics in tissue. Here we report the methods used in the rapid, low-cost development of a 3D model of a mucopolysaccharidosis type I patient's corpus callosum, which may be used for cell culture and drug release. The CAD model is developed from in vivo brain MRI tracing of the corpus callosum using open-source software, printed with poly (lactic-acid) on a Makerbot Replicator 5X, UV-sterilized, and coated with poly (lysine) for cellular adhesion. Adaptations of material and 3D printer for expanded applications are also discussed.

  18. Rapid Analysis of Apolar Low Molecular Weight Constituents in Wood Using High Pressure Liquid Chromatography with Evaporative Light Scattering Detection

    NARCIS (Netherlands)

    Claassen, F.W.; Haar, van de C.; Beek, van T.A.; Dorado, J.; Martinez-Inigo, M.; Sierra-Alvarez, R.

    2000-01-01

    A new high pressure liquid chromatographic method with evaporative light scattering detection was developed for the qualitative and quantitative analysis of apolar, low molecular weight constituents in wood. The wood extractives were obtained by means of a 6 h Soxhlet extraction with acetone. The

  19. Using rapid diagnostic tests as source of malaria parasite DNA for molecular analyses in the era of declining malaria prevalence

    DEFF Research Database (Denmark)

    Ishengoma, Deus S; Lwitiho, Sudi; Madebe, Rashid A

    2011-01-01

    continued molecular surveillance of malaria parasites is important to early identify emerging anti-malarial drug resistance, it is becoming increasingly difficult to obtain parasite samples from ongoing studies, such as routine drug efficacy trials. To explore other sources of parasite DNA, this study...

  20. Country-wide surveillance of molecular markers of antimalarial drug resistance in Senegal by use of positive Malaria Rapid Diagnostic Tests

    DEFF Research Database (Denmark)

    Ndiaye, Magatte; Sow, Doudou; Nag, Sidsel

    2017-01-01

    of drug resistance. Therefore, surveillance of drug resistance in the malaria parasites is essential. The objective of this pilot study was to test the feasibility of routinely sampled malaria rapid diagnostic tests (RDTs) at a national scale to assess the temporal changes in the molecular profiles...... of antimalarial drug resistance markers of Plasmodium falciparum parasites. Overall, 9,549 positive malaria RDTs were collected from 14 health facilities across the country. A limited random set of RDTs were analyzed regarding Pfcrt gene polymorphisms at codon 72-76. Overall, a high but varied prevalence (> 50...

  1. Pseudomonas aeruginosa intensive care unit outbreak: winnowing of transmissions with molecular and genomic typing.

    Science.gov (United States)

    Parcell, Benjamin J; Oravcova, Katarina; Pinheiro, Miguel; Holden, Matthew T G; Phillips, Gabby; Turton, Jane F; Gillespie, Stephen H

    2017-12-08

    Pseudomonas aeruginosa healthcare outbreaks can be time consuming and difficult to investigate. Guidance does not specify which typing technique is most practical to base decisions on. We explore the usefulness of whole genome sequencing (WGS) in the investigation of a Pseudomonas aeruginosa outbreak describing how it compares with pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) analysis. Six patient isolates and six environmental samples from an Intensive Care Unit (ICU) positive for P. aeruginosa over two years underwent VNTR, PFGE and WGS. VNTR and PFGE were required to fully determine the potential source of infection and rule out others. WGS results unambiguously distinguished linked isolates giving greater assurance of the transmission route between wash hand basin (WHB) water and two patients supporting control measures employed. WGS provided detailed information without need for further typing. When allied to epidemiological information it can be used to understand outbreak situations rapidly and with certainty. Implementation of WGS in real-time would be a major advance in day-to-day practice. It could become a standard of care as it becomes more widespread due to its reproducibility and reduction in costs. Copyright © 2017. Published by Elsevier Ltd.

  2. Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

    Science.gov (United States)

    Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; Garcia, K. Christopher; Müller, Thomas D.; Piehler, Jacob

    2017-07-01

    The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

  3. Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

    Science.gov (United States)

    Shahzad, Toufeeq; Zhan, Ming-Yue; Yang, Pei-Jin; Yu, Xiao-Qiang; Rao, Xiang-Jun

    2017-07-01

    C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities. © 2017 Wiley Periodicals, Inc.

  4. Molecular Dependence of the Large Seebeck Effect in τ-Type Organic Conductors

    Science.gov (United States)

    Aizawa, Hirohito; Kuroki, Kazuhiko; Yoshino, Harukazu; Mousdis, George A.; Papavassiliou, George C.; Murata, Keizo

    2014-10-01

    We study the Seebeck effect in the τ-type organic conductors, τ-(EDO-S,S-DMEDT-TTF)2(AuBr2)1+y and τ-(P-S,S-DMEDT-TTF)2(AuBr2)1+y, where EDO-S,S-DMEDT-TTF and P-S,S-DMEDT-TTF are abbreviated as OOSS and NNSS, respectively, both experimentally and theoretically. Theoretically in particular, we perform first-principles band calculation for the two materials and construct a two-orbital model, on the basis of which we calculate the Seebeck coefficient. We show that the calculated temperature dependence of the Seebeck coefficient S is semi-quantitatively consistent with the experimental observation. In both materials, the absolute value of the Seebeck coefficient is maximum at a certain temperature, and this temperature is lower for NNSS than for OOSS. From a band structure viewpoint, we find that this can be traced back to the narrowness of the band gap between the upper and the lower pudding-mold type bands. On the other hand, the Seebeck coefficient of NNSS in the low temperature regime steeply increases with increasing temperature, which is due to the narrowness of the upper band. These differences in thermoelectric properties demonstrate the effectiveness of controlling the band structure through molecular modification.

  5. Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

    Science.gov (United States)

    Vallari, A S; Hickman, R K; Hackett, J R; Brennan, C A; Varitek, V A; Devare, S G

    1998-12-01

    A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected

  6. Rapid Targeted Next-Generation Sequencing Platform for Molecular Screening and Clinical Genotyping in Subjects with Hemoglobinopathies

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    Xuan Shang

    2017-09-01

    Full Text Available Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

  7. Molecular Epidemiologic Analysis of Enterococcus faecalis Isolates in Cuba by Multilocus Sequence Typing

    Science.gov (United States)

    Kobayashi, Nobumichi; Nagashima, Shigeo

    2009-01-01

    We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6′)-aph(2″), aph(3′), ant(6), ant(3″)(9), aph(2″)-Id, aph(2″)-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6′)-Ie-aph(2″)-Ia, aph(3′), ant(6), ant(3″)(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits. PMID:19857135

  8. Molecular epidemiology of human immunodeficiency virus type 1 in Guangdong province of southern China.

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    Song Chen

    Full Text Available BACKGROUND: Although the outbreak of human immunodeficiency virus type 1 (HIV-1 in Guangdong has been documented for more than a decade, the molecular characteristics of such a regional HIV-1 epidemic remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: By sequencing of HIV-1 pol/env genes and phylogenetic analysis, we performed a molecular epidemiologic study in a representative subset (n  = 200 of the 508 HIV-1-seropositive individuals followed up at the center for HIV/AIDS care and treatment of Guangzhou Hospital of Infectious Diseases. Of 157 samples (54.1% heterosexual acquired adults, 20.4% needle-sharing drug users, 5.7% receivers of blood transfusion, 1.3% men who have sex with men, and 18.5% remained unknown with successful sequencing for both pol and env genes, 105 (66.9% HIV-1 subtype CRF01_AE and 24 (15.3% CRF07_BC, 9 (5.7% B', 5 (3.2% CRF08_BC, 5 (3.2% B, 1 (0.6% C, 3 (1.9% CRF02_AG, and 5 (3.2% inter-region recombinants were identified within pol/env sequences. Thirteen (8.3% samples (3 naïves, 6 and 5 received with antiretroviral treatment [ART] 1-21 weeks and ≥24 weeks respectively showed mutations conferring resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors or protease inhibitors. Among 63 ART-naïve patients, 3 (4.8% showed single or multiple drug resistant mutations. Phylogenetic analysis showed 8 small clusters (2-3 sequences/cluster with only 17 (10.8% sequences involved. CONCLUSION/SIGNIFICANCE: This study confirms that sexual transmission with dominant CRF01_AE strain is a major risk for current HIV-1 outbreak in the Guangdong's general population. The transmission with drug-resistant variants is starting to emerge in this region.

  9. Molecular typing of Leptospira interrogans serovar Hardjo isolates from leptospirosis outbreaks in Brazilian livestock.

    Science.gov (United States)

    Cosate, Maria Raquel V; Sakamoto, Tetsu; de Oliveira Mendes, Tiago Antônio; Moreira, Élvio C; Regis da Silva, Carlos G; Brasil, Bruno S A F; Oliveira, Camila S F; de Azevedo, Vasco Ariston; Ortega, José Miguel; Leite, Rômulo C; Haddad, João Paulo

    2017-06-15

    Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira spp. This zoonotic disease is distributed globally and affects domestic animals, including cattle. Leptospira interrogans serogroup Sejroe serovar Hardjo and Leptospira borgpetersenii serogroup Sejroe serovar Hardjo remain important species associated with this reproductive disease in livestock production. Previous studies on Brazilian livestock have reported that L. interrogans serovar Hardjo is the most prevalent leptospiral agent in this country and is related to clinical signs of leptospirosis, which lead to economic losses in production. Here, we described the isolation of three clinical strains (Norma, Lagoa and Bolivia) obtained from leptospirosis outbreaks that occurred in Minas Gerais state in 1994 and 2008. Serological and molecular typing using housekeeping (secY and 16SrRNA) and rfb locus (ORF22 and ORF36) genes were applied for the identification and comparative analysis of Leptospira spp. Our results identified the three isolates as L. interrogans serogroup Sejroe serovar Hardjo and confirmed the occurrence of this bacterial strain in Brazilian livestock. Genetic analysis using ORF22 and ORF36 grouped the Leptospira into serogroup Sejroe and subtype Hardjoprajitno. Genetic approaches were also applied to compare distinct serovars of L. interrogans strains by verifying the copy numbers of the IS1500 and IS1533 insertion sequences (ISs). The IS1500 copy number varied among the analyzed L. interrogans strains. This study provides evidence that L. interrogans serogroup Sejroe serovar Hardjo subtype Hardjoprajitno causes bovine leptospirosis in Brazilian production. The molecular results suggested that rfb locus (ORF22 and ORF36) could improve epidemiological studies by allowing the identification of Leptospira spp. at the serogroup level. Additionally, the IS1500 and IS1533 IS copy number analysis suggested distinct genomic features among closely related leptospiral strains.

  10. Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

    Science.gov (United States)

    2012-01-01

    Background In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Results Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for

  11. Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

    Directory of Open Access Journals (Sweden)

    Yang Huaan

    2012-07-01

    Full Text Available Abstract Background In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Results Twenty informative plants from a cross of RxS (disease resistant x susceptible in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM, and are now replacing the markers previously developed by a traditional DNA

  12. A reliable and accurate portable device for rapid quantitative estimation of iodine content in different types of edible salt.

    Science.gov (United States)

    Yadav, Kapil; Kumar, Rakesh; Chakrabarty, Arijit; Pandav, Chandrakant S

    2015-01-01

    Continuous monitoring of salt iodization to ensure the success of the Universal Salt Iodization (USI) program can be significantly strengthened by the use of a simple, safe, and rapid method of salt iodine estimation. This study assessed the validity of a new portable device, iCheck Iodine developed by the BioAnalyt GmbH to estimate the iodine content in salt. Validation of the device was conducted in the laboratory of the South Asia regional office of the International Council for Control of Iodine Deficiency Disorders (ICCIDD). The validity of the device was assessed using device specific indicators, comparison of iCheck Iodine device with the iodometric titration, and comparison between iodine estimation using 1 g and 10 g salt by iCheck Iodine using 116 salt samples procured from various small-, medium-, and large-scale salt processors across India. The intra- and interassay imprecision for 10 parts per million (ppm), 30 ppm, and 50 ppm concentrations of iodized salt were 2.8%, 6.1%, and 3.1%, and 2.4%, 2.2%, and 2.1%, respectively. Interoperator imprecision was 6.2%, 6.3%, and 4.6% for the salt with iodine concentrations of 10 ppm, 30 ppm, and 50 ppm respectively. The correlation coefficient between measurements by the two methods was 0.934 and the correlation coefficient between measurements using 1 g of iodized salt and 10 g of iodized salt by the iCheck Iodine device was 0.983. The iCheck Iodine device is reliable and provides a valid method for the quantitative estimation of the iodine content of iodized salt fortified with potassium iodate in the field setting and in different types of salt.

  13. Rifampin-Isoniazid Oligonucleotide Typing: an Alternative Format for Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis ▿ †

    Science.gov (United States)

    Hernández-Neuta, Iván; Varela, Andrés; Martin, Anandi; von Groll, Andrea; Jureen, Pontus; López, Beatriz; Imperiale, Belén; Šķenders, Ģirts; Ritacco, Viviana; Hoffner, Sven; Morcillo, Nora; Palomino, Juan Carlos; Del Portillo, Patricia

    2010-01-01

    A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources. PMID:20881173

  14. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

    Science.gov (United States)

    Minkoff, Benjamin B.; Stecker, Kelly E.; Sussman, Michael R.

    2015-01-01

    Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

  15. Preparation of hybrid molecularly imprinted polymer with double-templates for rapid simultaneous purification of theophylline and chlorogenic acid in green tea.

    Science.gov (United States)

    Tang, Weiyang; Li, Guizhen; Row, Kyung Ho; Zhu, Tao

    2016-05-15

    A novel double-templates technique was adopted for solid-phase extraction packing agent, and the obtained hybrid molecularly imprinted polymers with double-templates (theophylline and chlorogenic acid) were characterized by fourier transform infrared and field emission scanning electron microscope. The molecular recognition ability and binding capability for theophylline and chlorogenic acid of polymers was evaluated by static absorption and dynamic adsorption curves. A rapid and accurate approach was established for simultaneous purification of theophylline and chlorogenic acid in green tea by coupling hybrid molecularly imprinted solid-phase extraction with high performance liquid chromatography. With optimization of SPE procedure, a reliable analytical method was developed for highly recognition towards theophylline and chlorogenic acid in green tea with satisfactory extraction recoveries (theophylline: 96.7% and chlorogenic acid: 95.8%). The limit of detection and limit of quantity of the method were 0.01 μg/mL and 0.03 μg/mL for theophylline, 0.05 μg/mL and 0.17 μg/mL for chlorogenic acid, respectively. The recoveries of proposed method at three spiked levels analysis were 98.7-100.8% and 98.3-100.2%, respectively, with the relative standard deviation less than 1.9%. Hybrid molecularly imprinted polymers with double-templates showed good performance for two kinds of targets, and the proposed approach with high affinity of hybrid molecularly imprinted polymers might offer a novel method for the purification of complex samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Rapid molecular detection of invasive species in ballast and harbor water by integrating environmental DNA and light transmission spectroscopy.

    Science.gov (United States)

    Egan, Scott P; Grey, Erin; Olds, Brett; Feder, Jeffery L; Ruggiero, Steven T; Tanner, Carol E; Lodge, David M

    2015-04-07

    Invasive species introduced via the ballast water of commercial ships cause enormous environmental and economic damage worldwide. Accurate monitoring for these often microscopic and morphologically indistinguishable species is challenging but critical for mitigating damages. We apply eDNA sampling, which involves the filtering and subsequent DNA extraction of microscopic bits of tissue suspended in water, to ballast and harbor water sampled during a commercial ship's 1400 km voyage through the North American Great Lakes. Using a lab-based gel electrophoresis assay and a rapid, field-ready light transmission spectroscopy (LTS) assay, we test for the presence of two invasive species: quagga (Dreissena bugensis) and zebra (D. polymorpha) mussels. Furthermore, we spiked a set of uninfested ballast and harbor samples with zebra mussel tissue to further test each assay's detection capabilities. In unmanipulated samples, zebra mussel was not detected, while quagga mussel was detected in all samples at a rate of 85% for the gel assay and 100% for the LTS assay. In the spiked experimental samples, both assays detected zebra mussel in 94% of spiked samples and 0% of negative controls. Overall, these results demonstrate that eDNA sampling is effective for monitoring ballast-mediated invasions and that LTS has the potential for rapid, field-based detection.

  17. Leishmania OligoC-TesT as a simple, rapid, and standardized tool for molecular diagnosis of cutaneous leishmaniasis in Peru.

    Science.gov (United States)

    Espinosa, Diego; Boggild, Andrea K; Deborggraeve, Stijn; Laurent, Thierry; Valencia, Cristian; Pacheco, Rosa; Miranda-Verástegui, César; Llanos-Cuentas, Alejandro; Leclipteux, Thierry; Dujardin, Jean-Claude; Büscher, Philippe; Arévalo, Jorge

    2009-08-01

    Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru.

  18. Stimuli Responsive Systems Constructed Using Cucurbit[n]uril-Type Molecular Containers

    Science.gov (United States)

    2015-01-01

    Conspectus This Account focuses on stimuli responsive systems that function in aqueous solution using examples drawn from the work of the Isaacs group using cucurbit[n]uril (CB[n]) molecular containers as key recognition elements. Our entry into the area of stimuli responsive systems began with the preparation of glycoluril derived molecular clips that efficiently distinguish between self and nonself by H-bonds and π–π interactions even within complex mixtures and therefore undergo self-sorting. We concluded that the selectivity of a wide variety of H-bonded supramolecular assemblies was higher than previously appreciated and that self-sorting is not exceptional behavior. This lead us to examine self-sorting within the context of CB[n] host–guest chemistry in water. We discovered that CB[n] homologues (CB[7] and CB[8]) display remarkably high binding affinity (Ka up to 1017 M–1) and selectivity (ΔΔG) toward their guests, which renders CB[n]s prime components for the construction of stimuli responsive host–guest systems. The CB[7]·adamantaneammonium ion complex, which is particularly privileged (Ka = 4.2 × 1012 M–1), was introduced by us as a stimulus to trigger constitutional changes in multicomponent self-sorting systems. For example, we describe how the free energy associated with the formation of host–guest complexes of CB[n]-type receptors can drive conformational changes of included guests like triazene–arylene foldamers and cationic calix[4]arenes, as well as induced conformational changes (e.g., ammonium guest size dependent homotropic allostery, metal ion triggered folding, and heterochiral dimerization) of the hosts themselves. Many guests display large pKa shifts within their CB[n]–guest complexes, which we used to promote pH controlled guest swapping and thermal trans-to-cis isomerization of azobenzene derivatives. We also used the high affinity and selectivity of CB[7] toward its guests to outcompete an enzyme (bovine carbonic

  19. Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form.

    Science.gov (United States)

    Amine, Khadija; Miri, Lamia; Naimi, Adil; Saile, Rachid; El Kharrim, Abderrahmane; Mikou, Afaf; Kettani, Anass

    2015-01-01

    There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.

  20. Molecular Typing of Vibrio parahaemolyticus Strains Isolated from Mollusks in the North Adriatic Sea.

    Science.gov (United States)

    Rahman, Mohammad Shamsur; Carraro, Roberta; Cardazzo, Barbara; Carraro, Lisa; Meneguolo, Davide Boscolo; Martino, Maria Elena; Andreani, Nadia Andrea; Bordin, Paola; Mioni, Renzo; Barco, Lisa; Novelli, Enrico; Balzan, Stefania; Fasolato, Luca

    2017-08-01

    Vibrio parahaemolyticus is an emerging foodborne pathogen in the Mediterranean, usually associated with shellfish consumption. The increase in the number of outbreaks in Europe is primarily associated with the global warming of the ocean that has a great impact on the spread and genetic selection of waterborne pathogens. The primary role of Italy in Europe's mollusk production, together with the fact that cases of infections with V. parahaemolyticus are not always notified to the European community, highlighted the necessity of acquiring new information about the epidemiological involvement of shellfish products. The aim of the study was to provide useful insights into the first steps of the Risk Assessment associated with V. parahaemolyticus through the molecular characterization of isolates from commercialized mollusks. A total of 102 strains identified as V. parahaemolyticus were investigated as part of a larger sampling (1-year survey) from several shellfish species collected from the Venice lagoon and the North Adriatic sea. All strains were characterized by multilocus sequence typing and tested for the presence of virulence genes (trh and tdh). The study of sampling/environmental factors and epidemiological analyses was performed to describe the behaviors of the different genetic populations. The population structure analysis highlighted three genetic clusters that could be subject to temperature selection during cold (≤15°C) and warm (>16°C) seasons. Moreover, other factors, such as molluscan species (clams/mussels), probably played a role in the distribution of genetic clusters. Although few strains carried the virulence factors (n = 6 trh+), epidemiological links with clinical isolates and a local dissemination of some sequence types were underlined. This work provides a useful background on the genotype spread as a first step in the Hazard Identification in light of future climate changes.

  1. Molecular organization of the mating-type loci in the homothallic Ascomycete Eupenicillium crustaceum.

    Science.gov (United States)

    Pöggeler, Stefanie; O'Gorman, Céline M; Hoff, Birgit; Kück, Ulrich

    2011-07-01

    Eupenicillium species are the teleomorphic (sexual) forms of anamorphic (asexual) members of the genus Penicillium, which contains many species of industrial importance. Here we describe the first molecular analysis of the mating-type (MAT) locus from a homothallic (self-fertile) Eupenicillium species, E. crustaceum. This ascomycete is a sexual relative of the penicillin producer Penicillium chrysogenum, which while long considered asexual, was recently shown to possess the required genetic machinery for heterothallic breeding. The E. crustaceum genome contains two MAT loci, MAT1-1 and MAT1-2, in an arrangement characteristic of other known homothallic euascomycetes, such as Neosartorya fischeri. MAT1-1 is flanked by conserved APN2 (DNA lyase) and SLA2 (cytoskeleton assembly control) genes and encodes a homologue of the α-box domain protein MAT1-1-1. Conversely, MAT1-2 carries a HMG-domain gene MAT1-2-1, and is flanked by a degenerate SLA2 gene and an intact homologue of the P. chrysogenum ORF Pc20g08960. Here we demonstrate the transcriptional expression of both mating-type genes during vegetative development. Furthermore, the MAT1-1-1 and MAT1-2-1 sequences were used to resolve the phylogenetic relationship of E. crustaceum with other ascomycetes. Phylogenetic trees confirmed a very close relationship between the homothallic E. crustaceum and the supposedly heterothallic P. chrysogenum. This close taxonomic association makes E. crustaceum an ideal candidate for future expression and evolutionary studies of sexual reproduction, with the ultimate aim of inducing sex in P. chrysogenum. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  2. Molecular Characterization and Panton-Valentine Leucocidin Typing of Community-Acquired Methicillin-Sensitive Staphylococcus aureus Clinical Isolates

    Science.gov (United States)

    Sloan, Tim; Kearns, Angela M.; James, Richard

    2012-01-01

    Limited comprehensive molecular typing data exist currently for Panton-Valentine leucocidin (PVL)-positive, methicillin-sensitive Staphylococcus aureus (PVL-MSSA) clinical isolates. Characterization of PVL-MSSA isolates by multilocus sequence typing (MLST) and spa typing in this study showed a genetic similarity to PVL-positive, methicillin-resistant S. aureus (PVL-MRSA) strains, although three novel spa types and a novel MLST (ST1518) were detected. Furthermore, the detection of PVL phages and haplotypes in PVL-MSSA identical to those previously found in PVL-MRSA isolates highlights the role these strains may play as precursors of emerging lineages of clinical significance. PMID:22718937

  3. A molecular dynamics simulation study for the mechanical properties of different types of carbon nanotubes

    Science.gov (United States)

    Talukdar, Keka; Mitra, Apurba Krishna

    2012-09-01

    Carbon nanotubes have caught tremendous attention of the researchers during the last decade due to their excellent mechanical, electrical, optical and thermal properties. The exploitation of these fibers as reinforcing agents in making strong fiber composites has been a primary research topic in the recent investigations on composite materials. Although the theoretical results are rather optimistic, the goal of achieving high strength of the carbon nanotube composites is still not satisfactorily realized. We report here a comparative study of the mechanical properties of single-walled, multi-walled and bundle of single-walled carbon nanotubes. Their mechanical behavior is investigated by molecular dynamics simulation, considering Brenner's second generation reactive empirical bond order interatomic potential between the carbon atoms making a tube. For a long range interaction, we have defined a weak van der Waals force which acts between different layers of a multi-walled tube or between different tubes of a bundle. Samples of three isolated armchair single-wall carbon nanotubes of different diameters, a multi-wall armchair carbon nanotube and finally a bundle of three armchair single-walled nanotubes of same diameter are taken. Their fracture pattern and buckling behavior are modeled and compared. Significant changes are observed in the mechanical properties of the samples of different types of carbon nanotubes which arise due to the interaction between the shells of a multi-walled tube or the tubes in a bundle.

  4. Fast response dry-type artificial molecular muscles with [c2]daisy chains

    Science.gov (United States)

    Iwaso, Kazuhisa; Takashima, Yoshinori; Harada, Akira

    2016-06-01

    Hierarchically organized myosin and actin filaments found in biological systems exhibit contraction and expansion behaviours that produce work and force by consuming chemical energy. Inspired by these naturally occurring examples, we have developed photoresponsive wet- and dry-type molecular actuators built from rotaxane-based compounds known as [c2]daisy chains (specifically, [c2]AzoCD2 hydrogel and [c2]AzoCD2 xerogel). These actuators were prepared via polycondensation between four-armed poly(ethylene glycol) and a [c2]daisy chain based on α-cyclodextrin as the host component and azobenzene as a photoresponsive guest component. The light-induced actuation arises from the sliding motion of the [c2]daisy chain unit. Ultraviolet irradiation caused the gels to bend towards the light source. The response of the [c2]AzoCD2 xerogel, even under dry conditions, is very fast (7° every second), which is 10,800 times faster than the [c2]AzoCD2 hydrogel (7° every 3 h). In addition, the [c2]AzoCD2 xerogel was used as a crane arm to lift an object using ultraviolet irradiation to produce mechanical work.

  5. Controllable Molecular Packing Motif and Overlap Type in Organic Nanomaterials for Advanced Optical Properties

    Directory of Open Access Journals (Sweden)

    Taoyu Zou

    2018-01-01

    Full Text Available The optical properties of organic materials are very sensitive to subtle structural modification, and a proper understanding of the structure-property relationship is essential to improve the performance of organic electronic devices. The phase transitions of the η-CuPc to the α-CuPc, then to the β-CuPc were investigated using In situ X-ray diffraction and the differential scanning calorimetry (DSC. The five stages in the phase-transition process from low to high-temperature were observed, which consisted of (1 the η-CuPc; (2 a mixture of the η- and α-CuPc; (3 a mixture of the η-, α- and β-CuPc; (4 a mixture of the α- and β-CuPc; and (5 the β-CuPc. The vibrational and optical properties at different phase-transition stages were correlated to molecular packing motif and molecule overlap type through systematic analyses of the Fourier–transform infrared, Raman and UV-VIS spectra. Moreover, the mechanism for the morphology evolution was also discussed in detail.

  6. Molecular typing of canine parvovirus from Sulaimani, Iraq and phylogenetic analysis using partial VP2 gene

    Directory of Open Access Journals (Sweden)

    M.O.Baba Sheikh

    2017-09-01

    Full Text Available Canine parvovirus (CPV remains the most significant viral cause of haemorrhagic enteritis and bloody diarrhoea in puppies over the age of 12 weeks. The objective of the present study was to detect and genotype CPV-2 by polymerase chain reaction (PCR and to perform phylogenetic analysis using partial VP2 gene sequences. We analysed eight faecal samples of unvaccinated dogs with signs of vomiting and bloody diarrhoea during the period from December 2013 to May 2014 in different locations in Sulaimani, Kurdistan, Iraq. After PCR detection, we found that all viral sequences in our study were CPV-2b variants, which differed genetically by 0.8% to 3.6% from five commercially available vaccines. Alignment between eight nucleotides of field virus sequences showed 95% to 99.5% similarity. The phylogenetic analysis for the 8 field sequences formed two distinct clusters with two sequences belonging to strains from China and Thailand and the other six – with a strain from Egypt. Molecular characterisation and CPV typing are crucial in epidemiological studies for future prevention and control of the disease.

  7. Molecular typing of monophasic Salmonella 4,[5]:i:- strains isolated in Belgium (2008-2011).

    Science.gov (United States)

    Boland, Cécile; Bertrand, Sophie; Mattheus, Wesley; Dierick, Katelijne; Wattiau, Pierre

    2014-01-31

    To assess the distribution of Salmonella 4,[5]:i:- subtypes in the Belgian food chain and compare it to the subtypes associated with human infections, a molecular assessment was initiated. Two hundred fifty-three Salmonella isolates serotyped as 4,[5]:i:- during the period 2008-2011 in Belgium and originating from animal productions, food or human clinical samples were analysed by a specific duplex PCR. One hundred ninety-four isolates (76.7%) fit the profile of a S. Typhimurium monophasic variant as defined by the European Food Safety Authority. The other isolates possessed but did not express the phase II flagellin gene (23.3%). Multiple Locus Variable Number of Tandem Repeats Analysis (MLVA) revealed many but closely related profiles in the fljB-negative S. Typhimurium monophasic variant isolates. Some MLVA types were associated with both human and animal isolates but no unique source of human contamination could be demonstrated. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation.

    Science.gov (United States)

    Ripoll-Rozada, Jorge; García-Cazorla, Yolanda; Getino, María; Machón, Cristina; Sanabria-Ríos, David; de la Cruz, Fernando; Cabezón, Elena; Arechaga, Ignacio

    2016-06-01

    Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes. © 2016 John Wiley & Sons Ltd.

  9. The molecular mechanisms of zinc neurotoxicity and the pathogenesis of vascular type senile dementia.

    Science.gov (United States)

    Mizuno, Dai; Kawahara, Masahiro

    2013-11-07

    Zinc (Zn) is an essential trace element that is abundantly present in the brain. Despite its importance in normal brain functions, excess Zn is neurotoxic and causes neurodegeneration following transient global ischemia and plays a crucial role in the pathogenesis of vascular-type dementia (VD). We have investigated the molecular mechanisms of Zn-induced neurotoxicity using immortalized hypothalamic neurons (GT1-7 cells) and found that carnosine (β-alanyl histidine) and histidine (His) inhibited Zn2+-induced neuronal death. A DNA microarray analysis revealed that the expression of several genes, including metal-related genes (metallothionein and Zn transporter 1), endoplasmic reticulum (ER)-stress related genes (GADD34, GADD45, and p8), and the calcium (Ca)-related gene Arc (activity-related cytoskeleton protein), were affected after Zn exposure. The co-existence of carnosine or His inhibited the expression of GADD34, p8, and Arc, although they did not influence the expression of the metal-related genes. Therefore, ER-stress and the disruption of Ca homeostasis may underlie the mechanisms of Zn-induced neurotoxicity, and carnosine might be a possible drug candidate for the treatment of VD.

  10. The Molecular Mechanisms of Zinc Neurotoxicity and the Pathogenesis of Vascular Type Senile Dementia

    Directory of Open Access Journals (Sweden)

    Masahiro Kawahara

    2013-11-01

    Full Text Available Zinc (Zn is an essential trace element that is abundantly present in the brain. Despite its importance in normal brain functions, excess Zn is neurotoxic and causes neurodegeneration following transient global ischemia and plays a crucial role in the pathogenesis of vascular-type dementia (VD. We have investigated the molecular mechanisms of Zn-induced neurotoxicity using immortalized hypothalamic neurons (GT1-7 cells and found that carnosine (β-alanyl histidine and histidine (His inhibited Zn2+-induced neuronal death. A DNA microarray analysis revealed that the expression of several genes, including metal-related genes (metallothionein and Zn transporter 1, endoplasmic reticulum (ER-stress related genes (GADD34, GADD45, and p8, and the calcium (Ca-related gene Arc (activity-related cytoskeleton protein, were affected after Zn exposure. The co-existence of carnosine or His inhibited the expression of GADD34, p8, and Arc, although they did not influence the expression of the metal-related genes. Therefore, ER-stress and the disruption of Ca homeostasis may underlie the mechanisms of Zn-induced neurotoxicity, and carnosine might be a possible drug candidate for the treatment of VD.

  11. Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D.

    Science.gov (United States)

    DeAngelis, Paul L; White, Carissa L

    2002-03-01

    Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.

  12. Clinical, Biochemical and Molecular Characteristics of the Main Types of Porphyria.

    Science.gov (United States)

    Szlendak, Urszula; Bykowska, Ksenia; Lipniacka, Agnieszka

    2016-01-01

    Porphyrias are diverse disorders that arise from various inherited enzyme defects in the heme biosynthesis pathway, except for porphyria cutanea tarda (PCT), in which the enzyme deficiency in most cases is acquired. The biosynthetic blocks resulting from the defective enzymes are largely expressed either in the liver or bone marrow, the sites where the majority of heme is produced. Although the pathophysiologic mechanisms of the clinical manifestations of the porphyrias are not fully understood, two cardinal features prevail: skin photosensitivity and neurologic symptoms of intermittent autonomic neuropathy, acute neurovisceral attacks, and disorders of the nervous system. The primary diagnosis of the proband is based on biochemical testing, which is not always able to identify acute porphyrias, especially in asymptomatic family carriers when heme precursors and porphyrins excretion is normal, low-normal and high-reduced values of enzyme activity overlap, and hematological diseases responsible for abnormal blood cells distribution coexist. Molecular analysis of gene mutations responsible for each type of porphyria is the best diagnostic approach for symptomatic as well as presymptomatic gene carriers.

  13. Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy.

    Science.gov (United States)

    Pasmant, E; Louvrier, C; Luscan, A; Cohen, J; Laurendeau, I; Vidaud, M; Vidaud, D; Goutagny, S; Kalamarides, M; Parfait, B

    2015-06-11

    Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy. We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria. A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2). High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  14. Rapid identification of molecular changes in tulsi (Ocimum sanctum Linn) upon ageing using leaf spray ionization mass spectrometry.

    Science.gov (United States)

    Sarkar, Depanjan; Srimany, Amitava; Pradeep, T

    2012-10-07

    Tulsi or Holy Basil (Ocimum sanctum Linn) is a medicinally important plant. Ursolic acid (UA) and oleanolic acid (OA) are among its major constituents which account for many medicinal activities of the plant. In the present work, we deployed a new ambient ionization method, leaf spray ionization, for rapid detection of UA, OA and their oxidation products from tulsi leaves. Tandem electrospray ionization mass spectrometry (ESI-MS) has been performed on tulsi leaf extracts in methanol to establish the identity of the compounds. We probed changes occurring in the relative amounts of the parent compounds (UA and OA) with their oxidized products and the latter show an increasing trend upon ageing. The findings are verified by ESI-MS analysis of tulsi leaf extracts, which shows the same trend proving the reliability of the leaf spray method.

  15. Two mutations associated with macrolide resistance in Treponema pallidum: increasing prevalence and correlation with molecular strain type in Seattle, Washington.

    Science.gov (United States)

    Grimes, Matthew; Sahi, Sharon K; Godornes, B Charmie; Tantalo, Lauren C; Roberts, Neal; Bostick, David; Marra, Christina M; Lukehart, Sheila A

    2012-12-01

    Although azithromycin promised to be a safe and effective single-dose oral treatment of early syphilis, azithromycin treatment failure has been documented and is associated with mutations in the 23S rDNA of corresponding Treponema pallidum strains. The prevalence of strains harboring these mutations varies throughout the United States and the world. We examined T. pallidum strains circulating in Seattle, Washington, from 2001 to 2010 to determine the prevalence of 2 mutations associated with macrolide resistance and to determine whether these mutations were associated with certain T. pallidum strain types. Subjects were enrolled in a separate ongoing study of cerebrospinal fluid abnormalities in patients with syphilis. T. pallidum DNA purified from blood and T. pallidum strains isolated from blood or cerebrospinal fluid were analyzed for two 23S rDNA mutations and for the molecular targets used in an enhanced molecular stain typing system. Nine molecular strain types of T. pallidum were identified in Seattle from 2001 to 2010. Both macrolide resistance mutations were identified in Seattle strains, and the prevalence of resistant T. pallidum exceeded 80% in 2005 and increased through 2010. Resistance mutations were associated with discrete molecular strain types of T. pallidum. Macrolide-resistant T. pallidum strains are highly prevalent in Seattle, and each mutation is associated with discrete strain types. Macrolides should not be considered for treatment of syphilis in regions where prevalence of the mutations is high. Combining the resistance mutations with molecular strain typing permits a finer analysis of the epidemiology of syphilis in a community.

  16. Molecular characteristics of Human Endogenous Retrovirus type-W in schizophrenia and bipolar disorder.

    Science.gov (United States)

    Perron, H; Hamdani, N; Faucard, R; Lajnef, M; Jamain, S; Daban-Huard, C; Sarrazin, S; LeGuen, E; Houenou, J; Delavest, M; Moins-Teisserenc, H; Moins-Teiserenc, H; Bengoufa, D; Yolken, R; Madeira, A; Garcia-Montojo, M; Gehin, N; Burgelin, I; Ollagnier, G; Bernard, C; Dumaine, A; Henrion, A; Gombert, A; Le Dudal, K; Charron, D; Krishnamoorthy, R; Tamouza, R; Leboyer, M

    2012-12-04

    Epidemiological and genome-wide association studies of severe psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD), suggest complex interactions between multiple genetic elements and environmental factors. The involvement of genetic elements such as Human Endogenous Retroviruses type 'W' family (HERV-W) has consistently been associated with SZ. HERV-W envelope gene (env) is activated by environmental factors and encodes a protein displaying inflammation and neurotoxicity. The present study addressed the molecular characteristics of HERV-W env in SZ and BD. Hundred and thirty-six patients, 91 with BD, 45 with SZ and 73 healthy controls (HC) were included. HERV-W env transcription was found to be elevated in BD (P<10-4) and in SZ (P=0.012) as compared with HC, but with higher values in BD than in SZ group (P<0.01). The corresponding DNA copy number was paradoxically lower in the genome of patients with BD (P=0.0016) or SZ (P<0.0003) than in HC. Differences in nucleotide sequence of HERV-W env were found between patients with SZ and BD as compared with HC, as well as between SZ and BD. The molecular characteristics of HERV-W env also differ from what was observed in Multiple Sclerosis (MS) and may represent distinct features of the genome of patients with BD and SZ. The seroprevalence for Toxoplasma gondii yielded low but significant association with HERV-W transcriptional level in a subgroup of BD and SZ, suggesting a potential role in particular patients. A global hypothesis of mechanisms inducing such major psychoses is discussed, placing HERV-W at the crossroads between environmental, genetic and immunological factors. Thus, particular infections would act as activators of HERV-W elements in earliest life, resulting in the production of an HERV-W envelope protein, which then stimulates pro-inflammatory and neurotoxic cascades. This hypothesis needs to be further explored as it may yield major changes in our understanding and treatment of

  17. High-Performance Near-Infrared Phototransistor Based on n-Type Small-Molecular Organic Semiconductor

    KAUST Repository

    Li, Feng

    2016-12-13

    A solution-processed near-infrared (NIR) organic phototransistor (OPT) based on n-type organic small molecular material BODIPY-BF2 has been successfully fabricated. Its unprecedented performance, as well as its easy fabrication and good stability, mark this BODIPY-BF2 based OPT device as a very promising candidate for optoelectronic applications in the NIR regime.

  18. Screening and Rapid Molecular Diagnosis of Tuberculosis in Prisons in Russia and Eastern Europe: A Cost-Effectiveness Analysis

    Science.gov (United States)

    Winetsky, Daniel E.; Negoescu, Diana M.; DeMarchis, Emilia H.; Almukhamedova, Olga; Dooronbekova, Aizhan; Pulatov, Dilshod; Vezhnina, Natalia; Owens, Douglas K.; Goldhaber-Fiebert, Jeremy D.

    2012-01-01

    Background Prisons of the former Soviet Union (FSU) have high rates of multidrug-resistant tuberculosis (MDR-TB) and are thought to drive general population tuberculosis (TB) epidemics. Effective prison case detection, though employing more expensive technologies, may reduce long-term treatment costs and slow MDR-TB transmission. Methods and Findings We developed a dynamic transmission model of TB and drug resistance matched to the epidemiology and costs in FSU prisons. We evaluated eight strategies for TB screening and diagnosis involving, alone or in combination, self-referral, symptom screening, mass miniature radiography (MMR), and sputum PCR with probes for rifampin resistance (Xpert MTB/RIF). Over a 10-y horizon, we projected costs, quality-adjusted life years (QALYs), and TB and MDR-TB prevalence. Using sputum PCR as an annual primary screening tool among the general prison population most effectively reduced overall TB prevalence (from 2.78% to 2.31%) and MDR-TB prevalence (from 0.74% to 0.63%), and cost US$543/QALY for additional QALYs gained compared to MMR screening with sputum PCR reserved for rapid detection of MDR-TB. Adding sputum PCR to the currently used strategy of annual MMR screening was cost-saving over 10 y compared to MMR screening alone, but produced only a modest reduction in MDR-TB prevalence (from 0.74% to 0.69%) and had minimal effect on overall TB prevalence (from 2.78% to 2.74%). Strategies based on symptom screening alone were less effective and more expensive than MMR-based strategies. Study limitations included scarce primary TB time-series data in FSU prisons and uncertainties regarding screening test characteristics. Conclusions In prisons of the FSU, annual screening of the general inmate population with sputum PCR most effectively reduces TB and MDR-TB prevalence, doing so cost-effectively. If this approach is not feasible, the current strategy of annual MMR is both more effective and less expensive than strategies using self

  19. Screening and rapid molecular diagnosis of tuberculosis in prisons in Russia and Eastern Europe: a cost-effectiveness analysis.

    Directory of Open Access Journals (Sweden)

    Daniel E Winetsky

    Full Text Available Prisons of the former Soviet Union (FSU have high rates of multidrug-resistant tuberculosis (MDR-TB and are thought to drive general population tuberculosis (TB epidemics. Effective prison case detection, though employing more expensive technologies, may reduce long-term treatment costs and slow MDR-TB transmission.We developed a dynamic transmission model of TB and drug resistance matched to the epidemiology and costs in FSU prisons. We evaluated eight strategies for TB screening and diagnosis involving, alone or in combination, self-referral, symptom screening, mass miniature radiography (MMR, and sputum PCR with probes for rifampin resistance (Xpert MTB/RIF. Over a 10-y horizon, we projected costs, quality-adjusted life years (QALYs, and TB and MDR-TB prevalence. Using sputum PCR as an annual primary screening tool among the general prison population most effectively reduced overall TB prevalence (from 2.78% to 2.31% and MDR-TB prevalence (from 0.74% to 0.63%, and cost US$543/QALY for additional QALYs gained compared to MMR screening with sputum PCR reserved for rapid detection of MDR-TB. Adding sputum PCR to the currently used strategy of annual MMR screening was cost-saving over 10 y compared to MMR screening alone, but produced only a modest reduction in MDR-TB prevalence (from 0.74% to 0.69% and had minimal effect on overall TB prevalence (from 2.78% to 2.74%. Strategies based on symptom screening alone were less effective and more expensive than MMR-based strategies. Study limitations included scarce primary TB time-series data in FSU prisons and uncertainties regarding screening test characteristics.In prisons of the FSU, annual screening of the general inmate population with sputum PCR most effectively reduces TB and MDR-TB prevalence, doing so cost-effectively. If this approach is not feasible, the current strategy of annual MMR is both more effective and less expensive than strategies using self-referral or symptom screening alone

  20. Redundancy and molecular evolution: the rapid Induction of bone formation by the mammalian transforming growth factor-β3 isoform

    Directory of Open Access Journals (Sweden)

    Ugo Ripamonti

    2016-09-01

    Full Text Available The soluble osteogenic molecular signals of the transforming growth factor-β (TGF-β supergene family are the molecular bases of the induction of bone formation and postnatal bone tissue morphogenesis with translation into clinical contexts. The mammalian TGF-β3 isoform, a pleiotropic member of the family, controls a vast array of biological processes including the induction of bone formation. Recombinant hTGF-β3 induces substantial bone formation when implanted with either collagenous bone matrices or coral-derived macroporous bioreactors in the rectus abdominis muscle of the non-human primate Papio ursinus. In marked contrast, the three mammalian TGF-βs do not initiate the induction of bone formation in rodents and lagomorphs. The induction of bone by hTGF-β3/preloaded bioreactors is orchestrated by inducing fibrin-fibronectin rings that structurally organize tissue patterning and morphogenesis within the macroporous spaces. Induced advancing extracellular matrix rings provide the structural anchorage for hyper chromatic cells, interpreted as differentiating osteoblasts re-programmed by hTGF-β3 from invading myoblastic and/or pericytic differentiated cells. Runx2 and Osteocalcin expression are significantly up-regulated correlating to multiple invading cells differentiating into the osteoblastic phenotype. Bioreactors pre-loaded with recombinant human Noggin (hNoggin, a BMPs antagonist, show down-regulation of BMP-2 and other profiled osteogenic proteins’ genes resulting in minimal bone formation. Coral-derived macroporous constructs preloaded with binary applications of hTGF-β3 and hNoggin also show down-regulation of BMP-2 with the induction of limited bone formation. The induction of bone formation by hTGF-β3 is via the BMPs pathway and it is thus blocked by hNoggin. Our systematic studies in Papio ursinus with translational hTGF-β3 in large cranio-mandibulo-facial defects in humans are now requesting the re-evaluation of Bone

  1. DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

    DEFF Research Database (Denmark)

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong

    2011-01-01

    Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required....... In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification...

  2. A rapid molecular method for differentiating two special forms (lycopersici and radicis-lycopersici) of Fusarium oxysporum.

    Science.gov (United States)

    Attitalla, Idress H; Fatehi, Jamshid; Levenfors, Jens; Brishammar, Sture

    2004-07-01

    Two pathogenic special forms (f. sp.) of the Fusarium oxysporum species complex f. sp. lycopersici (Fol) and f. sp. radicis-lycopersici (Forl) are morphologically indistinguishable. Although they are pathogenic to the same host genus Lycopersicon (tomato), and infect the same tomato cultivar, they form distinct diseases; Fol causes wilt and Forl causes crown rot and root rot. These two special forms apparently exist as genetically isolated populations, based on vegetative compatibility and molecular variation at the DNA level. In seeking efficient diagnostic tools for differentiating Fol and Forl isolates, we examined three techniques: isozyme analysis, mitochondrial DNA (mtDNA) RFLP by HaeIII-digestion of total genomic DNA, and an osmotic method using high performance liquid chromatography (HPLC) to detect fungal pigments. The isolates were collected from geographically widespread locations. Distinct HPLC-profile differences were found between an endophytic non-pathogenic isolate and the other pathogenic isolates. However, the direct mtDNA RFLP technique proved to be an efficient diagnostic tool for routine differentiation of Fol and Forl isolates.

  3. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  4. On the Development of a Very Rapid Gas Chromatographic Method for the Analysis of High Molecular Weight Hydrocarbons in Cigarette Smoke

    Directory of Open Access Journals (Sweden)

    Coleman III WM

    2014-12-01

    Full Text Available A very rapid, flash-gas chromatographic (GC, quantitative method for the analysis of high molecular weight saturated hydrocarbons in cigarette smoke has been developed. The method was fast, accurate and precise. Sample turn around times were approximately six minutes, with an accompanying average percent relative standard deviation (%RSD of less than 10%. Four linear saturated hydrocarbons with 27, 29, 31 and 33 carbon atoms were quantitated in an array of reference cigarettes ranging in “tar” deliveries from approximately 2 to approximately 20 mg. By use of a cyclohexane extraction of cigarette smoke captured on Cambridge filter pads, the extraction efficiency was determined to be greater than 95% for each hydrocarbon. The approach represents a significant advance over current analytical procedures that require, on average, greater than 30-min sample turn around times.

  5. Uveal melanoma prognostication: from lesion size and cell type to molecular class.

    Science.gov (United States)

    Gill, Harmeet S; Char, Devron H

    2012-06-01

    To review the evidence for molecular genetic testing of uveal melanoma in the context of prognostic indicators of metastasis and tumour-related mortality. Review of the literature and personal experiences of the authors. We conducted a MEDLINE, Embase, and PubMed literature search (1980-2011) for English-language abstracts and full-text references regarding molecular genetic testing of uveal melanoma. Search terms included uveal, melanoma, cytogenetic, gene, and molecular. All studies in which patients with primary uveal melanoma underwent molecular genetic testing with survival data for disease-related metastasis and mortality were reviewed. From 176 identified articles, 40 were scientific studies of uveal melanomas that included histologic and molecular genetic analysis. Of those, 24 included survival data, correlation of molecular genetic features with other prognostic indicators, or both. Cytogenetic and microarray gene expression analysis allows uveal melanoma lesions to be classified as high risk or low risk for metastasis and disease-related mortality. Gene expression profiling supersedes clinical, histologic, and cytogenetic prognosticators. Uveal melanoma comprises a heterogeneous group of malignancies based on its molecular biology. Molecular class by gene expression profiling has the most strongly predictive value for uveal melanoma metastasis and mortality. Copyright © 2012 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.

  6. Molecular Aspects of Modulation of L-type Calcium Channels by Protein Kinase C.

    Science.gov (United States)

    Weiss, Sharon; Dascal, Nathan

    2015-01-01

    Ca(2+) influx via L-type Ca(2+) channel (L-VDCC; CaV1.2) is required for cardiac and smooth muscle contraction. These channels are located in the plasma membrane and along the T-tubules (in cardiomyocytes), along with various scaffold and trafficking proteins. CaV1.2 is modulated by different hormones and transmitters and was implicated in a variety of cardiovascular pathologies, many of which also involve protein kinase C (PKC). One of the prominent pathways of PKC activation in cardiac and smooth muscle cells is via activation of Gq-coupled receptors and subsequent activation of protein lipase C (PLC). CaV1.2 was shown to be modulated, phosphorylated by, and associated with PKC both in vitro and in vivo. Despite the well documented enhancing effect of vasoconstrictors operating via Gq on CaV1.2 channels, the molecular mechanism by which PKC affects the channel has not yet been resolved. Furthermore, the nature of PKC modulation of CaV1.2 appears to be species-, age- and tissue-dependent. Results from experiments in heterologous expression systems are often contradicting and are difficult to coalesce. The choice of both the heterologous expression system and the CaV1.2 isoform expressed are at the core of this conundrum. Complete reconstitution of the enhancing effect of PKC was successful only in Xenopus oocytes and only when the long N-terminus (NT) isoform of the channel was expressed. This review summarizes past and new findings regarding the mechanism by which activated PKC modulates CaV1.2 channels in native tissues and heterologous expression systems, and suggests perspectives for future research.

  7. Molecular typing of mycobacteria isolated from extrapulmonary tuberculosis patients at Debre Birhan Referral Hospital, central Ethiopia.

    Science.gov (United States)

    Garedew, Legesse; Mihret, Adane; Ameni, Gobena

    2013-07-01

    Extrapulmonary tuberculosis (EPTB) constitutes about 10% to 20% of all cases of tuberculosis in immunocompetent patients and more than 50% of the cases in HIV-positive individuals worldwide. Little information is available on the clonal diversity of Mycobacterium species in Ethiopia from EPTB. This study was carried out on smear-negative EPTB patients to molecularly characterize Mycobacterium tuberculosis complex strains. A questionnaire, smear staining, culture, deletion typing, and spoligotyping were employed. The proportional distribution of EPTB and isolates did not vary substantially (p > 0.05) amongst the socio-demographic parameters considered in the current investigation. Out of 98 fine needle aspirates processed for culture, 36.7% (36/98) were positive for mycobacterial growth. Further speciation of those culture-positive isolates showed that 88.9% were M. tuberculosis and the remaining could be non-tuberculous mycobacterial species. Spoligotyping revealed 16 clusters out of which 2 were new to the SITVIT database. The most dominant spoligotypes were SIT54, SIT53, and SIT149 in decreasing order. SIT54, SIT134, SIT173, SIT345, SIT357, SIT926, SIT91088, and SIT1580 were reported for the first time in Ethiopia. The family with the highest frequency identified was M. tuberculosis family T1, followed by family 33. Most of the strains belonged to Euro-American (61.4%) and Indo-Oceanic (36.3%) lineages. The present study shows the importance of M. tuberculosis as a major cause of EPTB in the study area. Moreover, the majority of isolates of M. tuberculosis were found in clusters, suggesting the possibility of the existence of recent transmission. This warrants strengthening of the control programs for EPTB in the study area.

  8. Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis.

    Science.gov (United States)

    Ben-Rebeh, Imen; Grati, Mhamed; Bonnet, Crystel; Bouassida, Walid; Hadjamor, Imen; Ayadi, Hammadi; Ghorbel, Abdelmonem; Petit, Christine; Masmoudi, Saber

    2016-01-01

    Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, cadherin-23, protocadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively. In the present study, we recruited four Tunisian families with a diagnosis of USH1, together with healthy unrelated controls. Affected members underwent detailed audiologic and ocular examinations. We used the North African Deafness (NADf) chip to search for known North African mutations associated with USH. Then, we selected microsatellite markers covering USH1 known loci to genotype the DNA samples. Finally, we performed DNA sequencing of three known USH1 genes: MYO7A, PCDH15, and USH1C. Four biallelic mutations, all single base changes, were found in the MYO7A, USH1C, and PCDH15 genes. These mutations consist of a previously reported splicing defect c.470+1G>A in MYO7A, three novel variants, including two nonsense (p.Arg3X and p.Arg134X) in USH1C and PCDH15, respectively, and one frameshift (p.Lys615Asnfs*6) in MYO7A. We found a remarkable genetic heterogeneity in the studied families with USH1 with a variety of mutations, among which three were novel. These novel mutations will be included in the NADf mutation screening chip that will allow a higher diagnosis efficiency of this extremely genetically heterogeneous disease. Ultimately, efficient molecular diagnosis of USH in a patient's early childhood is of utmost importance, allowing better educational and therapeutic management.

  9. [Xanthinuria type 1 in a woman with arthralgias: a combined clinical and molecular genetic investigation].

    Science.gov (United States)

    Därr, Roland Wolfgang; Lenzner, Steffen; Eggermann, Thomas; Därr, Wolfgang Hermann

    2016-04-01

    A 53-year old woman with recurrent polyarthralgias, negative test results in a recent rheumatologic work-up and an unmeasurably low uric acid serum concentration presented for suspected IgM paraproteinemia. Physical examination, abdominal ultrasound and routine laboratory test results were unremarkable. Repeat determination confirmed a markedly decreased uric acid (UA) serum concentration. Urinary xanthine and hypoxanthine concentrations were increased by 14-fold and 7.5-fold, respectively. Fractional urinary UA excretion was not increased and the allopurinol loading test was normal. Sequencing of the xanthine dehydrogenase (XDH) gene revealed the pathogenic deletion c.641delC in the homozygous state. Segregation analysis showed that the patient's mother and her two adult sons were carriers of the mutation but not a half-sister and a half-brother of her deceased father. There was no evidence of parental consanguinity. These results established xanthinuria type 1 as the cause of the patient's recurrent polyarthralgias due to a previously unreported homozygosity for the known mutation c.641delC of the XDH gene. The patient was advised to adhere to a low-purine diet and to ensure an increased daily fluid-intake of at least 2.5 l. She has since remained symptom free. Markedly lowered serum uric acid concentrations are a hallmark of xanthinuria and of hereditary renal hypouricemia, and in the absence of severe hepatic failure or evidence of an untoward drug effect should raise suspicion of these diseases. A targeted diagnostic work-up should then be initiated and factitious hypouricemia due to IgM paraproteinemia considered only in the case of equivocal test results. Molecular-genetic characterization and segregation analysis will ultimately establish the underlying genotype. © Georg Thieme Verlag KG Stuttgart · New York.

  10. Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Yvonne Ukamaka Ajamma

    2016-08-01

    -species. This approach can be employed for rapid identification of mosquitoes.

  11. GIANT MOLECULAR CLOUDS IN THE EARLY-TYPE GALAXY NGC 4526

    Energy Technology Data Exchange (ETDEWEB)

    Utomo, Dyas; Blitz, Leo [Department of Astronomy and Radio Astronomy Laboratory, University of California, Berkeley, CA 94720 (United States); Davis, Timothy [European Southern Observatory, Karl-Schwarzschild-Strasse 2, D-85748 Garching-bei-Muenchen (Germany); Rosolowsky, Erik [Department of Physics, University of Alberta, 4-181 CCIS, Edmonton, AB T6G 2E1 (Canada); Bureau, Martin; Cappellari, Michele [Sub-department of Astrophysics, Department of Physics, University of Oxford, Denys Wilkinson Building, Keble Road, Oxford OX1 3RH (United Kingdom); Sarzi, Marc, E-mail: dyas@berkeley.edu [Centre for Astrophysics Research, University of Hertfordshire, Hatfield, Herts AL1 9AB (United Kingdom)

    2015-04-10

    We present a high spatial resolution (≈20 pc) of {sup 12}CO(2 −1) observations of the lenticular galaxy NGC 4526. We identify 103 resolved giant molecular clouds (GMCs) and measure their properties: size R, velocity dispersion σ{sub v}, and luminosity L. This is the first GMC catalog of an early-type galaxy. We find that the GMC population in NGC 4526 is gravitationally bound, with a virial parameter α ∼ 1. The mass distribution, dN/dM ∝ M{sup −2.39±0.03}, is steeper than that for GMCs in the inner Milky Way, but comparable to that found in some late-type galaxies. We find no size–line width correlation for the NGC 4526 clouds, in contradiction to the expectation from Larson’s relation. In general, the GMCs in NGC 4526 are more luminous, denser, and have a higher velocity dispersion than equal-size GMCs in the Milky Way and other galaxies in the Local Group. These may be due to higher interstellar radiation field than in the Milky Way disk and weaker external pressure than in the Galactic center. In addition, a kinematic measurement of cloud rotation shows that the rotation is driven by the galactic shear. For the vast majority of the clouds, the rotational energy is less than the turbulent and gravitational energy, while the four innermost clouds are unbound and will likely be torn apart by the strong shear at the galactic center. We combine our data with the archival data of other galaxies to show that the surface density Σ of GMCs is not approximately constant, as previously believed, but varies by ∼3 orders of magnitude. We also show that the size and velocity dispersion of the GMC population across galaxies are related to the surface density, as expected from the gravitational and pressure equilibrium, i.e., σ{sub v} R{sup −1/2} ∝ Σ{sup 1/2}.

  12. MCL and mincle: C-type lectin receptors that sense damaged self and pathogen associated molecular patterns

    Directory of Open Access Journals (Sweden)

    Mark B Richardson

    2014-06-01

    Full Text Available MCL (macrophage C-type lectin and mincle (macrophage inducible C-type lectin comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage associated and pathogen associated molecular patterns. In this review we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure-activity relationships of activating ligands, particularly trehalose dimycolate (TDM and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies.

  13. Effect of polymer molecular weight and of polymer blends on the properties of rapidly gelling nasal inserts.

    Science.gov (United States)

    Bertram, Ulrike; Bodmeier, Roland

    2012-06-01

    The objective was to investigate the potential of polymer molecular weight (MW) and polymer blends for the control of drug release from in situ gelling nasal inserts prepared by lyophilization of solutions of model drugs (oxymetazoline HCl, diprophyllin) and polymers. Drug release, polymer solution viscosity, water uptake and mass loss, mechanical properties, and bioadhesion potential were measured. Sonication was effective to reduce the viscosity/polymer MW of carrageenan solutions. Nasal inserts prepared from sonicated carrageenan showed an insignificant reduction in water uptake with sonication time and no disintegration of the gel matrix. In contrast, inserts of different MW Na-alginates revealed a reduced water uptake and an increased mass loss with lower MW. Inserts prepared from carrageenan/low MW Na-alginate blends took up more water at a higher low MW Na-alginate content. Sonicated carrageenan inserts released oxymetazoline HCl independent of the sonication time and diprophyllin with only a slight reduction in the release rate. Release of both drugs from Na-alginate inserts was slow from high MW inserts because no insert dissolution occurred. Increasing the Na-alginate content of inserts prepared from polymer blends accelerated the drug release enabling release rates over a broad range. The bioadhesion potential of Na-alginate inserts was strongly reduced for the low MW grades because of dissolution of the inserts. Xanthan gum and Carbopol 971 blended with Na-alginate formed inserts with poor bioadhesion. The use of polymer blends to control the drug release from nasal inserts was superior to the use of polymers of different MW.

  14. Conformational Analysis of a High-Mannose-Type Oligosaccharide Displaying Glucosyl Determinant Recognised by Molecular Chaperones Using NMR-Validated Molecular Dynamics Simulation.

    Science.gov (United States)

    Suzuki, Tatsuya; Kajino, Megumi; Yanaka, Saeko; Zhu, Tong; Yagi, Hirokazu; Satoh, Tadashi; Yamaguchi, Takumi; Kato, Koichi

    2017-02-16

    Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high-mannose-type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses (13) C labelling and lanthanide tagging to detect conformation-dependent paramagnetic effects by NMR spectroscopy. The NMR-validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced-fit mechanism. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Molecular evidence of malaria and zoonotic diseases among rapid diagnostic test-negative febrile patients in low-transmission season, Mali

    DEFF Research Database (Denmark)

    Touré, Mahamoudou; Petersen, Pelle T; Bathily, Sidy N'd

    2017-01-01

    From November to December 2012 in Sélingué-Mali, blood samples from 88 febrile patients who tested negative by malaria Paracheck (®) rapid diagnostic tests (RDTs) were used to assess the presence of sub-RDT Plasmodium falciparum as well as Borrelia, Coxiella burnetii, and Babesia applying molecul...... the febrile patients call for further studies to assess the causes of fever among malaria RDT-negative patients in Sélingué.......From November to December 2012 in Sélingué-Mali, blood samples from 88 febrile patients who tested negative by malaria Paracheck (®) rapid diagnostic tests (RDTs) were used to assess the presence of sub-RDT Plasmodium falciparum as well as Borrelia, Coxiella burnetii, and Babesia applying molecular...... tools. Plasmodium sp. was present among 57 (60.2%) of the 88 malaria RDT-negative patients, whereas the prevalence of Borrelia, C. burnetii, and Babesia were 3.4% (N = 3), 1.1% (N = 1), and 0.0%, respectively. The additional diagnostic use of polymerase chain reaction (PCR) identified a high proportion...

  16. Testing mitochondrial sequences and anonymous nuclear markers for phylogeny reconstruction in a rapidly radiating group: molecular systematics of the Delphininae (Cetacea: Odontoceti: Delphinidae)

    Science.gov (United States)

    Kingston, Sarah E; Adams, Lara D; Rosel, Patricia E

    2009-01-01

    Background Many molecular phylogenetic analyses rely on DNA sequence data obtained from single or multiple loci, particularly mitochondrial DNA loci. However, phylogenies for taxa that have undergone recent, rapid radiation events often remain unresolved. Alternative methodologies for discerning evolutionary relationships under these conditions are desirable. The dolphin subfamily Delphininae is a group that has likely resulted from a recent and rapid radiation. Despite several efforts, the evolutionary relationships among the species in the subfamily remain unclear. Results Here, we compare a phylogeny estimated using mitochondrial DNA (mtDNA) control region sequences to a multi-locus phylogeny inferred from 418 polymorphic genomic markers obtained from amplified fragment length polymorphism (AFLP) analysis. The two sets of phylogenies are largely incongruent, primarily because the mtDNA tree provides very poor resolving power; very few species' nodes in the tree are supported by bootstrap resampling. The AFLP phylogeny is considerably better resolved and more congruent with relationships inferred from morphological data. Both phylogenies support paraphyly for the genera Stenella and Tursiops. The AFLP data indicate a close relationship between the two spotted dolphin species and recent ancestry between Stenella clymene and S. longirostris. The placement of the Lagenodelphis hosei lineage is ambiguous: phenetic analysis of the AFLP data is consistent with morphological expectations but the phylogenetic analysis is not. Conclusion For closely related, recently diverged taxa, a multi-locus genome-wide survey is likely the most comprehensive approach currently available for phylogenetic inference. PMID:19811651

  17. Testing mitochondrial sequences and anonymous nuclear markers for phylogeny reconstruction in a rapidly radiating group: molecular systematics of the Delphininae (Cetacea: Odontoceti: Delphinidae).

    Science.gov (United States)

    Kingston, Sarah E; Adams, Lara D; Rosel, Patricia E

    2009-10-07

    Many molecular phylogenetic analyses rely on DNA sequence data obtained from single or multiple loci, particularly mitochondrial DNA loci. However, phylogenies for taxa that have undergone recent, rapid radiation events often remain unresolved. Alternative methodologies for discerning evolutionary relationships under these conditions are desirable. The dolphin subfamily Delphininae is a group that has likely resulted from a recent and rapid radiation. Despite several efforts, the evolutionary relationships among the species in the subfamily remain unclear. Here, we compare a phylogeny estimated using mitochondrial DNA (mtDNA) control region sequences to a multi-locus phylogeny inferred from 418 polymorphic genomic markers obtained from amplified fragment length polymorphism (AFLP) analysis. The two sets of phylogenies are largely incongruent, primarily because the mtDNA tree provides very poor resolving power; very few species' nodes in the tree are supported by bootstrap resampling. The AFLP phylogeny is considerably better resolved and more congruent with relationships inferred from morphological data. Both phylogenies support paraphyly for the genera Stenella and Tursiops. The AFLP data indicate a close relationship between the two spotted dolphin species and recent ancestry between Stenella clymene and S. longirostris. The placement of the Lagenodelphis hosei lineage is ambiguous: phenetic analysis of the AFLP data is consistent with morphological expectations but the phylogenetic analysis is not. For closely related, recently diverged taxa, a multi-locus genome-wide survey is likely the most comprehensive approach currently available for phylogenetic inference.

  18. Testing mitochondrial sequences and anonymous nuclear markers for phylogeny reconstruction in a rapidly radiating group: molecular systematics of the Delphininae (Cetacea: Odontoceti: Delphinidae

    Directory of Open Access Journals (Sweden)

    Kingston Sarah E

    2009-10-01

    Full Text Available Abstract Background Many molecular phylogenetic analyses rely on DNA sequence data obtained from single or multiple loci, particularly mitochondrial DNA loci. However, phylogenies for taxa that have undergone recent, rapid radiation events often remain unresolved. Alternative methodologies for discerning evolutionary relationships under these conditions are desirable. The dolphin subfamily Delphininae is a group that has likely resulted from a recent and rapid radiation. Despite several efforts, the evolutionary relationships among the species in the subfamily remain unclear. Results Here, we compare a phylogeny estimated using mitochondrial DNA (mtDNA control region sequences to a multi-locus phylogeny inferred from 418 polymorphic genomic markers obtained from amplified fragment length polymorphism (AFLP analysis. The two sets of phylogenies are largely incongruent, primarily because the mtDNA tree provides very poor resolving power; very few species' nodes in the tree are supported by bootstrap resampling. The AFLP phylogeny is considerably better resolved and more congruent with relationships inferred from morphological data. Both phylogenies support paraphyly for the genera Stenella and Tursiops. The AFLP data indicate a close relationship between the two spotted dolphin species and recent ancestry between Stenella clymene and S. longirostris. The placement of the Lagenodelphis hosei lineage is ambiguous: phenetic analysis of the AFLP data is consistent with morphological expectations but the phylogenetic analysis is not. Conclusion For closely related, recently diverged taxa, a multi-locus genome-wide survey is likely the most comprehensive approach currently available for phylogenetic inference.

  19. Rapid disease progression in human immunodeficiency virus type 1-infected individuals with adverse reactions to trimethoprim-sulfamethoxazole prophylaxis

    NARCIS (Netherlands)

    Veenstra, J.; Veugelers, P. J.; Keet, I. P.; van der Ven, A. J. A. M.; Miedema, F.; Lange, J. M.; Coutinho, R. A.

    1997-01-01

    We studied the relation between the occurrence of adverse reactions to trimethoprim-sulfamethoxazole (TMP-SMZ) prophylaxis and the subsequent course of human immunodeficiency virus (HIV) infection in a cohort of homosexual men. Adverse reactions to TMP-SMZ were associated with a more rapid

  20. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay.

    Science.gov (United States)

    Singh, A K; Maurya, A K; Kant, S; Umrao, J; Kushwaha, R A S; Nag, V L; Dhole, T N

    2013-01-01

    The emergence of extensively drug-resistant tuberculosis (XDR-TB) is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. We evaluated 98 multidrug-resistant (MDR) M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system) and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. A total of seven (17.4%) were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V) in seven (41.2%) and gyrA + WT1-3 + MUT1 in four (23.5%); rrs MUT1 (A1401G) in 11 (64.7%), and rrs WT1-2 + MUT1 in eight (47.1%); and embB MUT1B (M306V) in 11 (64.7%) strains. These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  1. A novel molecularly imprinted electrochemical sensor based on graphene quantum dots coated on hollow nickel nanospheres with high sensitivity and selectivity for the rapid determination of bisphenol S.

    Science.gov (United States)

    Rao, Hanbing; Zhao, Xun; Liu, Xin; Zhong, Ji; Zhang, Zhaoyi; Zou, Ping; Jiang, Yuanyuan; Wang, Xianxiang; Wang, Yanying

    2018-02-15

    In this paper, a novel molecularly imprinted electrochemical sensor (MIECS) based on a glassy carbon electrode (GCE) modified with graphene quantum dots (GQDs) coated on hollow nickel nanospheres (hNiNS) for the rapid determination of bisphenol S (BPS) was proposed for the first time. HNiNS and GQDs as electrode modifications were used to enlarge the active area and electron-transport ability for amplifying the sensor signal, while molecularly imprinted polymer (MIP) film was electropolymerized by using pyrrole as monomer and BPS as template to detect BPS via cyclic voltammetry (CV). Scanning electron microscope (SEM), energy-dispersive spectrometry (EDS), CV and differential pulse voltammetry (DPV) were employed to characterize the fabricated sensor. Experimental conditions, such as molar ratio of monomer to template, electropolymerization cycles, pH, incubation time and elution time were optimized. The DPV response of the MIECS to BPS was obtained in the linear range from 0.1 to 50μM with a low limit of detection (LOD) of 0.03μM (S/N = 3) under the optimized conditions. The MIECS exhibited excellent response towards BPS with high sensitivity, selectivity, good reproducibility, and stability. In addition, the proposed MIECS was also successfully applied for the determination of BPS in the plastic samples with simple sample pretreatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

    Science.gov (United States)

    Howson, E L A; Armson, B; Madi, M; Kasanga, C J; Kandusi, S; Sallu, R; Chepkwony, E; Siddle, A; Martin, P; Wood, J; Mioulet, V; King, D P; Lembo, T; Cleaveland, S; Fowler, V L

    2017-06-01

    Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV. © 2015 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.

  3. Molecular Epidemiology Study in Xuanwei: the Relationship among
Coal Type, Genotype and Lung Cancer Risk

    Directory of Open Access Journals (Sweden)

    Jihua LI

    2015-01-01

    Full Text Available Background and objective It has been proven that the lung cancer mortality rate in Xuanwei County, China was among the highest in the country and has been associated with exposure to indoor smoky coal emissions that contain high levels of polycyclic aromatic hydrocarbons. This risk may be modified by variation in genetic polymorphisms and coal subtypes. Our objective was to use molecular epidemiological techniques to investigate the relationship among genetic polymorphisms, coal subtype and lung cancer risk in Xuanwei County. Methods On the basis of two population-based case-control studies in residents of Xuanwei County, China, questionnaires covering demographic information, smoking history, family and personal medical history, and information on other variables were administered and buccal cells and sputum samples were collected separately from each subject enrolled to extract DNA. GST superfamily, AKR1C3 superfamily, OGG1 superfamily and other genotype were scanned by useing PCR method. ORs and 95%CIs were used to estimate the association between genotypes, coal subtypes and lung cancer risk factors by conditional Logistic regression using Statistical Analysis Software. Results Compared with subjects who using smokeless coal or wood, smoky coal use was statistically significantly associated with lung cancer risk (OR=7.7, 95%CI: 4.5-13.3. There was marked heterogeneity in risk estimates for specific subtypes of smoky coal. Estimates were highest for coal from the Laibin (OR=24.8, Longtan (OR=11.6 and Baoshan (OR=6.0 coal types, and lower for coal from other types; the risk within the same subtype of coal in male and female were similar. The GSTM1-null genotype, the AKR1C3 (Ex1-70C>G, OGG1 (Ex6-315C>G genotypes were closely associated with increased risk of lung cancer in Xuanwei County, and their odds ratios (95%CI were 2.3 (1.3-4.2, 1.8 (1.0-3.5 and 1.9 (1.1-3.3, respectively. Compared to subjects who with GSTM1-positive and used less than

  4. Development of magnetic molecularly imprinted polymers with double templates for the rapid and selective determination of amphenicol antibiotics in water, blood, and egg samples.

    Science.gov (United States)

    Wei, Shoulian; Li, Jianwen; Liu, Yong; Ma, Jinkui

    2016-11-18

    A magnetic mesoporous dual-template molecularly imprinted polymer (Fe3O4@mSiO2 @DMIP) with a specific recognition capability for chloramphenicol (CAP) and florfenicol (FF) was synthesised. CAP and FF were used as dual-template molecules, α-methacrylic acid and Fe3O4@mSiO2@-CHCH2 as dual functional monomers, and ethylene glycol dimethyl methacrylate as a crosslinking agent. For comparison, a magnetic mesoporous non-molecularly imprinted polymer (Fe3O4@mSiO2@NIP) was also prepared using the same synthesis procedure, but without the dual templates. The prepared polymers were characterised using scanning electron microscopy, Fourier-transform infrared spectroscopy and adsorption experiments. Results indicated that both the Fe3O4@mSiO2@DMIP and the Fe3O4@mSiO2 @NIP were microspherical nanoparticles, and the surface of the Fe3O4@mSiO2@DMIP was rougher than that of the Fe3O4@mSiO2@NIP. In addition, the prepared Fe3O4@mSiO2@DMIP possessed a higher adsorption capacity and better selectivity for CAP and FF than the Fe3O4@mSiO2@NIP. The maximum static adsorption capacities of the Fe3O4@mSiO2@ DMIP for CAP and FF were 146.5 and 190.1mgg-1, respectively, whereas those of the Fe3O4@mSiO2 @NIP were 50.0 and 44.0mgg-1, respectively. The obtained Fe3O4@mSiO2@DMIP particles were applied as a magnetic solid-phase extraction sorbent for the rapid and selective extraction of CAP, FF, and thiamphenicol (TAP) in water, chicken blood and egg samples. The method of magnetic molecularly imprinted solid-phase extraction (M-MISPE) coupled to high-performance liquid chromatography with UV detection (HPLC-UV) was conducted to detect CAP, FF, and TAP. The limits of detection for CAP, FF, and TAP were 0.16, 0.08, and 0.08μgkg-1, respectively. The average recovery and precision values for the spiked water, chicken blood, and egg samples ranged from 88.3% to 99.1% and 2.7% to 7.9%, respectively. Given its rapidity, selectivity, and sensitivity, the developed method of M-MISPE coupled to HPLC

  5. Evaluation of the HerpeSelect Express rapid test in the detection of herpes simplex virus type 2 antibodies in patients with genital ulcer disease.

    Science.gov (United States)

    Al-Shobaili, Hani; Hassanein, Khaled M; Mostafa, Marwa Salah; Al Duways, Ali Saleh

    2015-01-01

    A rapid point-of-care test with high sensitivity and specificity is required in order to fulfill the need for early detection and screening of Herpes simplex virus type 2 (HSV-2) infection among patients with genital ulcer disease (GUD), for better management and control of virus transmission. The goal of this study is to evaluate the performance of the commercially available HerpeSelect Express rapid test in comparison with three ELISA assays: HerpeSelect ELISA, Kalon HSV-2 glycoprotein G2 assay, and monoclonal antibody blocking enzyme immunoassay, which was used as the gold standard for the detection of HSV-2 antibodies. This study showed high sensitivity (ranging from 82.6 to 100%) and specificity (100%) of the HerpeSelect Express rapid test when compared to the three ELISA assays. The agreement between the HerpeSelect Express rapid test with the three ELISAs ranged from 93.3 to 100%. The HerpeSelect Express rapid test has adequate sensitivity and specificity for confirming HSV-2 infection in patients with GUD, indicating its suitability for epidemiological studies. © 2014 Wiley Periodicals, Inc.

  6. Dissecting the molecular signatures of apical cell-type shoot meristems from two ancient land plant lineages.

    Science.gov (United States)

    Frank, Margaret H; Edwards, Molly B; Schultz, Eric R; McKain, Michael R; Fei, Zhangjun; Sørensen, Iben; Rose, Jocelyn K C; Scanlon, Michael J

    2015-08-01

    Shoot apical meristem (SAM) structure varies markedly within the land plants. The SAMs of many seedless vascular plants contain a conspicuous inverted, pyramidal cell called the apical cell (AC), which is unidentified in angiosperms. In this study, we use transcriptomic sequencing with precise laser microdissections of meristem subdomains to define the molecular signatures of anatomically distinct zones from the AC-type SAMs of a lycophyte (Selaginella moellendorffii) and a monilophyte (Equisetum arvense). The two model species for this study represent vascular plant lineages that diverged > 400 million yr ago. Our data comprise comprehensive molecular signatures for the distinct subdomains within AC-type SAMs, an anatomical anomaly whose functional significance has been debated in the botanical literature for over two centuries. Moreover, our data provide molecular support for distinct gene expression programs between the AC-type SAMs of Selaginella and Equisetum, as compared with the SAM transcriptome of the angiosperm maize. The results are discussed in light of the functional significance and evolutionary success of the AC-type SAM within the embryophytes. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  7. Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster

    DEFF Research Database (Denmark)

    Williamson, M; Lenz, C; Winther, A M

    2001-01-01

    The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins...

  8. T2D-Db: An integrated platform to study the molecular basis of Type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Sriram S

    2008-07-01

    Full Text Available Abstract Background Type 2 Diabetes Mellitus (T2DM is a non insulin dependent, complex trait disease that develops due to genetic predisposition and environmental factors. The advanced stage in type 2 diabetes mellitus leads to several micro and macro vascular complications like nephropathy, neuropathy, retinopathy, heart related problems etc. Studies performed on the genetics, biochemistry and molecular biology of this disease to understand the pathophysiology of type 2 diabetes mellitus has led to the generation of a surfeit of data on candidate genes and related aspects. The research is highly progressive towards defining the exact etiology of this disease. Results T2D-Db (Type 2 diabetes Database is a comprehensive web resource, which provides integrated and curated information on almost all known molecular components involved in the pathogenesis of type 2 diabetes mellitus in the three widely studied mammals namely human, mouse and rat. Information on candidate genes, SNPs (Single Nucleotide Polymorphism in candidate genes or candidate regions, genome wide association studies (GWA, tissue specific gene expression patterns, EST (Expressed Sequence Tag data, expression information from microarray data, pathways, protein-protein interactions and disease associated risk factors or complications have been structured in this on line resource. Conclusion Information available in T2D-Db provides an integrated platform for the better molecular level understanding of type 2 diabetes mellitus and its pathogenesis. Importantly, the resource facilitates graphical presentation of the gene/genome wide map of SNP markers and protein-protein interaction networks, besides providing the heat map diagram of the selected gene(s in an organism across microarray expression experiments from either single or multiple studies. These features aid to the data interpretation in an integrative way. T2D-Db is to our knowledge the first publicly available resource that

  9. A Rapidly Fatal Sepsis Caused by Listeria Monocytogenes Type-4b in A Patient with Chronic Renal Failure

    OpenAIRE

    Kose, Adem; Yakupogullari, Yusuf

    2015-01-01

    Introduction: Listeria monocytogenes is a significant zoonosis causing invasive infections in the susceptible persons. The current paper presented a patient who died due to a rapidly-progressing multiple organ failure (MOF) as a result of severe sepsis caused by L. monocytogenes. Case Presentation: A 70-years-old patient with chronic renal failure was admitted to the infectious diseases clinic due to diarrhea for one day. He was hospitalized and the body fluid samples were collected for labor...

  10. HLA –DRB1*, DQB1* Alleles In Hydatid Patients By Molecular Typing

    Directory of Open Access Journals (Sweden)

    mehdi Mosayebi

    2007-10-01

    Full Text Available Mosayebi M1, Dalimi Asl A2, Moazeni M3, Mosayebi Gh4 1. Ph.D Student, Department of Parasitology, Faculty of medicine, Tarbiat Modarres University 2. Professor, Department of Parasitology, Faculty of medicine, Tarbiat Modarres University 3. Professor, Department of Immunology, Faculty of medicine, Tarbiat Modarres University 4. Assistant professor, Department of Immunology, Faculty of medicine, Arak Medical Sciences University Abstract Background: Hydatidosis is a important disease that results from infection with larvae of the dog tape worm , Echinococcus granulosus in human and farm animals .Resistance or susceptibility to infectious diseases , for example , cystic and alveolar echinococcosis is restricted by individual host factors and immunologic responses,in many surveys has been shown.The target of this study that is the first survey dealing with the correlation between HLA-DRB1*& DQB1* alleles and cystic echinococcosis in Iranian patient,is investigation HLA-DRB1*and DQB1* allelic polymorphism in Iranian patient with hydatidosis . Materials and methods: The study was carried out on 56 patients with confirmed cystic echinococcosis and 30 apparently healthy individuals living on Arak area by HLA-DRB1*& DQB1* typing with PCR-SSP method.The first step was founding patients and blood sampling .DNA was prepared from whole blood and we used PCR-SSP with 31 primer mixes for per sample . PCR reaction mixtures were loaded in agarose gels and after electrophoresis , geles were examine under UV illumination and gel document . Analyse of results carried out with specific software and frequency& interpretation tables and homogeneity test for calculation of P-value in χ2 test with fisher΄s exact test . significant samples with logistic regression analysed and Odds-ratio calculate . Results: A statistically significant positive association was found between HLA-DQB1*02 and the occurrence of cystic echinococcosis(P<0.05,(Odds-ratio=2.87 Conclusion: The

  11. The molecular adsorption-type endotoxin detection system using immobilized ɛ-polylysine

    Science.gov (United States)

    Ooe, Katsutoshi; Tsuji, Akihito; Nishishita, Naoki; Hirano, Yoshiaki

    2007-12-01

    mass incrementation is occurred, and the existence of endtoxin can be detected immediately, by using of Quartz Crystal Microbalance (QCM). In this report, the immobilization of ɛ-polylysine onto the Au and Si substrate and its adsorptive activity are described. We use X-ray Photoelectron Spectroscopy (XPS) to confirm the ɛ-polylysine immobilization, and the adsorptive activity of immobilized ɛ-polylysine is measured by AFM and QCM. This molecular adsorption type endotoxin sensor aims to the realization of "real-time endotoxin detection system".

  12. Finding a Potential Dipeptidyl Peptidase-4 (DPP-4 Inhibitor for Type-2 Diabetes Treatment Based on Molecular Docking, Pharmacophore Generation, and Molecular Dynamics Simulation

    Directory of Open Access Journals (Sweden)

    Harika Meduru

    2016-06-01

    Full Text Available Dipeptidyl peptidase-4 (DPP-4 is the vital enzyme that is responsible for inactivating intestinal peptides glucagon like peptide-1 (GLP-1 and Gastric inhibitory polypeptide (GIP, which stimulates a decline in blood glucose levels. The aim of this study was to explore the inhibition activity of small molecule inhibitors to DPP-4 following a computational strategy based on docking studies and molecular dynamics simulations. The thorough docking protocol we applied allowed us to derive good correlation parameters between the predicted binding affinities (pKi of the DPP-4 inhibitors and the experimental activity values (pIC50. Based on molecular docking receptor-ligand interactions, pharmacophore generation was carried out in order to identify the binding modes of structurally diverse compounds in the receptor active site. Consideration of the permanence and flexibility of DPP-4 inhibitor complexes by means of molecular dynamics (MD simulation specified that the inhibitors maintained the binding mode observed in the docking study. The present study helps generate new information for further structural optimization and can influence the development of new DPP-4 inhibitors discoveries in the treatment of type-2 diabetes.

  13. Finding a Potential Dipeptidyl Peptidase-4 (DPP-4) Inhibitor for Type-2 Diabetes Treatment Based on Molecular Docking, Pharmacophore Generation, and Molecular Dynamics Simulation.

    Science.gov (United States)

    Meduru, Harika; Wang, Yeng-Tseng; Tsai, Jeffrey J P; Chen, Yu-Ching

    2016-06-13

    Dipeptidyl peptidase-4 (DPP-4) is the vital enzyme that is responsible for inactivating intestinal peptides glucagon like peptide-1 (GLP-1) and Gastric inhibitory polypeptide (GIP), which stimulates a decline in blood glucose levels. The aim of this study was to explore the inhibition activity of small molecule inhibitors to DPP-4 following a computational strategy based on docking studies and molecular dynamics simulations. The thorough docking protocol we applied allowed us to derive good correlation parameters between the predicted binding affinities (pKi) of the DPP-4 inhibitors and the experimental activity values (pIC50). Based on molecular docking receptor-ligand interactions, pharmacophore generation was carried out in order to identify the binding modes of structurally diverse compounds in the receptor active site. Consideration of the permanence and flexibility of DPP-4 inhibitor complexes by means of molecular dynamics (MD) simulation specified that the inhibitors maintained the binding mode observed in the docking study. The present study helps generate new information for further structural optimization and can influence the development of new DPP-4 inhibitors discoveries in the treatment of type-2 diabetes.

  14. Development of a one-step probe based molecular assay for rapid immunodiagnosis of infection with M. tuberculosis using dried blood spots.

    Directory of Open Access Journals (Sweden)

    Thomas Blauenfeldt

    Full Text Available BACKGROUND: Antigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with M. tuberculosis. Compared to Interferon-γ release assays (IGRA, IP-10 is released in high levels enabling novel approaches such as field friendly dried blood spots (DBS and molecular detection. AIM: To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS. METHOD: We developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay was validated and applied for the diagnosis of M. tuberculosis infection in DBS samples from 43 patients with confirmed TB, 13 patients with latent TB and 96 presumed uninfected controls. In parallel, IP-10 and INF-γ levels were measured in Quantiferon (QFT-TB plasma supernatants. RESULTS: IP-10 mRNA upregulation was detectable at 4 hours after stimulation (6 fold upregulation peaking at 8 hours (108 fold upregulation. IFN-γ expression occurred in concert but levels were lower (peak 6.7 fold upregulation. IP-10 gene expression level was significantly higher in patients with tuberculosis (median 31.2, IQR 10.7-67.0 and persons with latent tuberculosis infection (LTBI (41.2, IQR 9.8-64.9 compared to healthy controls (1.6, IQR 1.1-2.4; p<0.0001. The IP-10 mRNA and protein based tests had comparable diagnostic accuracy to QFT-TB, sensitivity (85% and 88% vs 85% and specificity (96% and 96% vs 97%, p = ns.. CONCLUSION: We developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce the local technological requirements everywhere and make it possible to offer highly accurate immunodiagnostic tests in low resource settings.

  15. Wild-type catalase peroxidase vs G279D mutant type: Molecular basis of Isoniazid drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Singh, Aishwarya; Singh, Aditi; Grover, Sonam; Pandey, Bharati; Kumari, Anchala; Grover, Abhinav

    2018-01-30

    Mycobacterium tuberculosis katG gene is responsible for production of an enzyme catalase peroxidase that peroxidises and activates the prodrug Isoniazid (INH), a first-line antitubercular agent. INH interacts with catalase peroxidase enzyme within its heme pocket and gets converted to an active form. Mutations occurring in katG gene are often linked to reduced conversion rates for INH. This study is focussed on one such mutation occurring at residue 279, where glycine often mutates to aspartic acid (G279D). In the present study, several structural analyses were performed to study the effect of this mutation on functionality of KatG protein. On comparison, mutant protein exhibited a lower docking score, smaller binding cavity and reduced affinity towards INH. Molecular dynamics analysis revealed the mutant to be more rigid and less compact than the native protein. Essential dynamics analysis determined correlated motions of residues within the protein structure. G279D mutant was found to have many residues that showed related motions and an undesirable effect on the functionality of protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Rapid and high-resolution distinction of community-acquired and nosocomial Staphylococcus aureus isolates with identical pulsed-field gel electrophoresis patterns and spa types.

    Science.gov (United States)

    Glasner, Corinna; Sabat, Artur J; Dreisbach, Annette; Larsen, Anders R; Friedrich, Alexander W; Skov, Robert L; van Dijl, Jan Maarten

    2013-03-01

    Methicillin-resistant Staphylococcus aureus (MRSA) represent a serious threat for public health worldwide. Of particular concern is the emergence of community-acquired MRSA, which is often difficult to distinguish from nosocomial MRSA due to a lack of suitable typing methods for early detection. For example, the USA300 pulsed-field gel electrophoresis (PFGE) pattern includes both the 'classical' community-acquired USA300 clone with spa type t008 and an epidemiologically unrelated nosocomial clone with spa type t024. Likewise, spa typing cannot distinguish the classic USA300 from nosocomial MRSA with the spa type t008. Since the fast and high-resolution distinction of these S. aureus types is important for infection prevention and surveillance, we investigated whether multiple-locus variable number tandem repeat fingerprinting (MLVF) can be applied to overcome these limitations. Indeed, MLVF correctly grouped 91 MRSA isolates belonging to the classic USA300 lineage, nosocomial MRSA isolates with the USA300 PFGE profile and spa type t024, and nosocomial MRSA isolates with spa type t008 into 3 distinct clusters. Importantly, several sub-clusters were also identified, reflecting epidemiological relationships between the respective isolates. We conclude that MLVF has the discriminatory power needed to rapidly distinguish very similar community-acquired and nosocomial MRSA isolates and that MLVF-based sub-clustering of isolates is highly useful for epidemiological investigations, outbreak prevention, and control. Copyright © 2012 Elsevier GmbH. All rights reserved.

  17. Exercise and type 2 diabetes: molecular mechanisms regulating glucose uptake in skeletal muscle

    National Research Council Canada - National Science Library

    Stanford, Kristin I; Goodyear, Laurie J

    2014-01-01

    Exercise is a well-established tool to prevent and combat type 2 diabetes. Exercise improves whole body metabolic health in people with type 2 diabetes, and adaptations to skeletal muscle are essential for this improvement...

  18. Validation of a rapid type 1 diabetes autoantibody screening assay for community-based screening of organ donors to identify subjects at increased risk for the disease.

    Science.gov (United States)

    Wasserfall, C; Montgomery, E; Yu, L; Michels, A; Gianani, R; Pugliese, A; Nierras, C; Kaddis, J S; Schatz, D A; Bonifacio, E; Atkinson, M A

    2016-07-01

    The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)-positive] for the disease. This necessitated the establishment of a type 1 diabetes-specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme-linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma-associated protein-2 (IA-2A) and zinc transporter-8 (ZnT8A) were modified to identify AAb-positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98-100% specificity for all three assays, including sensitivities of 64-82% (GADA), 60-64% (IA-2A) and 62-68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow-up studies (14 GADA+, two IA-2A(+) , four multiple AAb-positive). Rapid screening for type 1 diabetes-associated AAb in organ donors is feasible, allowing for identification of non-diabetic, high-risk individuals and procurement of valuable tissues for natural history studies of this disease. © 2016 British Society for Immunology.

  19. Overview of molecular typing methods for outbreak detection and epidemiological surveillance

    NARCIS (Netherlands)

    Sabat, A. J.; Budimir, A.; Nashev, D.; Sa-Leao, R.; van Dijl, J. M.; Laurent, F.; Grundmann, H.; Friedrich, A. W.

    2013-01-01

    Typing methods for discriminating different bacterial isolates of the same species are essential epidemiological tools in infection prevention and control. Traditional typing systems based on phenotypes, such as serotype, biotype, phage-type, or antibiogram, have been used for many years. However,

  20. Temperature dependence of electron mobility in N-type organic molecular crystals: Theoretical study

    Science.gov (United States)

    Lin, Lili; Fan, Jianzhong; Jiang, Supu; Wang, Zhongjie; Wang, Chuan-Kui

    2017-11-01

    The temperature dependence of electron mobility in three Fx-TCNQ molecular crystals is studied. The electron mobility calculated based on Marcus charge transfer rate for all three molecules increases, as the temperature becomes high. Nevertheless, the electron mobility calculated based on quantum charge transfer rate shows opposite temperature dependence and indicates bandlike transport mechanism. Similar intrinsic transport properties are obtained for three systems. The different temperature dependence for Fx-TCNQ molecules detected should be induced by different transfer paths or external factors. Our investigation could help one better understand experimental results and provide intuitive view on the transfer mechanism in molecular crystals.

  1. Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

    Science.gov (United States)

    1988-10-31

    the disease, dengue hemorrhagic fever (DHF), occurs in children, which could lead to denglic * shock syndrome (DSS). Recently the pathogenesis of...CC AX .121. G C E I H C 1) Q. G C C S A N W I I A Y H C A A I K 7 r. 5 A INCA ICCA TCC ACC CCC ANC GAN aIc ICC ANA c CIT ACN AMCA CCI TC CAC CTC MCC...laistead, S.B. : Pathogenesis of dengue: challitnges to molecular biology. Science 239 (1988) 476-481. 1 lenikoff, S.: .tnidirectional digestion with

  2. A rapidly fatal sepsis caused by listeria monocytogenes type-4b in a patient with chronic renal failure.

    Science.gov (United States)

    Kose, Adem; Yakupogullari, Yusuf

    2015-03-01

    Listeria monocytogenes is a significant zoonosis causing invasive infections in the susceptible persons. The current paper presented a patient who died due to a rapidly-progressing multiple organ failure (MOF) as a result of severe sepsis caused by L. monocytogenes. A 70-years-old patient with chronic renal failure was admitted to the infectious diseases clinic due to diarrhea for one day. He was hospitalized and the body fluid samples were collected for laboratory analyses. Within few hours, his vital findings worsened, and he developed respiratory arrest. Ceftriaxone and gentamycin were administrated. However, he died due to disseminated intravascular coagulation, septic shock and meningoencephalitis at the 22nd hour of admission. Causative agent was identified as L. monocytogenes serotype-4b in post-mortem period. L. monocytogenes can cause progressive and rapidly fatal infections in the vulnerable persons, with multisystem involvement. Since this bacterium is not susceptible to cephalosporines, it will be better to consider effective antimicrobials in the treatment of the possible cases.

  3. A handheld flow genetic analysis system (FGAS): towards rapid, sensitive, quantitative and multiplex molecular diagnosis at the point-of-care level.

    Science.gov (United States)

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2015-06-21

    A handheld flow genetic analysis system (FGAS) is proposed for rapid, sensitive, multiplex and real-time quantification of nucleic acids at the point-of-care (POC) level. The FGAS includes a helical thermal-gradient microreactor and a microflow actuator, as well as control circuitry for temperature, fluid and power management, and smartphone fluorescence imaging. All of these features are integrated into a field-portable and easy-to-use molecular diagnostic platform powered by lithium batteries. Due to the unique design of the microreactor, not only steady temperatures for denaturation and annealing/extension but also a linear thermal gradient for spatial high-resolution melting can be achieved through simply maintaining a single heater at constant temperature. The smartphone fluorescence imaging system has a wide field of view that captures all PCR channels of the microreactor in a single snapshot without the need for any mechanical scanning. By these designs, the FGAS enables real-time monitoring of the temporal and spatial fluorescence signatures of amplicons during continuous-flow amplification. On the current FGAS, visual detection of as little as 10 copies per μL of genomic DNA of Salmonella enterica was achieved in 15 min, with real-time quantitative detection of the DNA over 6 orders of magnitude concentration from 10(6) to 10(1) copies per μL also completed in 7.5-15 min. In addition, multiple pathogenic DNA targets could be simultaneously discriminated with direct bar-chart readout or multiplex spatial melting in serial flow. We anticipate that the FGAS has great potential to become a next-generation gene analyzer for POC molecular diagnostics.

  4. Informing Antibiotic Treatment Decisions: Evaluating Rapid Molecular Diagnostics To Identify Susceptibility and Resistance to Carbapenems against Acinetobacter spp. in PRIMERS III.

    Science.gov (United States)

    Evans, Scott R; Hujer, Andrea M; Jiang, Hongyu; Hill, Carol B; Hujer, Kristine M; Mediavilla, Jose R; Manca, Claudia; Tran, Thuy Tien T; Domitrovic, T Nicholas; Higgins, Paul G; Seifert, Harald; Kreiswirth, Barry N; Patel, Robin; Jacobs, Michael R; Chen, Liang; Sampath, Rangarajan; Hall, Thomas; Marzan, Christine; Fowler, Vance G; Chambers, Henry F; Bonomo, Robert A

    2017-01-01

    The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen. Copyright © 2016 American Society for Microbiology.

  5. Targeting the Immunogenetic Diseases with the Appropriate HLA Molecular Typing: Critical Appraisal on 2666 Patients Typed in One Single Centre

    Directory of Open Access Journals (Sweden)

    M. Guarene

    2013-01-01

    Full Text Available We compared the immunogenetic data from 2666 patients affected by HLA-related autoimmune diseases with those from 4389 ethnically matched controls (3157 cord blood donors CBD, 1232 adult bone marrow donors BMD, to verify the appropriateness of HLA typing requests received in the past decade. The frequency of HLA-B*27 phenotype was 10.50% in 724 ankylosing spondylitis, 16.80% in 125 uveitis (3.41% BMD, 4.24% CBD, P<0.0001; HLA-B*51 allele was 15.57% in 212 Behçet’s disease (12.91% BMD, 9.88% CBD, P<0.0001; the HLA-DRB1-rheumatoid arthritis (RA shared epitope was 13.72% in 554 RA (10.85% BMD, 13.48% CBD, P=0.016; the carriers of almost one of HLA-DQB1 susceptibility alleles were 84.91% in 795 celiac disease (CD and 59.37% in 256 insulin-dependent diabetes mellitus (IDDM (46.06% in 875 CBD, 42.75% in 662 BMD P<0.0001. Overall, our results show that the HLA marker frequencies were higher in patients than controls, but lower than expected from the literature data (excluding CD and IDDM and demonstrate that, in complex immunogenetic conditions, a substantial number of genetic analyses are redundant and inappropriate, burdening to the public health costs. For this reason, we suggest the Italian Scientific Society of Immunogenetics to establish guidelines to improve the appropriateness of typing requests.

  6. Immobilization of ɛ-polylysine onto the probe surface for molecular adsorption type endotoxin detection system

    Science.gov (United States)

    Ooe, Katsutoshi; Tsuji, Akihito; Nishishita, Naoki; Hirano, Yoshiaki

    2007-04-01

    adsorption reaction between ɛ-polylysine and endotoxin. ɛ-polylysine has the structure of straight chain molecule composed by 25-30 residues made by lysine, and it is used as an antimicrobial agent, moreover, cellulose beads with immobilized ɛ-polylysine is used as the barrier filter for endotoxin removal. Therefore, it is expected that the endotoxin be adsorbed to the immobilized ɛ-polylysine onto the probe. As the result of this reaction, the mass of the probe is increased, and endotoxin can be detected by using of Quartz Crystal Microbalance (QCM). In our previous research, we have already acquired the proteins immobilization technique onto Au and Si surface. In this report, the proposal of molecular adsorption type endotoxin detection system, and the immobilization of ɛ-polylysine onto the probe are described. We use X-ray Photoelectron Spectroscopy (XPS) to confirm the ɛ-polylysine immobilization, and the adsorptive activity of immobilized ɛ-polylysine is measured by XPS and AFM. The purpose of this study is to bring about the realization of "Real-time endotoxin detection system".

  7. Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications

    NARCIS (Netherlands)

    Wang, G.; van Dam, A. P.; Schwartz, I.; Dankert, J.

    1999-01-01

    Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the

  8. Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis

    Energy Technology Data Exchange (ETDEWEB)

    Feswick, April; Loughery, Jennifer R.; Isaacs, Meghan A.; Munkittrick, Kelly R.; Martyniuk, Christopher J., E-mail: cmartyni@yahoo.ca

    2016-12-15

    testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

  9. Molecular typing of Staphylococcus aureus of different origins based on the polymorphism of the spa gene: characterization of a novel spa type.

    Science.gov (United States)

    Hakimi Alni, Reza; Mohammadzadeh, Abdolmajid; Mahmoodi, Pezhman

    2018-01-01

    The present study was conducted to determine the molecular diversity of Staphylococcus aureus strains isolated from human, bovine and food samples based on the polymorphism of the spa gene. A total of 208 S. aureus isolated from human, bovine raw milk and food samples were assessed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single locus sequence typing (SLST) methods, followed by determination of spa types using Ridom SpaServer. Altogether, 15 distinct RFLP patterns were recorded (I-XV). The highest heterogeneity was observed among S. aureus isolated from humans, whereas most of bovine and food S. aureus isolates indicated certain RFLP patterns. Although most of the isolates from patients showed RFLP pattern I, none of the S. aureus isolated from carriers had this spa pattern. Besides, the results of SLST led to the characterization of 16 spa types, and one of them was a novel spa type which has been registered in Ridom SpaServer for the first time and designated as type t16929. Determination of a high number of shared RFLP patterns between human and food S. aureus isolates indicated possible transmission of S. aureus and the source of food contamination. Thus, effective hygiene measures should be taken to break transmission routes. However, it seems that S. aureus isolated from patients, carriers and bovine should be considered in a different way, since some isolates had similar patterns, while the others showed their own specific pattern.

  10. Replica exchange molecular dynamics simulations reveal the structural and molecular properties of levan-type fructo-oligosaccharides of various chain lengths.

    Science.gov (United States)

    Kanjanatanin, Pongsakorn; Pichyangkura, Rath; Chunsrivirot, Surasak

    2016-08-17

    Levan and levan-type fructo-oligosaccharides (LFOs) have various potential applications in pharmaceutical and food industries due to their beneficial properties such as their low intrinsic viscosity and high water solubility. Previous studies showed that they exhibited prebiotic effects, anti-inflammatory and anti-tumor activities against Sarcoma-180 tumor cells of human. Despite their various potential applications, the structural and molecular properties of LFOs of various chain lengths are not well understood. We employed the replica-exchange molecular dynamics simulations method (REMD) in AMBER14 to elucidate structural and molecular properties of LFOs with chain lengths of 5 (LFO5), 10 (LFO10) and 15 (LFO15) residues in two models of generalized Born implicit solvent (GBHCT and GBOBC1). For LFO10 and LFO15, four distinct conformations (helix-like, partial helix, zig-zag and random structures) were characterized by their upper-middle and lower-middle torsions. For LFO5, two distinct conformations (partial helix and random structures) were characterized by their middle torsion and molecular angle of residues 1, 3 and 5. To determine hydrogen bonds important for the formation of helix-like structures of LFO10 and LFO15, occurrence frequencies of hydrogen bonds were analyzed, and the O6(i)--H3O(i+1) hydrogen bond was found with the highest frequency, suggesting its importance in helix formation. Among three dihedral angles between two fructosyl units [ϕ (O5'-C2'-O6-C6), ψ (C2'-O6-C6-C5) and ω (O6-C6-C5-C4)], dihedral angle distributions showed that ω was the most flexible dihedral angle and probably responsible for conformational differences of LFOs. Our study provides important insights into the structural and molecular properties of LFOs, which tend to form helical structures as the chain length increases from 5 to 15 residues. This information could be beneficial for the selection of LFOs with appropriate lengths and properties for pharmaceutical and

  11. Rapid detection of vip1-type genes from Bacillus cereus and characterization of a novel vip binary toxin gene.

    Science.gov (United States)

    Yu, Xiumei; Liu, Tao; Liang, Xiaoxing; Tang, Changqing; Zhu, Jun; Wang, Shiquan; Li, Shuangcheng; Deng, Qiming; Wang, Linxia; Zheng, Aiping; Li, Ping

    2011-12-01

    A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. A bioorganometallic approach for rapid electrochemical analysis of human immunodeficiency virus type-1 reverse transcriptase in serum

    Energy Technology Data Exchange (ETDEWEB)

    Labib, Mahmoud; Shipman, Patrick O.; Martic, Sanela [Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, ON, N6A 5B7 (Canada); Kraatz, Heinz-Bernhard, E-mail: hkraatz@uwo.ca [Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, ON, N6A 5B7 (Canada)

    2011-05-30

    Highlights: > Two-dimensional electrochemical assay of HIV-1 reverse transcriptase in serum is presented. > XPS and ToF-SIMS confirmed binding of synthetic ferrocene-lipoic acid to gold. > Dynamic range was 100-500 pg mL{sup -1} with an LOD of 50 pg mL{sup -1}. > The biosensor allows sensitive and selective detection of reverse transcriptase in 20 s. - Abstract: Determination of low-abundance human immunodeficiency virus (HIV) enzymes is essential for early detection of the viral infection. Designed for the rapid detection of the virus in human serum, a rapid and ultrasensitive electrochemical assay for HIV-1 reverse transcriptase (HIV-1 RT) is presented in this article. The assay format is based on the formation of a self-assembled monolayer (SAM) of a synthesized ferrocene (Fc)-labeled lipoic acid onto a gold electrode. X-ray photoelectron spectroscopy (XPS) and Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) were employed to confirm the binding of the Fc-labeled lipoic acid to the gold via the Au-S bond. A short RT-specific peptide, VEAIIRILQQLLFIH, was covalently attached to the Fc-labeled lipoic acid. Square wave voltammetry (SWV) offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RT in the range of 100-500 pg mL{sup -1}, equivalent to 85.5-427.4 fM, with a detection limit of 50 pg mL{sup -1} (42.7 fM). The developed biosensor is inexpensive, easy to prepare and operate, and allows a highly selective detection of RT in 20 s.

  13. Detection of molecular paths associated with insulitis and type 1 diabetes in non-obese diabetic mouse.

    Directory of Open Access Journals (Sweden)

    Erno Lindfors

    Full Text Available Recent clinical evidence suggests important role of lipid and amino acid metabolism in early pre-autoimmune stages of type 1 diabetes pathogenesis. We study the molecular paths associated with the incidence of insulitis and type 1 diabetes in the Non-Obese Diabetic (NOD mouse model using available gene expression data from the pancreatic tissue from young pre-diabetic mice. We apply a graph-theoretic approach by using a modified color coding algorithm to detect optimal molecular paths associated with specific phenotypes in an integrated biological network encompassing heterogeneous interaction data types. In agreement with our recent clinical findings, we identified a path downregulated in early insulitis involving dihydroxyacetone phosphate acyltransferase (DHAPAT, a key regulator of ether phospholipid synthesis. The pathway involving serine/threonine-protein phosphatase (PP2A, an upstream regulator of lipid metabolism and insulin secretion, was found upregulated in early insulitis. Our findings provide further evidence for an important role of lipid metabolism in early stages of type 1 diabetes pathogenesis, as well as suggest that such dysregulation of lipids and related increased oxidative stress can be tracked to beta cells.

  14. [New facts about the molecular background of isovanilline-type sweeteners].

    Science.gov (United States)

    Kálmán, Noémi; Magyarné-Jeszenszki, Erzsébet; Kurtán, Tibor; Antus, Sándor

    2014-01-01

    As a continuation of our studies on the relationship between structure and sweet taste of isovanilline-type sweeteners, (3-hydroxy-4-methoxy)benzyl-benzoate (17) and -salicylate (17c), analogues of dihydrochalcone-type sweetener analogues of (+)-phyllodulcin [(+)-5] and CH-401 (14c) have been synthesized. Surprisingly, 17c has been found to be tastless, while 17e was slightly sweet. These observations could be explained by the current hypothesis on the receptor model for isovanilline-type sweet derivatives.

  15. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven

    2010-01-01

    ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...

  16. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen Jacques

    2010-01-01

    ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18 is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...

  17. Prevalence and molecular typing of Clostridium perfringens in captive wildlife in India.

    Science.gov (United States)

    Milton, Arockiasamy Arun Prince; Agarwal, Rajesh Kumar; Bhuvana Priya, Govindarajan; Saminathan, Mani; Aravind, Manivasagam; Reddy, Avinash; Athira, C K; Ramees, Thadiyampuram; Sharma, Anil Kumar; Kumar, Ashok

    2017-04-01

    The prevalence of Clostridium perfringens in captive wildlife in India has not been reported. The objective of the study was to determine the fecal prevalence of C. perfringens in captive wildlife in India. The prevalence in captive wild ruminants, non-ruminants, birds and caretakers were 34.1%, 36%, 22.5% and 6.7%, respectively. Toxinotyping of C. perfringens indicated that the predominant type was type A with a prevalence rate of 69.7%, followed by type A with cpb2 gene (28.3%) and type B (2.%). Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India.

    Science.gov (United States)

    Maurya, Anand Kumar; Umrao, Jyoti; Singh, Amresh Kumar; Kant, Surya; Kushwaha, Ram Awadh Singh; Dhole, Tapan N

    2013-01-01

    The problem of multi-drug resistance tuberculosis (MDR-TB) is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST) of MDR-TB cases in Northern India. A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%), 52 (94.5%) and 17 (30.9%) strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05). The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3%) in S531L, 52 (94.5%) in S315T1 and 11 (20%) in C15T regions respectively (P < 0.05). Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

  19. In Vivo Selective Capture and Rapid Identification of Luteolin and Its Metabolites in Rat Livers by Molecularly Imprinted Solid-Phase Microextraction.

    Science.gov (United States)

    Gao, Die; Wang, Dan-Dan; Zhang, Qian; Yang, Feng-Qing; Xia, Zhi-Ning; Zhang, Qi-Hui; Yuan, Chun-Su

    2017-02-15

    A method based on molecularly imprinted solid-phase microextraction (MIP-SPME) coupled with liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) was developed for the detection of luteolin and its metabolites in vivo. The MIP-SPME fibers were first fabricated by dopamine and silane, and then luteolin MIPs-coated fibers were successfully prepared using luteolin, acrylamide (AM), and ethylene glycol dimethacrylate (EGDMA) as the template, functional monomer and cross-linker, respectively. The characterizations of polymers were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and the Brunauer-Emmett-Teller method (BET). The properties involving adsorption and selective experiments were evaluated, and these results revealed that MIP fibers presented high adsorption capacity and selectivity to luteolin. Furthermore, the developed MIP-SPME coupled with the LC-QTOF-MS/MS method was adopted to capture and identify luteolin and its metabolites in rat livers in vivo, and eventually, apigenin, chrysoeriol, and diosmetin were rapidly identified as metabolites.

  20. Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows.

    Science.gov (United States)

    Marques, S; Huss, V A R; Pfisterer, K; Grosse, C; Thompson, G

    2015-05-01

    The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Development of molecularly imprinted column-on line-two dimensional liquid chromatography for rapidly and selectively monitoring estradiol in cosmetics.

    Science.gov (United States)

    Guo, Pengqi; Xu, Xinya; Xian, Liang; Ge, Yanhui; Luo, Zhimin; Du, Wei; Jing, Wanghui; Zeng, Aiguo; Chang, Chun; Fu, Qiang

    2016-12-01

    Nowadays, the illegal use of estradiol in cosmetics has caused a series of events which endangering public health seriously. Therefore, it is imperative to establish a simple, fast and specific method for monitoring the illegal use of estradiol in cosmetics. In current study, we developed a molecular imprinted monolithic column two dimensional liquid chromatography method (MIMC-2D-LC) for rapid and selective determination of estradiol in various cosmetic samples. The best polymerization, morphology, structure property, surface groups, and the adsorption performance of the prepared material were investigated. The MIMC-2D-LC was validated and successfully used for detecting estradiol in cosmetic samples with good selectivity, sensitivity, efficiency and reproducibility. The linear range of the MIMC-2D-LC for estradiol was 0.5-50μgg -1 with the limit of detection of 0.08μgg -1 . Finally, six batches of cosmetic samples obtained from local markets were tested by the proposed method. The test results showed that the illegal use of estradiol still existed in the commercially available samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Rapid and accurate assessment of GPCR-ligand interactions Using the fragment molecular orbital-based density-functional tight-binding method.

    Science.gov (United States)

    Morao, Inaki; Fedorov, Dmitri G; Robinson, Roger; Southey, Michelle; Townsend-Nicholson, Andrea; Bodkin, Mike J; Heifetz, Alexander

    2017-09-05

    The reliable and precise evaluation of receptor-ligand interactions and pair-interaction energy is an essential element of rational drug design. While quantum mechanical (QM) methods have been a promising means by which to achieve this, traditional QM is not applicable for large biological systems due to its high computational cost. Here, the fragment molecular orbital (FMO) method has been used to accelerate QM calculations, and by combining FMO with the density-functional tight-binding (DFTB) method we are able to decrease computational cost 1000 times, achieving results in seconds, instead of hours. We have applied FMO-DFTB to three different GPCR-ligand systems. Our results correlate well with site directed mutagenesis data and findings presented in the published literature, demonstrating that FMO-DFTB is a rapid and accurate means of GPCR-ligand interactions. © 2017 Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. © 2017 Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  3. Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

    Science.gov (United States)

    Christner, Martin; Trusch, Maria; Rohde, Holger; Kwiatkowski, Marcel; Schlüter, Hartmut; Wolters, Manuel; Aepfelbacher, Martin; Hentschke, Moritz

    2014-01-01

    Background In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification. Methods Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak. Results Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates. Conclusions MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow. PMID:25003758

  4. Herpes simplex virus type 2 induces rapid cell death and functional impairment of murine dendritic cells in vitro

    NARCIS (Netherlands)

    Jones, CA; Fernandez, M; Herc, K; Bosnjak, L; Miranda-Saksena, M; Boadle, RA; Cunningham, A

    2003-01-01

    Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h

  5. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

    Directory of Open Access Journals (Sweden)

    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  6. Development of new-type rapid analysis technology of polychlorinated biphenyls by using liquid chromatographic clean-up material (polyvinyl alcohol gel).

    Science.gov (United States)

    Sawatsubashi, Tetsuya; Tsukahara, Chisato; Baba, Keigo; Ohi, Etsumasa; Shinoda, Akiko; Miura, Norio

    2008-01-04

    We developed a new-type rapid polychlorinated biphenyl (PCB) analysis technology on the basis of a liquid chromatographic clean-up system combined with a large-volume injection GC-LRMS. Among 18 kinds of materials such as polymer gels, normal-phase silica gels, reversed-phase silica gels, carbon material and ion-exchange material, polyvinyl alcohol (PVA) gel and poly (hydroxylmethacrylate) gel were found to give rather good separation performance for insulating oil. Especially, PVA gel was confirmed to be the most suitable for rapid PCB analysis because of its least required quantity of fraction liquid as well as the highest resolution. Then, we confirmed elution characteristics of all PCB isomers and removal efficiency of insulating oil on PVA gel under an optimized condition, and established high-performance clean-up system using a combination of octadecyl silica gel (ODS), porous graphite carbon (PGC) and PVA gel. In this system, we applied newly valve-switching method that could remove other impurities. In addition, it was demonstrated that the proposed clean-up system could become highly sensitive and rapid PCB analysis technology with 2-h analysis time, lower measurement limit of less than 0.05 mg/kg, and a variation coefficient of less than 5%, by coupling with a large-volume injection type GC-LRMS. Thus, we can conclude that this rapid PCB analysis technology has not only good correlativity (R2>0.999) with standard analysis method but also high durability and can be fully applied to actual PCB-treatment plants.

  7. Acyclic cucurbit[n]uril-type molecular containers: influence of glycoluril oligomer length on their function as solubilizing agents.

    Science.gov (United States)

    Gilberg, Laura; Zhang, Ben; Zavalij, Peter Y; Sindelar, Vladimir; Isaacs, Lyle

    2015-04-07

    We present the synthesis of a series of six new glycoluril derived molecular clips and acyclic CB[n]-type molecular containers (1–3) that all feature SO3(−) solubilizing groups but differ in the number of glycoluril rings between the two terminal dialkoxyaromatic sidewalls. We report the X-ray crystal structure of 3b which shows that its dialkoxynaphthalene sidewalls actively define a hydrophobic cavity with high potential to engage in π–π interactions with insoluble aromatic guests. Compounds 1–3 possess very good solubility characteristics (≥38 mM) and undergo only very weak self-association (Ks containers 3a and 3b which feature three glycoluril rings between the terminal dialkoxy-o-xylylene and dialkoxynaphthalene sidewalls are less efficient solubilizing agents than 4a and 4b because of their smaller hydrophobic cavities. Containers 1 and 2 behave as molecular clip type receptors and therefore possess the ability to bind to and thereby solubilize aromatic drugs like camptothecin, ziprasidone, and PBS-1086.

  8. Mapping the binding site of a large set of quinazoline type EGF-R inhibitors using molecular field analyses and molecular docking studies.

    Science.gov (United States)

    Hou, Tingjun; Zhu, Lili; Chen, Lirong; Xu, Xiaojie

    2003-01-01

    In the current work, three-dimensional QSAR studies for one large set of quinazoline type epidermal growth factor receptor (EGF-R) inhibitors were conducted using two types of molecular field analysis techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). These compounds belonging to six different structural classes were randomly divided into a training set of 122 compounds and a test set of 13 compounds. The statistical results showed that the 3D-QSAR models derived from CoMFA were superior to those generated from CoMSIA. The most optimal CoMFA model after region focusing bears significant cross-validated r(2)(cv) of 0.60 and conventional r(2) of 0.92. The predictive power of the best CoMFA model was further validated by the accurate estimation to these compounds in the external test set, and the mean agreement of experimental and predicted log(IC(50)) values of the inhibitors is 0.6 log unit. Separate CoMFA models were conducted to evaluate the influence of different partial charges (Gasteiger-Marsili, Gasteiger-Hückel, MMFF94, ESP-AM1, and MPA-AM1) on the statistical quality of the models. The resulting CoMFA field map provides information on the geometry of the binding site cavity and the relative weights of various properties in different site pockets for each of the substrates considered. Moreover, in the current work, we applied MD simulations combined with MM/PBSA (Molecular mechanics/Possion-Boltzmann Surface Area) to determine the correct binding mode of the best inhibitor for which no ligand-protein crystal structure was present. To proceed, we define the following procedure: three hundred picosecond molecular dynamics simulations were first performed for the four binding modes suggested by DOCK 4.0 and manual docking, and then MM/PBSA was carried out for the collected snapshots. The most favorable binding mode identified by MM/PBSA has a binding free energy about 10 kcal/mol more

  9. Molecular typing of Escherichia coli strains isolated from food in Casablanca (Morocco).

    Science.gov (United States)

    Badri, S; Fassouane, A; Filliol, I; Hassar, M; Cohen, N

    2009-06-15

    Serotyping of O- and H- antigens is regarded as the gold standard in classification of E. coli for taxonomic and epidemiological purposes similar to the Kaufmann-White scheme for Salmonella enterica. Molecular methods to replace or to support the serotyping were recently applied. Using the molecular polymorphism of the somatic (O- antigen) gene rfb cluster and flagella (H- antigen) gene fliC, 74 E. coli strains carrying the virulent genes isolated from food were characterised by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results showed that 49 (66%) of the isolates revealed a reproducible and clear cut classification, with a very good correlation to the collection of reference strains, were found.

  10. Exercise and Type 2 Diabetes: Molecular Mechanisms Regulating Glucose Uptake in Skeletal Muscle

    Science.gov (United States)

    Stanford, Kristin I.; Goodyear, Laurie J.

    2014-01-01

    Exercise is a well-established tool to prevent and combat type 2 diabetes. Exercise improves whole body metabolic health in people with type 2 diabetes, and adaptations to skeletal muscle are essential for this improvement. An acute bout of exercise increases skeletal muscle glucose uptake, while chronic exercise training improves mitochondrial…

  11. Molecular typing of Treponema pallidum: a 5-year surveillance in Shanghai, China.

    Science.gov (United States)

    Dai, Ting; Li, Kang; Lu, Haikong; Gu, Xin; Wang, Qianqiu; Zhou, Pingyu

    2012-11-01

    Previously, a small study showed that 14f was the predominant subtype of Treponema pallidum in Shanghai, China. The result was quite different from the genotype distribution in other areas of China. This study aimed to identify the strain types of Treponema pallidum in samples collected over a 5-year period in Shanghai. From 2007 to 2011, genital swabs were collected from patients with syphilis from the Shanghai Skin Disease Hospital. Positive specimens were typed by the enhanced typing method by adding a tp0548 gene to the existing arp and tpr genotype system. In total, 304 of the 372 enrolled patients yielded fully typeable DNA. Ten arp types (4, 6, 8, 9, 11, 12, 13, 14, 15, and 19), 3 tpr types (a, d, and o), and 5 tp0548 types (a, c, f, g, and i) were identified. In total, 12 subtypes were identified with a combination of the arp and tpr genes. Subtype 14d was found in 270 samples (88.8%). When the combination included the tp0548 gene, the 12 CDC subtypes identified were divided into 14 strain types. The predominant type was 14d/f (88.8%), followed by 15d/f (3.6%), 13d/f (1.3%), and 19d/c (1.3%). Two of the 44 14d/f-infected patients and both of the 19d/c-infected patients who underwent a lumbar puncture were diagnosed with neurosyphilis. This study showed that the predominant type in Shanghai was 14d/f. While this is in keeping with data from other areas in China, it is different from an earlier report showing that 14f is the most common genotype in Shanghai. Further studies are needed to better understand the association between strain types and neurosyphilis.

  12. Familial Amyloid Polyneuropathy Type IV (FINNISH with Rapid Clinical Progression in an Iranian Woman: A Case Report

    Directory of Open Access Journals (Sweden)

    Arash Babaei-Ghazani

    2016-05-01

    Full Text Available Familial amyloid polyneuropathy (FAP type IV (FINNISH is a rare clinical entity with challenging neuropathy and cosmetic deficits. Amyloidosis can affect peripheral sensory, motor, or autonomic nerves. Nerve lesions are induced by deposits of amyloid fibrils and treatment approaches for neuropathy are challenging. Involvement of cranial nerves and atrophy in facial muscles is a real concern in daily life of such patients. Currently, diagnosis of neuropathy can be made by electrodiagnostic studies and diagnosis of amyloidosis can be made by genetic testing or by detection of amyloid deposition in abdominal fat pad, rectal, or nerve biopsies. It is preferable to consider FAP as one of the differential diagnosis of a case presented with multiple cranial nerves symptoms. The authors present a case of familial amyloid polyneuropathy (FAP type IV with severe involvement of multiple cranial nerves, peripheral limb neuropathy, and orthostatic hypotension.

  13. Rapid Assessment of Key Structural Elements of Different Vegetation Types of West African Savannas in Burkina Faso

    Directory of Open Access Journals (Sweden)

    Qasim Mohammad

    2016-01-01

    Full Text Available Estimations of Leaf Area Index (LAI have recently gained attention due to the sensitivity to the effects of climate change and its impact on forest ecosystems. Hence, a study was conducted on the LAI estimation of four vegetation types: (i gallery forests, (ii woodland savannas, (iii tree savannas, and (iv shrub savannas, at two protected areas of Nazinga Game Ranch and Bontioli Nature Reserve, Burkina Faso. A relationship between LAI and Crown Diameter was also investigated at these two sites. Digital hemispherical photography was used for the LAI estimation. Crown diameters (CD were determined perpendicular to each other and averaged for each tree and shrub. Overall results revealed that LAI ranged from 0-1.33 and the CD was recorded in the range of 0.46-11.01 m. The gallery forests recorded the highest mean LAI 1.33 ± 0.32 as well as the highest mean CD 7.69 ± 1.90 m. The LAI for the vegetation types were at their lower ends as the study was conducted in summer season, higher values are therefore expected in the wet season, as a significant correlation between LAI and precipitation has been emphasized by various studies. Continuous LAI monitoring and studies on various growth parameters of different vegetation types at the study sites are recommended towards enhanced monitoring and an ecologically feasible forest- and savanna-use and management to maintain essential ecosystem functions and services.

  14. Molecular dynamics simulation of gas models of Lennard-Jones type interactions: Extensivity associated with interaction range and external noise

    Science.gov (United States)

    Kadijani, M. Nouri; Abbasi, H.; Nezamipour, S.

    2017-06-01

    Statistics of a two-dimensional gas model interacting through a Lennard-Jones type potential, is considered. The goal is to examine the extensivity of internal energy in respect to the potential range and external white noise through molecular dynamics simulation. Accordingly a molecular dynamics simulation model is designed that provides reasonable evidence, in this respect. It is shown that for the long range potential the internal energy scales according to non-extensive thermodynamics expectation and the criteria is specified. Besides, for the short range case we demonstrate that the external noise drastically modifies the statistics of gas and makes the internal energy non-extensive. The relation between the non-extensive parameter, q, and the relaxation time and the noise intensity is obtained.

  15. Near-Infrared Spectroscopy, a Rapid Method for Predicting the Age of Male and Female Wild-Type and Wolbachia Infected Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Maggy T Sikulu-Lord

    2016-10-01

    Full Text Available Estimating the age distribution of mosquito populations is crucial for assessing their capacity to transmit disease and for evaluating the efficacy of available vector control programs. This study reports on the capacity of the near-infrared spectroscopy (NIRS technique to rapidly predict the ages of the principal dengue and Zika vector, Aedes aegypti. The age of wild-type males and females, and males and females infected with wMel and wMelPop strains of Wolbachia pipientis were characterized using this method. Calibrations were developed using spectra collected from their heads and thoraces using partial least squares (PLS regression. A highly significant correlation was found between the true and predicted ages of mosquitoes. The coefficients of determination for wild-type females and males across all age groups were R2 = 0.84 and 0.78, respectively. The coefficients of determination for the age of wMel and wMelPop infected females were 0.71 and 0.80, respectively (P< 0.001 in both instances. The age of wild-type female Ae. aegypti could be identified as < or ≥ 8 days old with an accuracy of 91% (N = 501, whereas female Ae. aegypti infected with wMel and wMelPop were differentiated into the two age groups with an accuracy of 83% (N = 284 and 78% (N = 229, respectively. Our results also indicate NIRS can distinguish between young and old male wild-type, wMel and wMelPop infected Ae. aegypti with accuracies of 87% (N = 253, 83% (N = 277 and 78% (N = 234, respectively. We have demonstrated the potential of NIRS as a predictor of the age of female and male wild-type and Wolbachia infected Ae. aegypti mosquitoes under laboratory conditions. After field validation, the tool has the potential to offer a cheap and rapid alternative for surveillance of dengue and Zika vector control programs.

  16. Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis.

    Science.gov (United States)

    Larrasa, José; García-Sánchez, Alfredo; Ambrose, Nicholas C; Parra, Alberto; Alonso, Juan M; Rey, Joaquín M; Hermoso-de-Mendoza, Miguel; Hermoso-de-Mendoza, Javier

    2004-11-01

    This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.

  17. [Molecular pathology: applications of molecular biology in pathological anatomy].

    Science.gov (United States)

    Wistuba, I I

    2001-07-01

    The rapid development of molecular biology techniques as well as recent progress in the understanding of genetic and molecular basis of human diseases have had enormous impact in the practice of clinical pathology. Since new diagnostic (molecular) tools are now available, the concept of Molecular Pathology is emerging. Molecular Pathology is defined by the use of molecular biology techniques and the type of specimens that are involved in its practice, basically ARN and ADN, extracted from cytological and tissue specimens. Although most methods used in molecular pathology and their applications are still under investigation and clinical validation they have great potential in several areas of pathological diagnosis, particularly on infectious and neoplastic diseases. Introduction of these techniques in pathology laboratories in our country should significantly enhance the diagnostic and research skills in the field.

  18. Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains

    NARCIS (Netherlands)

    Baleiras Couto, M.M.; Eijsma, B.; Hofstra, H.; Huis in 't Veld, J.H.J.; Vossen, J.M.B.M. van der

    1996-01-01

    Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite

  19. Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

    Directory of Open Access Journals (Sweden)

    McEwan Alastair G

    2010-06-01

    Full Text Available Abstract Background Catheter-associated urinary tract infection (CAUTI is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD from 39 strains revealed they clustered into five distinct clades (A-E ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

  20. Molecular Dissection of Isolated Disease Features in Mosaic Neurofibromatosis Type 1

    OpenAIRE

    Maertens, Ophélia; De Schepper, Sofie; Vandesompele, Jo; Brems, Hilde; Heyns, Ine; Janssens, Sandra; Speleman, Frank; Legius, Eric; Messiaen, Ludwine

    2007-01-01

    Elucidation of the biological framework underlying the development of neurofibromatosis type 1 (NF1)-related symptoms has proved to be difficult. Complicating factors include the large size of the NF1 gene, the presence of several NF1 pseudogenes, the complex interactions between cell types, and the NF1-haploinsufficient state of all cells in the body. Here, we investigate three patients with distinct NF1-associated clinical manifestations (neurofibromas only, pigmentary changes only, and ass...

  1. Transcriptomic analysis to elucidate the molecular mechanisms that underlie feed efficiency in meat-type chickens.

    Science.gov (United States)

    Lee, Jeeyoung; Karnuah, Arthur B; Rekaya, Romdhane; Anthony, Nicholas B; Aggrey, Samuel E

    2015-10-01

    Feed efficiency phenotypes defined by genotypes or gene markers are unknown. To date, there are only limited studies on global gene expression profiling on feed efficiency. The objective of this study was to identify genes and pathways associated with residual feed intake (RFI) through transcriptional profiling of duodenum at two different ages in a chicken population divergently selected for low (LRFI) or high (HRFI) RFI. The global gene expression differences in LRFI and HRFI were assessed by the Affymetrix GeneChip(®) Chicken Genome Array and RT-PCR using duodenal tissue on days 35 and 42. The Ingenuity Pathway Analysis program was used to identify canonical and gene network pathways associated with RFI. A global view of gene expression differences between LRFI and HRFI suggest that RFI can be explained by differences in cell division, growth, proliferation and apoptosis, protein synthesis, lipid metabolism, and molecular transport of cellular molecules. Chickens selected for improved RFI achieve efficiency by reducing feed intake with a nominal or no change in weight gain by either up-regulating CD36, PPARα, HMGCS2, GCG or down-regulating PCSK2, CALB1, SAT1, and SGK1 genes within the lipid metabolism, small molecule biochemistry, molecular transport, cell death, and protein synthesis molecular and cellular functions. Chickens selected for reduced RFI via reduced feed intake with no change in weight gain achieve feed efficiency for growth by the up-regulation of genes that reduce appetite with increased cellular oxidative stress, prolonged cell cycle, DNA damage, and apoptosis in addition to increased oxidation of dietary fat and efficient fatty acids transported from the intestines.

  2. DNA microarrays immobilized on unmodified plastics in a microfluidic biochip for rapid typing of Avian Influenza Virus

    DEFF Research Database (Denmark)

    Yi, Sun; Perch-Nielsen, Ivan R.; Dufva, Martin

    2011-01-01

    to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on non-modified plastic surfaces. This one-step, cost-effective process provides very high immobilization and hybridization efficiencies and is applicable to many different types of polymers. Using this microarray fabrication technique......, a portable cyclic olefin copolymer (COC) microarray device containing eight individually addressable microfluidic channels was developed for fast identification of Avian Influenza Virus (AIV) by DNA hybridization. This plastic biochip offers benefits of low fabrication cost and parallel processing...

  3. DES14X3taz: A TYPE I SUPERLUMINOUS SUPERNOVA SHOWING A LUMINOUS, RAPIDLY COOLING INITIAL PRE-PEAK BUMP

    Energy Technology Data Exchange (ETDEWEB)

    Smith, M.; Sullivan, M.; D’Andrea, C. B.; Castander, F. J.; Casas, R.; Prajs, S.; Papadopoulos, A.; Nichol, R. C.; Karpenka, N. V.; Bernard, S. R.; Brown, P.; Cartier, R.; Cooke, J.; Curtin, C.; Davis, T. M.; Finley, D. A.; Foley, R. J.; Gal-Yam, A.; Goldstein, D. A.; González-Gaitán, S.; Gupta, R. R.; Howell, D. A.; Inserra, C.; Kessler, R.; Lidman, C.; Marriner, J.; Nugent, P.; Pritchard, T. A.; Sako, M.; Smartt, S.; Smith, R. C.; Spinka, H.; Thomas, R. C.; Wolf, R. C.; Zenteno, A.; Abbott, T. M. C.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Buckley-Geer, E.; Rosell, A. Carnero; Kind, M. Carrasco; Carretero, J.; Crocce, M.; Cunha, C. E.; Costa, L. N. da; Desai, S.; Diehl, H. T.; Doel, P.; Estrada, J.; Evrard, A. E.; Flaugher, B.; Fosalba, P.; Frieman, J.; Gerdes, D. W.; Gruen, D.; Gruendl, R. A.; James, D. J.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Marshall, J. L.; Martini, P.; Miller, C. J.; Miquel, R.; Nord, B.; Ogando, R.; Plazas, A. A.; Reil, K.; Romer, A. K.; Roodman, A.; Rykoff, E. S.; Sanchez, E.; Scarpine, V.; Schubnell, M.; Sevilla-Noarbe, I.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Walker, A. R.; Wester, W.

    2016-02-03

    We present DES14X3taz, a new hydrogen-poor superluminous supernova (SLSN-I) discovered by the Dark Energy Survey (DES) supernova program, with additional photometric data provided by the Survey Using DECam for Superluminous Supernovae. Spectra obtained using Optical System for Imaging and low-Intermediate-Resolution Integrated Spectroscopy on the Gran Telescopio CANARIAS show DES14X3taz is an SLSN-I at z = 0.608. Multi-color photometry reveals a double-peaked light curve: a blue and relatively bright initial peak that fades rapidly prior to the slower rise of the main light curve. Our multi-color photometry allows us, for the first time, to show that the initial peak cools from 22,000 to 8000 K over 15 rest-frame days, and is faster and brighter than any published core-collapse supernova, reaching 30% of the bolometric luminosity of the main peak. No physical Ni-56-powered model can fit this initial peak. We show that a shock-cooling model followed by a magnetar driving the second phase of the light curve can adequately explain the entire light curve of DES14X3taz. Models involving the shock-cooling of extended circumstellar material at a distance of similar or equal to 400 R-circle dot are preferred over the cooling of shock-heated surface layers of a stellar envelope. We compare DES14X3taz to the few double-peaked SLSN-I events in the literature. Although the rise. times and characteristics of these initial peaks differ, there exists the tantalizing possibility that they can be explained by one physical interpretation

  4. In Situ D-periodic Molecular Structure of Type II Collagen

    Energy Technology Data Exchange (ETDEWEB)

    Antipova, Olga; Orgel, Joseph P.R.O. (IIT)

    2010-05-06

    Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.

  5. Intrachromosomal mitotic nonallelic homologous recombination is the major molecular mechanism underlying type-2 NF1 deletions.

    Science.gov (United States)

    Roehl, Angelika C; Vogt, Julia; Mussotter, Tanja; Zickler, Antje N; Spöti, Helene; Högel, Josef; Chuzhanova, Nadia A; Wimmer, Katharina; Kluwe, Lan; Mautner, Victor-Felix; Cooper, David N; Kehrer-Sawatzki, Hildegard

    2010-10-01

    Nonallelic homologous recombination (NAHR) is responsible for the recurrent rearrangements that give rise to genomic disorders. Although meiotic NAHR has been investigated in multiple contexts, much less is known about mitotic NAHR despite its importance for tumorigenesis. Because type-2 NF1 microdeletions frequently result from mitotic NAHR, they represent a good model in which to investigate the features of mitotic NAHR. We have used microsatellite analysis and SNP arrays to distinguish between the various alternative recombinational possibilities, thereby ascertaining that 17 of 18 type-2 NF1 deletions, with breakpoints in the SUZ12 gene and its highly homologous pseudogene, originated via intrachromosomal recombination. This high proportion of intrachromosomal NAHR causing somatic type-2 NF1 deletions contrasts with the interchromosomal origin of germline type-1 NF1 microdeletions, whose breakpoints are located within the NF1-REPs (low-copy repeats located adjacent to the SUZ12 sequences). Further, meiotic NAHR causing type-1 NF1 deletions occurs within recombination hotspots characterized by high GC-content and DNA duplex stability, whereas the type-2 breakpoints associated with the mitotic NAHR events investigated here do not cluster within hotspots and are located within regions of significantly lower GC-content and DNA stability. Our findings therefore point to fundamental mechanistic differences between the determinants of mitotic and meiotic NAHR. Hum Mutat 31:1163-1173, 2010. © 2010 Wiley-Liss, Inc.

  6. Investigations to improve carbon dioxide control with amine and molecular sieve type sorbers

    Science.gov (United States)

    Bertrand, J. F.; Brose, H. F.; Kester, F. L.; Lunde, P. J.

    1972-01-01

    The optimization trends and operating parameters of an integral molecular sieve bed heat exchanger were investigated. The optimum combination of substrate and coating for the HS-B porous polymer was determined based on the CO2 dynamic capacity in the presence of water vapor. Full size HS-B canister performance was evaluated. An Amine CO2 Concentrator utilizing IR-45 sorber material and available Manned Orbiting Laboratory hardware was designed, fabricated and tested for use as an experiment in the NASA 90-day space simulator test of 1970. It supported four men in the simulator for 71 days out of the 90-day test duration.

  7. The molecular puzzle of C-type lectin like natural killer cell receptors

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; Kolenko, Petr; Skálová, Tereza; Hašek, Jindřich; Vaněk, O.; Rozbeský, D.; Kotýnková, Kristýna; Novák, Petr; Bezouška, K.

    2011-01-01

    Roč. 18, č. 1 (2011), s. 15 ISSN 1211-5894. [Discussions in Structural Molecular Biology /9./. 24.03.2011-26.03.2011, Nové Hrady] R&D Projects: GA ČR GA303/09/0477; GA ČR GAP207/10/1040; GA ČR GAP302/11/0855 EU Projects: European Commission(XE) 31220 - SPINE2-COMPLEXES Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Keywords : protein structure * X-ray diffraction * natural killer cell Subject RIV: CE - Biochemistry

  8. Indirect rapid prototyping of sol-gel hybrid glass scaffolds for bone regeneration - Effects of organic crosslinker valence, content and molecular weight on mechanical properties.

    Science.gov (United States)

    Hendrikx, Stephan; Kascholke, Christian; Flath, Tobias; Schumann, Dirk; Gressenbuch, Mathias; Schulze, F Peter; Hacker, Michael C; Schulz-Siegmund, Michaela

    2016-04-15

    We present a series of organic/inorganic hybrid sol-gel derived glasses, made from a tetraethoxysilane-derived silica sol (100% SiO2) and oligovalent organic crosslinkers functionalized with 3-isocyanatopropyltriethoxysilane. The material was susceptible to heat sterilization. The hybrids were processed into pore-interconnected scaffolds by an indirect rapid prototyping method, described here for the first time for sol-gel glass materials. A large panel of polyethylene oxide-derived 2- to 4-armed crosslinkers of molecular weights ranging between 170 and 8000Da were incorporated and their effect on scaffold mechanical properties was investigated. By multiple linear regression, 'organic content' and the 'content of ethylene oxide units in the hybrid' were identified as the main factors that determined compressive strength and modulus, respectively. In general, 3- and 4-armed crosslinkers performed better than linear molecules. Compression tests and cell culture experiments with osteoblast-like SaOS-2 cells showed that macroporous scaffolds can be produced with compressive strengths of up to 33±2MPa and with a pore structure that allows cells to grow deep into the scaffolds and form mineral deposits. Compressive moduli between 27±7MPa and 568±98MPa were obtained depending on the hybrid composition and problems associated with the inherent brittleness of sol-gel glass materials could be overcome. SaOS-2 cells showed cytocompatibility on hybrid glass scaffolds and mineral accumulation started as early as day 7. On day 14, we also found mineral accumulation on control hybrid glass scaffolds without cells, indicating a positive effect of the hybrid glass on mineral accumulation. We produced a hybrid sol-gel glass material with significantly improved mechanical properties towards an application in bone regeneration and processed the material into macroporous scaffolds of controlled architecture by indirect rapid prototyping. We were able to produce macroporous materials

  9. Assessment of the dental and skeletal effects of fan-type rapid maxillary expansion screw and Hyrax screw on craniofacial structures

    Directory of Open Access Journals (Sweden)

    Umarevathi Gopalakrishnan

    2017-01-01

    Full Text Available Aims and Objectives: The purpose of the study was to assess the skeletal and dental effects of fan-type rapid maxillary expansion (RME appliance and Hyrax RME appliance on the craniofacial structures. Materials and Methods: The sample of the study included 12 patients with constricted maxillary arches. Acrylic bonded type of attachment was used for both groups. Changes in sagittal, vertical, and transverse relationship were assessed with lateral and frontal cephalograms, respectively. Intercanine and intermolar widths were measured with stone models. Pre- and immediate post-treatment records were statistically analyzed with Wilcoxon signed-rank test. The differences between the groups were evaluated using Mann–Whitney U-test. Since the data pertaining to intercanine width and intermolar width were normally distributed, parametric test of signifi cance (unpaired t-test was used to compare them. Results: Results showed that Hyrax presented with signifi cantly greater increments for both nasal cavity width and maxillary width when compared to fan-type RME. Both groups had retroclination of incisors. The increase in the intercanine width was almost similar in both groups. Conclusion: Fan-type RME caused only minimal expansion of the intermolar width when compared to the Hyrax. The ratio between the intercanine and intermolar width expansion was nearly 4:1 in the fan-type RME and 0.75:1 in Hyrax.

  10. Molecular evolution of the Bovini tribe (Bovidae, Bovinae: Is there evidence of rapid evolution or reduced selective constraint in Domestic cattle?

    Directory of Open Access Journals (Sweden)

    McCulloch Alan

    2009-04-01

    Full Text Available Abstract Background If mutation within the coding region of the genome is largely not adaptive, the ratio of nonsynonymous (dN to synonymous substitutions (dS per site (dN/dS should be approximately equal among closely related species. Furthermore, dN/dS in divergence between species should be equivalent to dN/dS in polymorphisms. This hypothesis is of particular interest in closely related members of the Bovini tribe, because domestication has promoted rapid phenotypic divergence through strong artificial selection of some species while others remain undomesticated. We examined a number of genes that may be involved in milk production in Domestic cattle and a number of their wild relatives for evidence that domestication had affected molecular evolution. Elevated rates of dN/dS were further queried to determine if they were the result of positive selection, low effective population size (Ne or reduced selective constraint. Results We have found that the domestication process has contributed to higher dN/dS ratios in cattle, especially in the lineages leading to the Domestic cow (Bos taurus and Mithan (Bos frontalis and within some breeds of Domestic cow. However, the high rates of dN/dS polymorphism within B. taurus when compared to species divergence suggest that positive selection has not elevated evolutionary rates in these genes. Likewise, the low rate of dN/dS in Bison, which has undergone a recent population bottleneck, indicates a reduction in population size alone is not responsible for these observations. Conclusion The effect of selection depends on effective population size and the selection coefficient (Nes. Typically under domestication both selection pressure for traits important in fitness in the wild and Ne are reduced. Therefore, reduced selective constraint could be responsible for the observed elevated evolutionary ratios in domesticated species, especially in B. taurus and B. frontalis, which have the highest dN/dS in the

  11. Molecular Analysis of Anisakis Type I Larvae in Marine Fish from Three Different Sea Areas in Korea

    OpenAIRE

    Sohn, Woon-Mok; Kang, Jung-Mi; Na, Byoung-Kuk

    2014-01-01

    Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA ...

  12. Synthesis, SAR and molecular docking study of novel non-β-lactam inhibitors of TEM type β-lactamase.

    Science.gov (United States)

    Antipin, Roman L; Beshnova, Daria A; Petrov, Rostislav A; Shiryaeva, Anna S; Andreeva, Irina P; Grigorenko, Vitaly G; Rubtsova, Maya Yu; Majouga, Alexander G; Lamzin, Victor S; Egorov, Alexey M

    2017-04-01

    The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type β-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed. Copyright © 2017. Published by Elsevier Ltd.

  13. Observation of heptamethylbenzenium cation over SAPO-type molecular sieve DNL-6 under real MTO conversion conditions.

    Science.gov (United States)

    Li, Jinzhe; Wei, Yingxu; Chen, Jingrun; Tian, Peng; Su, Xiong; Xu, Shutao; Qi, Yue; Wang, Quanyi; Zhou, You; He, Yanli; Liu, Zhongmin

    2012-01-18

    The heptamethylbenzenium cation (heptaMB(+)) has been speculated to be one of the most important active intermediates involved in the "hydrocarbon pool" mechanism of methanol-to-olefin (MTO) conversion. By the use of DNL-6, a newly synthesized SAPO-type molecular sieve with large cavities, heptaMB(+) has for the first time been directly observed during methanol conversion under real working conditions. (13)C-labeling experiments suggested that olefin formation mediated by heptaMB(+) mainly follows the side-chain mechanism. © 2011 American Chemical Society

  14. Molecular typing and virulence characteristic of methicillin-resistant Staphylococcus aureus isolates from pediatric patients in Bucaramanga, Colombia.

    Directory of Open Access Journals (Sweden)

    Mayra Alejandra Machuca

    Full Text Available BACKGROUND: Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins. METHODS: A total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates. RESULTS: Four CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%, sek (92% and pvl (88% were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%, sek-hlg (22.6%, seb-seq-sek-hlg (18.9% and seb-sek-hlg (15.1%. The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%, clf-B (87%, fnb-A (83% and ica (83%. The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found. CONCLUSION: Four major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV

  15. Molecular typing and virulence characteristic of methicillin-resistant Staphylococcus aureus isolates from pediatric patients in Bucaramanga, Colombia.

    Science.gov (United States)

    Machuca, Mayra Alejandra; Sosa, Luis Miguel; González, Clara Isabel

    2013-01-01

    Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC) of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins. A total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates. Four CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%), sek (92%) and pvl (88%) were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%), sek-hlg (22.6%), seb-seq-sek-hlg (18.9%) and seb-sek-hlg (15.1%). The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%), clf-B (87%), fnb-A (83%) and ica (83%). The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found. Four major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV) was the most frequent, confirming the presence

  16. Molecular and morphological comparison of two different types of Habronema muscae (Nematoda: Habronematidae) in horse.

    Science.gov (United States)

    Rakhshandehroo, Ehsan; Sharifiyazdi, Hassan; Shayegh, Hossein; Ahmadi, Amin

    2014-12-01

    Habronema muscae is a spirurid nematode that undergoes developmental stages in the stomach of equids, causing chronic catarrhal gastritis. Despite preceding investigations have developed polymerase chain reaction (PCR)-based assays for molecular diagnosis, we aimed to assess the applicability of cytochrome c oxidase subunit 1 (cox1) sequences to identify the H. muscae infection and to assess the level of intraspecific variations in this parasite obtained from affected horses in Southern Iran. According to the morphological characterizations, two different isolates of H. muscae were identified. Although the majority of the recovered specimens had normal characterizations of H. muscae, a number of parasites showed an abnormal feature as large, asymmetrical, and thick cuticular extensions was observed at their anterior end (head region) in gross and histologic examinations. Unexpectedly, molecular assay disclosed that both morphologically distinct samples were completely identical to each other based on cox1 sequence. Multiple alignment of the cox1 amino acid sequences showed that all polymorphism sites were silent. Also, phylogenetic analysis provided strong support that H. muscae form a sister group to Spirocerca lupi and Thelazia callipaeda.

  17. Metaplastic Breast Cancer: Molecular Typing and Identification of Potential Targeted Therapies at a Single Institution.

    Science.gov (United States)

    Edenfield, Jessica; Schammel, Christine; Collins, Justin; Schammel, David; Edenfield, W Jeff

    2017-02-01

    Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic subtype of breast cancer comprising approximately 0.5% to 5.0% of all invasive breast cancers with a poor prognosis and limited therapeutic options. We investigated MBC at our institution to evaluate outcomes and investigate the molecular profile of our cohort to determine the presence of mutations for which there are targeted therapies. We found our cohort to consist mainly of the matrix-producing variant (72%) with 48% having the stereotypical estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor-2-negative phenotype. While the overall survival of our cohort was an average of 1679 days (4.6 years), we had a surprising number of patients with second primaries (40%) and distant metastases (40%), yet few recurrences (12%). Molecular analysis of the tumors indicated that one gene mutation, CSFIR, was significantly associated with outcome (P = .021); however, the cohort was defined by frequent mutations in ERBB4 (36%), PIK3CA (48%), and FLT3 (60%), for which there are now targeted therapies. While surgery is the appropriate first step in the management of this aggressive malignancy, the collection of data pertaining to the use of targeted agents, although anecdotal, may provide clues to better treatment for these patients. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Evaluation of Rapid Molecular Detection Assays for Salmonella in Challenging Food Matrices at Low Inoculation Levels and Using Difficult-to-Detect Strains.

    Science.gov (United States)

    Ryan, Gina; Roof, Sherry; Post, Laurie; Wiedmann, Martin

    2015-09-01

    Assays for detection of foodborne pathogens are generally initially evaluated for performance in validation studies carried out according to guidelines provided by validation schemes (e.g., AOAC International or the International Organization for Standardization). End users often perform additional validation studies to evaluate the performance of assays in specific matrices (e.g., specific foods or raw material streams of interest) and with specific pathogen strains. However, these types of end-user validations are typically not well defined. This study was conducted to evaluate a secondary end user validation of four AOAC-validated commercial rapid detection assays (an isothermal nucleic acid amplification, an immunoassay, and two PCR-based assays) for their ability to detect Salmonella in two challenging matrices (dry pet food and dark chocolate). Inclusivity was evaluated with 68 diverse Salmonella strains at low population levels representing the limit of detection (LOD) for each assay. One assay detected all strains at the LOD, two assays detected multiple strains only at 10 times the LOD, and the fourth assay failed to detect two strains (Salmonella bongori and S. enterica subsp. houtenae) even at 1,000 times the LOD; this assay was not further evaluated. The three remaining assays were subsequently evaluated for their ability to detect five selected Salmonella strains in food samples contaminated at fractional levels. Unpaired comparisons revealed no significant difference between the results for each given assay and the results obtained with the reference assay. However, analysis of paired culture-confirmed results revealed assay false-negative rates of 4 to 26% for dry pet food and 12 to 16% for dark chocolate. Overall, our data indicate that rapid assays may have high false-negative rates when performance is evaluated under challenging conditions, including low-moisture matrices, strains that are difficult to detect, injured cells, and low inoculum

  19. The Requirement of L-Type Voltage-Dependent Calcium Channel (L-VDCC) in the Rapid-Acting Antidepressant-Like Effects of Scopolamine in Mice.

    Science.gov (United States)

    Yu, Hanjie; Li, Mengmeng; Shen, Xinbei; Lv, Dan; Sun, Xin; Wang, Jinting; Gu, Xinmei; Hu, Jingning; Wang, Chuang

    2018-02-01

    Previous studies have shown that a low dose of scopolamine produces rapid-acting antidepressant-like actions in rodents. Understanding the mechanisms underlying this effect and the dose-dependent variations of drug responses remains an important task. L-type voltage-dependent calcium channels were found to mediate rapid-acting antidepressant effects of certain medications (e.g., ketamine). Therefore, it is of great interest to determine the involvement of L-type voltage-dependent calcium channels in the action of scopolamine. Herein, we investigated the mechanisms underlying behavioral responses to various doses of scopolamine in mice to clarify the involvement of L-type voltage-dependent calcium channels in its modes of action. Open field test, novel object recognition test, and forced swimming test were performed on mice administered varied doses of scopolamine (0.025, 0.05, 0.1, 1, and 3 mg/kg, i.p.) alone or combined with L-type voltage-dependent calcium channel blocker verapamil (5 mg/kg, i.p.). Then, the changes in brain-derived neurotrophic factor and neuropeptide VGF (nonacronymic) levels in the hippocampus and prefrontal cortex of these mice were analyzed. Low doses of scopolamine (0.025 and 0.05 mg/kg) produced significant antidepressant-like effects in the forced swimming test, while higher doses (1 and 3 mg/kg) resulted in significant memory deficits and depressive-like behaviors. Moreover, the behavioral changes in responses to various doses may be related to the upregulation (0.025 and 0.05 mg/kg) and downregulation (1 and 3 mg/kg) of brain-derived neurotrophic factor and VGF in the hippocampus and prefrontal cortex in mice. We further found that the rapid-acting antidepressant-like effects and the upregulation on brain-derived neurotrophic factor and VGF produced by a low dose of scopolamine (0.025 mg/kg) were completely blocked by verapamil. These results indicate that L-type voltage-dependent calcium channels are likely involved in the behavioral

  20. Recent research on inherent molecular structure, physiochemical properties, and bio-functions of food and feed-type Avena sativa oats and processing-induced changes revealed with molecular microspectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Prates, Luciana Louzada [Department of Animal and Poultry Science, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Yu, Peiqiang [Department of Animal and Poultry Science, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    2017-05-16

    Avena sativa oat is a cereal widely used as human food and livestock feed. However, the low metabolized energy and the rapid rumen degradations of protein and starch have limited the use of A. sativa oat grains. To overcome this disadvantage, new A. sativa oat varieties have been developed. Additionally, heat-related processing has been performed to decrease the degradation rate and improve the absorption of amino acids in the small intestine. The nutritive value is reflected by both chemical composition and inherent molecular structure conformation. However, the traditional wet chemical analysis is not able to detect the inherent molecular structures within an intact tissue. The advanced synchrotron-radiation and globar-based molecular microspectroscopy have been developed recently and applied to study internal molecular structures and the processing induced structure changes in A. sativa oats and reveal how molecular structure changes in relation to nutrient availability. This review aimed to obtain the recent information regarding physiochemical properties, molecular structures, metabolic characteristics of protein, and the heat-induced changes in new A. sativa oat varieties. The use of the advanced vibrational molecular spectroscopy was emphasized, synchrotron- and globar-based (micro)spectroscopy, to reveal the inherent structure of A. sativa oats at cellular and molecular levels and to reveal the heat processing effect on the degradation characteristics and the protein molecular structure in A. sativa oats. The relationship between nutrient availability and protein molecular inherent structure was also presented. Information described in this review gives better insight in the physiochemical properties, molecular structure, and the heat-induced changes in A. sativa oat detected with advanced molecular spectroscopic techniques in combinination with conventional nutrition study techniques.

  1. Realization of Cu-Doped p-Type ZnO Thin Films by Molecular Beam Epitaxy.

    Science.gov (United States)

    Suja, Mohammad; Bashar, Sunayna B; Morshed, Muhammad M; Liu, Jianlin

    2015-04-29

    Cu-doped p-type ZnO films are grown on c-sapphire substrates by plasma-assisted molecular beam epitaxy. Photoluminescence (PL) experiments reveal a shallow acceptor state at 0.15 eV above the valence band edge. Hall effect results indicate that a growth condition window is found for the formation of p-type ZnO thin films, and the best conductivity is achieved with a high hole concentration of 1.54 × 10(18) cm(-3), a low resistivity of 0.6 Ω cm, and a moderate mobility of 6.65 cm(2) V(-1) s(-1) at room temperature. Metal oxide semiconductor capacitor devices have been fabricated on the Cu-doped ZnO films, and the characteristics of capacitance-voltage measurements demonstrate that the Cu-doped ZnO thin films under proper growth conditions are p-type. Seebeck measurements on these Cu-doped ZnO samples lead to positive Seebeck coefficients and further confirm the p-type conductivity. Other measurements such as X-ray diffraction, X-ray photoelectron, Raman, and absorption spectroscopies are also performed to elucidate the structural and optical characteristics of the Cu-doped p-type ZnO films. The p-type conductivity is explained to originate from Cu substitution of Zn with a valency of +1 state. However, all p-type samples are converted to n-type over time, which is mostly due to the carrier compensation from extrinsic defects of ZnO.

  2. Molecular typing of methicillin-resistant Staphylococcus aureus strains by PCR-RFLP of SPA gene: A reference laboratory perspective

    Directory of Open Access Journals (Sweden)

    Mehndiratta P

    2009-01-01

    Full Text Available Purpose: To characterize methicillin-resistant Staphylococcus aureus (MRSA strains by molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP of spa gene and to assess the utility of spa genotyping over bacteriophage typing in the discrimination of the strains. Materials and Methods: Studies were undertaken on 125 MRSA strains representing the most predominant phage types and the non phage typeable strains. Strains were typed by bacteriophage typing and PCR-RFLP of spa gene. DNA sequence analysis of the amplified spa gene fragment of the representative RFLP patterns was performed using standard protocols. Results: All the strains resistant to oxacillin were found to contain mec A gene. Fifty-two per cent of these strains were typeable by the international basic set of 23 phages. Five different PCR-RFLP patterns were observed among 125 MRSA strains. Non phage typeable strains were differentiated into four PCR-RFLP patterns. Sequencing of the spa gene from the representative strains of each RFLP pattern confirmed the length of these restriction fragments due to variation in the 24 bp and the 174 bp tandem repeats. It also revealed the presence of three new spa repeat patterns. Conclusion: The study demonstrates the importance of spa genotyping in the discrimination of MRSA strains, which were otherwise indistinguishable by bacteriophage typing. spa genotyping allowed differentiation of strains within a particular phage type. Nucleotide sequencing of isolates of different PCR-RFLP patterns indicated a correlation between the RFLP patterns of a variable number of tandem repeats and the phage type. The study provides valuable information on the epidemiological characterization of MRSA strains.

  3. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico

    Directory of Open Access Journals (Sweden)

    Fernando Bastida-González

    2016-01-01

    Full Text Available Nucleoprotein (N gene from rabies virus (RABV is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs via indirect fluorescent antibody (IFA test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers, for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing.

  4. Glycine receptor in rat hippocampal and spinal cord neurons as a molecular target for rapid actions of 17-β-estradiol

    Directory of Open Access Journals (Sweden)

    Liu Chun-Feng

    2009-01-01

    Full Text Available Abstract Glycine receptors (GlyRs play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP and spinal dorsal horn (SDH neurons, 17-β-estradiol (E2 rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly. In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERα and ERβ antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing α2, α2β or α3β subunits, suggesting that either α2-containing or α3β-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR α2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

  5. Rapid rise and fall of selfish sex-ratio X chromosomes in Drosophila simulans: spatiotemporal analysis of phenotypic and molecular data.

    Science.gov (United States)

    Bastide, Héloïse; Cazemajor, Michel; Ogereau, David; Derome, Nicolas; Hospital, Frédéric; Montchamp-Moreau, Catherine

    2011-09-01

    Sex-ratio drive, which has been documented in several Drosophila species, is induced by X-linked segregation distorters. Contrary to Mendel's law of independent assortment, the sex-ratio chromosome (X(SR)) is inherited by more than half the offspring of carrier males, resulting in a female-biased sex ratio. This segregation advantage allows X(SR) to spread in populations, even if it is not beneficial for the carriers. In the cosmopolitan species D. simulans, the Paris sex-ratio is caused by recently emerged selfish X(SR) chromosomes. These chromosomes have triggered an intragenomic conflict, and their propagation has been halted over a large area by the evolution of complete drive suppression. Previous molecular population genetics analyses revealed a selective sweep indicating that the invasion of X(SR) chromosomes was very recent in Madagascar (likely less than 100 years ago). Here, we show that X(SR) chromosomes are now declining at this location as well as in Mayotte and Kenya. Drive suppression is complete in the three populations, which display little genetic differentiation and share swept haplotypes, attesting to a common and very recent ancestry of the X(SR) chromosomes. Patterns of DNA sequence variation also indicate a fitness cost of the segmental duplication involved in drive. The data suggest that X(SR) chromosomes started declining first on the African continent, then in Mayotte, and finally in Madagascar and strongly support a scenario of rapid cycling of X chromosomes. Once drive suppression has evolved, standard X(ST) chromosomes locally replace costly X(SR) chromosomes in a few decades.

  6. BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF SALMONELLA-ENTERICA SEROVAR BERTA, AND COMPARISON OF METHODS FOR TYPING

    DEFF Research Database (Denmark)

    Olsen, J. E.; Brown, D. J.; Baggesen, Dorte Lau

    1992-01-01

    Strains of Salmonella enterica serovar berta (S. berta) from Denmark and seven other countries have been characterized with the aim of developing a rational typing strategy in connection with outbreak investigations, Biotyping divided the strains into H2S-positive (90 %) and H2S-negative (10...... with restriction enzyme analysis of plasmids seemed to be the most rational typing strategy for S. berta. The results indicated that S. berta strains regardless of geographical source or host are possibly clonal in nature....

  7. Toward molecular pathogenesis of an autoimmune disease: Refined genetic mapping of autoimmune polyglandular disease type I (APECED)

    Energy Technology Data Exchange (ETDEWEB)

    Aaltonen, J.; Bjoerses, P.; Peltonen, L. [National Public Health Institute, Helsinki (Finland)] [and others

    1994-09-01

    Autoimmune reactions encoupled to many human diseases are still only partially understood. Unravelling the molecular pathogenesis of inherited diseases with a strong autoimmune component in their clinical expression could help to dissect individual components in the molecular background of abnormal immune response. One such genetic disorder is autosomal recessive autoimmune polyglandular disease type I (PGD I), also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, MIM 240300). The disease is especially enriched in the genetically isolated population of Finland and we have assigned the APECED locus to human chromosome 21q22.3 in 14 Finnish families by linkage analyses. The best positional lod score of 6.49 was observed with marker D21S49. Based on the history of the Finns, the gene pool of this population clearly demonstrates the consequences of a founder effect and consequent isolation. In the Finnish population, we can take advantage of linkage disequilibrium and allelic association studies to more precisely define the critical DNA region for our disease gene of interest than would be possible by linkage analyses alone. We are now able to define the chromosomal region of interest between two flanking markers locating 1 cM apart. Linkage disequilibrium is observed with three of the markers used in the analyses and this suggests a distance of less than 500 kb to the disease locus, well approachable with molecular cloning techniques. Overlapping YAC and cosmid clones spanning our region of interest will facilitate the cloning of APECED gene in the near future.

  8. The effects of capsaicin and capsaicinoid analogs on metabolic molecular targets in highly energetic tissues and cell types.

    Science.gov (United States)

    Gannon, Nicholas P; Lambalot, Emily L; Vaughan, Roger A

    2016-05-01

    There is increasing interest in dietary chemicals that may provide benefits for pathologies such as diabetes and obesity. Capsaicinoids found in chili peppers and pepper extracts, are responsible for the "hot" or "spicy" sensation associated with these foods. Capsaicinoid consumption is also associated with enhanced metabolism, making them potentially therapeutic for metabolic disease by promoting weight loss. This review summarizes much of the current experimental evidence (ranging from basic to applied investigations) of the biochemical and molecular metabolic effects of capsaicinoids in metabolically significant cell types. Along with influencing metabolic rate, findings demonstrate capsaicinoids appear to alter molecular metabolic signaling, regulate hunger and satiety, blood metabolites, and catecholamine release. Notably, the majority of the experiments summarized herein utilized isolated supplemental or research grade capsaicinoids rather than natural food sources for experimental interventions. Additional work should be conducted using primary food sources of capsaicin to explore pharmacological, physiological, and metabolic benefits of both chronic and acute capsaicin consumption. © 2016 BioFactors, 42(3):229-246, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

  9. A Comparison of Molecular Typing Methods Applied to Enterobacter cloacae complex: hsp60 Sequencing, Rep-PCR, and MLST

    Directory of Open Access Journals (Sweden)

    Roberto Viau

    2017-02-01

    Full Text Available Molecular typing using repetitive sequenced-based PCR (rep-PCR and hsp60 sequencing were applied to a collection of diverse Enterobacter cloacae complex isolates. To determine the most practical method for reference laboratories, we analyzed 71 E. cloacae complex isolates from sporadic and outbreak occurrences originating from 4 geographic areas. While rep-PCR was more discriminating, hsp60 sequencing provided a broader and a more objective geographical tracking method similar to multilocus sequence typing (MLST. In addition, we suggest that MLST may have higher discriminative power compared to hsp60 sequencing, although rep-PCR remains the most discriminative method for local outbreak investigations. In addition, rep-PCR can be an effective and inexpensive method for local outbreak investigation.

  10. Possible hypoglycemic effect of Aloe vera L. high molecular weight fractions on type 2 diabetic patients

    Science.gov (United States)

    Yagi, Akira; Hegazy, Sahar; Kabbash, Amal; Wahab, Engy Abd-El

    2009-01-01

    Aloe vera L. high molecular weight fractions (AHM) containing less than 10 ppm of barbaloin and polysaccharide (MW: 1000 kDa) with glycoprotein, verectin (MW: 29 kDa), were prepared by patented hyper-dry system in combination of freeze–dry technique with microwave and far infrared radiation. AHM produced significant decrease in blood glucose level sustained for 6 weeks of the start of the study. Significant decrease in triglycerides was only observed 4 weeks after treatment and continued thereafter. No deterious effects on kidney and liver functions were apparent. Treatment of diabetic patients with AHM may relief vascular complications probably via activation of immunosystem. PMID:23964163

  11. Isoforms of wild type proteins often appear as low molecular weight bands on SDS-PAGE.

    Science.gov (United States)

    Zhang, Ju; Lou, Xiaomin; Shen, Haihong; Zellmer, Lucas; Sun, Yuan; Liu, Siqi; Xu, Ningzhi; Liao, D Joshua

    2014-08-01

    Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE), is a technique commonly used to detect specific proteins. SDS-PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography - mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non-malignant HEK293 and cancerous MDA-MB231 (MB231) cells separated using SDS-PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS-PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer-therapy studies. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Prognostic clinical and molecular biomarkers of renal disease in type 2 diabetes

    DEFF Research Database (Denmark)

    Pena, Michelle J; de Zeeuw, Dick; Mischak, Harald

    2015-01-01

    Diabetic kidney disease occurs in ∼ 25-40% of patients with type 2 diabetes. Given the high risk of progressive renal function loss and end-stage renal disease, early identification of patients with a renal risk is important. Novel biomarkers may aid in improving renal risk stratification. In thi...

  13. Specific populations of the yeast Geotrichum candidum revealed by molecular typing.

    Science.gov (United States)

    Jacques, Noémie; Mallet, Sandrine; Laaghouiti, Fatima; Tinsley, Colin R; Casaregola, Serge

    2017-04-01

    Geotrichum candidum is a ubiquitous yeast and an essential component in the production of many soft cheeses. We developed a multilocus sequence typing (MLST) scheme with five retained loci (NUP116, URA1, URA3, SAPT4 and PLB3) which were sufficiently divergent to distinguish 40 sequence types (STs) among the 67 G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or the dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a polymerase chain reaction inter-long terminal repeat scheme, a fast and reproducible random amplification of polymorphic DNA-like method for G. candidum, to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analysed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Epigenetics: a molecular link between environmental factors and type 2 diabetes

    National Research Council Canada - National Science Library

    Ling, Charlotte; Groop, Leif

    2009-01-01

    ... some insights into epigenetic mechanisms associated with type 2 diabetes. An overview of epigenetic regulation. Although there is no uniform definition of epigenetics, it has been described as heritable changes in gene function that occur without a change in the nucleotide sequence (6). Epigenetic modifications can be passed from one cell generation to...

  15. Molecular genetic analysis of Type II diabetes associated m.3243A ...

    African Journals Online (AJOL)

    Background: Type II diabetes is the most often considered as maternally inherited disease and A>G transition at position 3243 of mitochondrial DNA (m.3243A>G) in the encoding tRNALeu(UUR) gene is thought to be strongly responsible for the pathogenesis of the disease in number of cases. Aim: Current study was ...

  16. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2

    NARCIS (Netherlands)

    Kamsteeg, E.J.; Kress, W.; Catalli, C.; Hertz, J.M.; Witsch-Baumgartner, M.; Buckley, M.F.; Engelen, B.G.M. van; Schwartz, M.; Scheffer, H.

    2012-01-01

    Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and congenital forms, which frequently result in death because of respiratory deficiency, through to

  17. Molecular typing by pulsed-field gel electrophoresis of Flavobacterium psychrophilum isolates derived from Japanese fish.

    Science.gov (United States)

    Arai, H; Morita, Y; Izumi, S; Katagiri, T; Kimura, H

    2007-06-01

    Sixty-four isolates of Flavobacterium psychrophilum from ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel), and other fish (n=16) in Japan and the type strain (NCIMB 1947(T)) were typed using pulsed-field gel electrophoresis (PFGE) with endonuclease BlnI and XhoI. These isolates were classified into 20 clusters and 42 genotypes by PFGE analysis. The most predominant cluster of isolates from ayu was cluster XII (n=20), followed by clusters XVII, XVI, XX, XI, IX, X, XIII and XV; the remaining 17 isolates from other fish were divided into clusters I, II, III, IV, V, VI, VII, VIII, XIV, XVIII and XIX. The PFGE genotype of isolates from ayu clearly differed from those of other fish. The isolates from ayu in Gunma Prefecture belonged to clusters XII, XVI, XVII and XX, and the strains of three of these clusters (XII, XVII and XX) were isolated from ayu in 15 of 19 prefectures. PFGE typing enabled more accurate classification of isolates into clusters than previously achieved by analysing the restriction fragment length polymorphism of PCR products. These results suggest that F. psychrophilum isolated from ayu and other fish are genetically different and strains with several PFGE types have spread within Japan.

  18. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin

    DEFF Research Database (Denmark)

    Hoadley, Katherine A; Yau, Christina; Wolf, Denise M

    2014-01-01

    Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform o...

  19. Molecular typing of Campylobacter jejuni isolates involved in a neonatal outbreak indicates nosocomial transmission

    DEFF Research Database (Denmark)

    Llovo, J.; Mateo, E.; Munoz, A.

    2003-01-01

    Genotypic typing by restriction fragment length polymorphism and pulsed-field gel electrophoresis showed that two neonates in a neonatal ward were infected with the same Campylobacter jejuni strain. Isolates from the mother and brother of the index patient were identical to each other but distinc...

  20. Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

    NARCIS (Netherlands)

    Wang, G.; van Dam, A. P.; Spanjaard, L.; Dankert, J.

    1998-01-01

    To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA

  1. Obesity and type 2 diabetes : a systems biology perspective of a molecular mechanism

    NARCIS (Netherlands)

    Ciapaite, J.

    2007-01-01

    People who have excess body weight have a higher risk of insulin resistance and type 2 diabetes, because fat interferes with the body's ability to make and use insulin. Insulin, a hormone released by pancreatic beta-cells, is needed to move diet-derived glucose from blood into fat and muscle cells

  2. Prognostic clinical and molecular biomarkers of renal disease in type 2 diabetes

    NARCIS (Netherlands)

    Pena, Michelle J.; de Zeeuw, Dick; Mischak, Harald; Jankowski, Joachim; Oberbauer, Rainer; Woloszczuk, Wolfgang; Benner, Jacqueline; Dallmann, Guido; Mayer, Bernd; Mayer, Gert; Rossing, Peter; Lambers Heerspink, Hiddo J.

    Diabetic kidney disease occurs in similar to 25-40% of patients with type 2 diabetes. Given the high risk of progressive renal function loss and end-stage renal disease, early identification of patients with a renal risk is important. Novel biomarkers may aid in improving renal risk stratification.

  3. Molecular Epidemiology of Adenovirus Type 7 in the United States, 1966-2000

    Science.gov (United States)

    2002-03-01

    Med Trop Sao Paulo 1997;39:185-9. GF, et al. Extrapulmonary manifestations of adenovirus type 7 pneumonia 31. Golovina GI, Zolotaryov FN, Yurlova TI...residues. J Virol 1996;70:1836-44. 22. McNeill KM, Ridgely Benton F, Monteith SC, Tuchscherer MA, Gaydos 44. Kajon AE, Murtagh P, Garcia Franco S, Freire

  4. Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein.

    Science.gov (United States)

    Watts, Joel C; Giles, Kurt; Stöhr, Jan; Oehler, Abby; Bhardwaj, Sumita; Grillo, Sunny K; Patel, Smita; DeArmond, Stephen J; Prusiner, Stanley B

    2012-02-28

    Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ∼35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ∼185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD.

  5. Molecular Modeling Studies of 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors through Receptor-Based 3D-QSAR and Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Haiyan Qian

    2016-09-01

    Full Text Available 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1 is a potential target for the treatment of numerous human disorders, such as diabetes, obesity, and metabolic syndrome. In this work, molecular modeling studies combining molecular docking, 3D-QSAR, MESP, MD simulations and free energy calculations were performed on pyridine amides and 1,2,4-triazolopyridines as 11β-HSD1 inhibitors to explore structure-activity relationships and structural requirement for the inhibitory activity. 3D-QSAR models, including CoMFA and CoMSIA, were developed from the conformations obtained by docking strategy. The derived pharmacophoric features were further supported by MESP and Mulliken charge analyses using density functional theory. In addition, MD simulations and free energy calculations were employed to determine the detailed binding process and to compare the binding modes of inhibitors with different bioactivities. The binding free energies calculated by MM/PBSA showed a good correlation with the experimental biological activities. Free energy analyses and per-residue energy decomposition indicated the van der Waals interaction would be the major driving force for the interactions between an inhibitor and 11β-HSD1. These unified results may provide that hydrogen bond interactions with Ser170 and Tyr183 are favorable for enhancing activity. Thr124, Ser170, Tyr177, Tyr183, Val227, and Val231 are the key amino acid residues in the binding pocket. The obtained results are expected to be valuable for the rational design of novel potent 11β-HSD1 inhibitors.

  6. Data for molecular dynamics simulations of B-type cytochrome c oxidase with the Amber force field

    Directory of Open Access Journals (Sweden)

    Longhua Yang

    2016-09-01

    Full Text Available Cytochrome c oxidase (CcO is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. This article presents parameters for the cofactors of ba3-type CcO that are compatible with the all-atom Amber ff12SB and ff14SB force fields. Specifically, parameters were developed for the CuA pair, heme b, and the dinuclear center that consists of heme a3 and CuB bridged by a hydroperoxo group. The data includes geometries in XYZ coordinate format for cluster models that were employed to compute proton transfer energies and derive bond parameters and point charges for the force field using density functional theory. Also included are the final parameter files that can be employed with the Amber leap program to generate input files for molecular dynamics simulations with the Amber software package. Based on the high resolution (1.8 Å X-ray crystal structure of the ba3-type CcO from Thermus thermophilus (Protein Data Bank ID number PDB: 3S8F, we built a model that is embedded in a POPC lipid bilayer membrane and solvated with TIP3P water molecules and counterions. We provide PDB data files of the initial model and the equilibrated model that can be used for further studies.

  7. Phenotypic variability of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA): clinical, molecular and biochemical delineation

    Science.gov (United States)

    2011-01-01

    Background The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA) (OMIM 225400) is a rare inheritable connective tissue disorder characterized by a deficiency of collagen lysyl hydroxylase 1 (LH1; EC 1.14.11.4) due to mutations in PLOD1. Biochemically this results in underhydroxylation of collagen lysyl residues and, hence, an abnormal pattern of lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) crosslinks excreted in the urine. Clinically the disorder is characterized by hypotonia and kyphoscoliosis at birth, joint hypermobility, and skin hyperelasticity and fragility. Severe hypotonia usually leads to delay in gross motor development, whereas cognitive development is reported to be normal. Methods We describe the clinical, biochemical and molecular characterisation, as well as electron microscopy findings of skin, in 15 patients newly diagnosed with this rare type of Ehlers-Danlos syndrome. Results Age at diagnosis ranged from 5 months to 27 years, with only 1/3 of the patients been diagnosed correctly in the first year of life. A similar disease frequency was found in females and males, however a broad disease severity spectrum (intra- and interfamilial), independent of molecular background or biochemical phenotype, was observed. Kyphoscoliosis, one of the main clinical features was not present at birth in 4 patients. Importantly we also noted the occurrence of vascular rupture antenatally and postnatally, as well as developmental delay in 5 patients. Conclusion In view of these findings we propose that EDS VIA is a highly variable clinical entity, presenting with a broad clinical spectrum, which may also be associated with cognitive delay and an increased risk for vascular events. Genotype/phenotype association studies and additional molecular investigations in more extended EDS VIA populations will be necessary to further elucidate the cause of the variability of the disease severity. PMID:21699693

  8. Phenotypic variability of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA: clinical, molecular and biochemical delineation

    Directory of Open Access Journals (Sweden)

    Kariminejad Ariana

    2011-06-01

    Full Text Available Abstract Background The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA (OMIM 225400 is a rare inheritable connective tissue disorder characterized by a deficiency of collagen lysyl hydroxylase 1 (LH1; EC 1.14.11.4 due to mutations in PLOD1. Biochemically this results in underhydroxylation of collagen lysyl residues and, hence, an abnormal pattern of lysyl pyridinoline (LP and hydroxylysyl pyridinoline (HP crosslinks excreted in the urine. Clinically the disorder is characterized by hypotonia and kyphoscoliosis at birth, joint hypermobility, and skin hyperelasticity and fragility. Severe hypotonia usually leads to delay in gross motor development, whereas cognitive development is reported to be normal. Methods We describe the clinical, biochemical and molecular characterisation, as well as electron microscopy findings of skin, in 15 patients newly diagnosed with this rare type of Ehlers-Danlos syndrome. Results Age at diagnosis ranged from 5 months to 27 years, with only 1/3 of the patients been diagnosed correctly in the first year of life. A similar disease frequency was found in females and males, however a broad disease severity spectrum (intra- and interfamilial, independent of molecular background or biochemical phenotype, was observed. Kyphoscoliosis, one of the main clinical features was not present at birth in 4 patients. Importantly we also noted the occurrence of vascular rupture antenatally and postnatally, as well as developmental delay in 5 patients. Conclusion In view of these findings we propose that EDS VIA is a highly variable clinical entity, presenting with a broad clinical spectrum, which may also be associated with cognitive delay and an increased risk for vascular events. Genotype/phenotype association studies and additional molecular investigations in more extended EDS VIA populations will be necessary to further elucidate the cause of the variability of the disease severity.

  9. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

    Science.gov (United States)

    Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

    2014-01-01

    A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Electrical characteristics and interfacial reactions of rapidly annealed Pt/Ru Schottky contacts on n-type GaN

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, N.N.K.; Rajagopal Reddy, V. [Department of Physics, Sri Venkateswara University, Tirupati (India); Choi, C.J. [School of Semiconductor and Chemical Engineering, Semiconductor Physics Research Center (SPRC), Chonbuk National University, Jeonju (Korea, Republic of)

    2011-07-15

    The electrical properties and interfacial reactions of Pt/Ru Schottky contacts on n-type gallium nitride (GaN) have been investigated as a function of annealing temperature. The calculated Schottky barrier height (SBH) of the as-deposited Pt/Ru Schottky contact is found to be 0.69 eV current-voltage (I-V) and 0.76 eV capacitance-voltage (C-V). Experimental results showed that the SBHs are increased on increasing the annealing temperature. When the contact is annealed at 600 C, a maximum barrier height is obtained and the corresponding values are 0.87 eV (I-V) and 0.99 eV (C-V). The Norde method was also employed to extract the barrier height of Pt/Ru Schottky contacts and the values are 0.70 and 0.86 eV for the samples as-deposited and annealed at 600 C, which are in good agreement with those obtained from the I-V measurement. Shifts of the surface Fermi level are measured with the change in position of the Ga 2p core level peak. Based on the X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) studies, the formation of gallide phases at the Ru/Pt/n-GaN interface could be the reason for the increase in SBH at elevated temperatures. Atomic force microscopy (AFM) results showed that the surface morphology of the Pt/Ru Schottky contact did not change significantly even after annealing at 600 C. These results point out that a Pt/Ru Schottky contact may be a suitable candidate for the fabrication of GaN-based high-temperature device applications. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  11. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2

    DEFF Research Database (Denmark)

    Kamsteeg, Erik-Jan; Kress, Wolfram; Catalli, Claudio

    2012-01-01

    , but cardiac conduction abnormalities as in DM1 are observed and patients with DM2 additionally have muscle pain and stiffness. Both DM1 and DM2 are caused by unstable DNA repeats in untranslated regions of different genes: A (CTG)n repeat in the 3'-UTR of the DMPK gene and a (CCTG)n repeat in intron 1......Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and congenital forms, which frequently result in death because of respiratory deficiency, through to late......-onset baldness and cataract. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age and have a more severe course (anticipation). In myotonic dystrophy type 2 (DM2), no anticipation is described...

  12. VNTR molecular typing of salmonella enterica serovar typhi isolates in Kathmandu valley

    Directory of Open Access Journals (Sweden)

    B Acharya

    2012-03-01

    Full Text Available Background: Typhoid fever continues to be a worldwide health problem, especially in developing countries. Effective epidemiological surveillance is needed to monitor the presence and spread of disease. Materials and Methods: Variable number tandem repeats (VNTR was performed for Salmonella enterica serovar typhi by multiplex-PCR in 28 Nepalese isolates of sporadic typhoid fever. Results: From all 28 total isolates, we could identify 12 VNTR profiles among the isolates, signifying multiple variants in circulation within the region. Conclusion: The VNTR-based typing assay for serovar typhi isolates can be used during an outbreak of enteric fever. The typing could eventually form the basis of an effective epidemiological surveillance system for developing rational strategies to control typhoid fever. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6026 JPN 2012; 2(3: 220-223

  13. Elucidation of the Inhibitory Effect of Phytochemicals with Kir6.2 Wild-Type and Mutant Models Associated in Type-1 Diabetes through Molecular Docking Approach

    Directory of Open Access Journals (Sweden)

    Manaswini Jagadeb

    2014-12-01

    Full Text Available Among all serious diseases globally, diabetes (type 1 and type 2 still poses a major challenge to the world population. Several target proteins have been identified, and the etiology causing diabetes has been reasonably well studied. But, there is still a gap in deciding on the choice of a drug, especially when the target is mutated. Mutations in the KCNJ11 gene, encoding the kir6.2 channel, are reported to be associated with congenital hyperinsulinism, having a major impact in causing type 1 diabetes, and due to the lack of its 3D structure, an attempt has been made to predict the structure of kir6.2, applying fold recognition methods. The current work is intended to investigate the affinity of four phytochemicals namely, curcumin (Curcuma longa, genistein (Genista tinctoria, piperine (Piper nigrum, and pterostilbene (Vitis vinifera in a normal as well as in a mutant kir6.2 model by adopting a molecular docking methodology. The phytochemicals were docked in both wild and mutated kir6.2 models in two rounds: blind docking followed by ATP-binding pocket-specific docking. From the binding pockets, the common interacting amino acid residues participating strongly within the binding pocket were identified and compared. From the study, we conclude that these phytochemicals have strong affinity in both the normal and mutant kir6.2 model. This work would be helpful for further study of the phytochemicals above for the treatment of type 1 diabetes by targeting the kir6.2 channel.

  14. Elucidation of the Inhibitory Effect of Phytochemicals with Kir6.2 Wild-Type and Mutant Models Associated in Type-1 Diabetes through Molecular Docking Approach.

    Science.gov (United States)

    Jagadeb, Manaswini; Konkimalla, V Badireenath; Rath, Surya Narayan; Das, Rohit Pritam

    2014-12-01

    Among all serious diseases globally, diabetes (type 1 and type 2) still poses a major challenge to the world population. Several target proteins have been identified, and the etiology causing diabetes has been reasonably well studied. But, there is still a gap in deciding on the choice of a drug, especially when the target is mutated. Mutations in the KCNJ11 gene, encoding the kir6.2 channel, are reported to be associated with congenital hyperinsulinism, having a major impact in causing type 1 diabetes, and due to the lack of its 3D structure, an attempt has been made to predict the structure of kir6.2, applying fold recognition methods. The current work is intended to investigate the affinity of four phytochemicals namely, curcumin (Curcuma longa), genistein (Genista tinctoria), piperine (Piper nigrum), and pterostilbene (Vitis vinifera) in a normal as well as in a mutant kir6.2 model by adopting a molecular docking methodology. The phytochemicals were docked in both wild and mutated kir6.2 models in two rounds: blind docking followed by ATP-binding pocket-specific docking. From the binding pockets, the common interacting amino acid residues participating strongly within the binding pocket were identified and compared. From the study, we conclude that these phytochemicals have strong affinity in both the normal and mutant kir6.2 model. This work would be helpful for further study of the phytochemicals above for the treatment of type 1 diabetes by targeting the kir6.2 channel.

  15. Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation

    OpenAIRE

    Ripoll-Rozada, Jorge; Garc��a-Cazorla, Yolanda; Getino, Mar��a; Mach��n, Cristina; Sanabria-R��os, David; de la Cruz, Fernando; Cabez��n, Elena; Arechaga, Ignacio

    2016-01-01

    Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic AT...

  16. Kinetics and Thermochemistry of the Reactions of Propargyl-Type Radicals with Molecular Oxygen

    OpenAIRE

    Pekkanen, Timo Theodor

    2016-01-01

    The oxygen reactions of hydrocarbon radicals are important both in combustion and atmospheric chemistry. In this thesis I have studied the oxygen reactions of propagyl-type radicals (propargyl, 1-methylpropargyl, 3-methylpropargyl and 3-ethylpropargyl). Propargyl radical (2-propyn-1-yl) is an alkyl radical with the structure H3C≡C–CH2●. Because these radicals are resonance stabilised, their oxygen reactions are relatively slow. This means that in a combustion environment they might reach ...

  17. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2

    OpenAIRE

    Kamsteeg, Erik-Jan; Kress, Wolfram; Catalli, Claudio; Hertz, Jens M; Witsch-Baumgartner, Martina; Buckley, Michael F.; van Engelen, Baziel G.M.; Schwartz, Marianne; Scheffer, Hans

    2012-01-01

    Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and congenital forms, which frequently result in death because of respiratory deficiency, through to late-onset baldness and cataract. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age an...

  18. Molecular Epidemiology of Pasteurella multocida Circulating in India by Multilocus Sequence Typing.

    Science.gov (United States)

    Sarangi, L N; Thomas, P; Gupta, S K; Kumar, S; Viswas, K N; Singh, V P

    2016-04-01

    Multilocus sequence typing (MLST), a sequence-based typing method for bacterial pathogens, is currently the best method for long-term epidemiological study and to understand the population structure of the bacteria. This investigation was carried out to study the diversity of Pasteurella multocida isolates circulating in India. Ten different sequence types (ST) identified in this study are ST 122 from cattle, goat, mithun and pig; ST 50 from pig; ST 9 from cattle and sheep; ST 229 from cattle and goat; ST 71 and ST 277 from cattle; and ST 129, ST 280, ST 281 and ST 282 from avian species. Of these, ST 277, ST 280, ST 281 and ST 282 were identified for the first time. The analysis of results provides novel epidemiological information on the circulation of multiple STs across India. The majority of STs or their variants identified in this study have already been reported from different parts of the globe. This suggests that probably transboundary spread of strains across countries and continents has occurred across evolutionary time and is still happening. The isolation of ST 122 from small ruminants and pigs suggests that these species may be included in the preventive vaccination policy for effective control of haemorrhagic septicaemia in India. © 2014 Blackwell Verlag GmbH.

  19. Molecular Typing of Staphylococcus aureus Isolated from Patients with Autosomal Dominant Hyper IgE Syndrome

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    Inka Sastalla

    2017-06-01

    Full Text Available Autosomal dominant hyper IgE syndrome (AD-HIES is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3. This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST, protein A (spa typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13 found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL, and staphylococcal enterotoxins K and Q (SEK, SEQ. Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors.

  20. Accuracy of identification of tissue types in endoscopic esophageal mucosal biopsies used for molecular biology studies.

    Science.gov (United States)

    Beck, Plauto; Mayne, George C; Astill, David; Irvine, Tanya; Watson, David I; Dijckmeester, Willem A; Wijnhoven, Bas Pl; Hussey, Damian J

    2009-01-01

    To determine if histopathologic assessment of esophageal biopsies harvested for research study is justified due to the heterogeneity of tissues in the esophagus, and the consequent histopathologic mis-matches with the clinical histopathology of biopsies taken at the same level. Since 2004, patients undergoing upper endoscopy for a variety of clinical conditions were invited to provide additional esophageal biopsies; those were collected for research purpose at the same level as biopsies collected for clinical histopathology. Research biopsies were cut in two parts: one part was submitted to research histopathology and the other stored for molecular analysis. Results of clinical histopathology for each patient were summarized per biopsy level and compared to results obtained from research biopsies at the corresponding level. A total of 377 level summaries were obtained from 137 patients. Clinical histopathology summaries classified 123 levels (32.6%) as squamous epithelium, 84 levels (22.3%) as metaplastic columnar-lined epithelium, 135 levels (35.8%) as columnar-lined epithelium with intestinal metaplasia, 30 levels (8%) as dysplasia, and 5 levels (1.3%) as adenocarcinoma. Research histopathology matched to clinical summaries on 120 of 123 (97.5%) levels for squamous epithelium, 52 of 84 (61.9%) for metaplastic columnar-lined epithelium, and 94 of 135 (69.5%) for columnar-lined epithelium with intestinal metaplasia. There were no matches for dysplasia between the groups; however, they agreed on all five cases of AC. On 59 (70.2%) metaplastic columnar-lined epithelium levels and on 62 (46%) columnar-lined epithelium with intestinal metaplasia levels, tissue heterogeneity was observed in clinical histopathology, with portions of squamous epithelium within the samples. Matches with pure tissue samples in both clinical and research histopathology levels were observed on 22 (26.2%) levels of metaplastic columnar-lined epithelium and in 55 (40.7%) levels of columnar

  1. Accuracy of identification of tissue types in endoscopic esophageal mucosal biopsies used for molecular biology studies

    Directory of Open Access Journals (Sweden)

    Plauto Beck

    2009-02-01

    Full Text Available Plauto Beck1, George C Mayne1, David Astill2, Tanya Irvine1, David I Watson1, Willem A Dijckmeester1, Bas PL Wijnhoven1, Damian J Hussey11Department of Surgery, 2Department of Anatomical Pathology, Flinders University, Flinders Medical Centre, Bedford Park, South Australia, AustraliaObjectives: To determine if histopathologic assessment of esophageal biopsies harvested for research study is justified due to the heterogeneity of tissues in the esophagus, and the consequent histopathologic mis-matches with the clinical histopathology of biopsies taken at the same level.Methods: Since 2004, patients undergoing upper endoscopy for a variety of clinical conditions were invited to provide additional esophageal biopsies; those were collected for research purpose at the same level as biopsies collected for clinical histopathology. Research biopsies were cut in two parts: one part was submitted to research histopathology and the other stored for molecular analysis. Results of clinical histopathology for each patient were summarized per biopsy level and compared to results obtained from research biopsies at the corresponding level.Results: A total of 377 level summaries were obtained from 137 patients. Clinical histopathology summaries classified 123 levels (32.6% as squamous epithelium, 84 levels (22.3% as metaplastic columnar-lined epithelium, 135 levels (35.8% as columnar-lined epithelium with intestinal metaplasia, 30 levels (8% as dysplasia, and 5 levels (1.3% as adenocarcinoma. Research histopathology matched to clinical summaries on 120 of 123 (97.5% levels for squamous epithelium, 52 of 84 (61.9% for metaplastic columnar-lined epithelium, and 94 of 135 (69.5% for columnar-lined epithelium with intestinal metaplasia. There were no matches for dysplasia between the groups; however, they agreed on all five cases of AC. On 59 (70.2% metaplastic columnar-lined epithelium levels and on 62 (46% columnar-lined epithelium with intestinal metaplasia levels

  2. Molecular typing of Lactobacillus brevis isolates from Korean food using repetitive element-polymerase chain reaction.

    Science.gov (United States)

    Kaur, Jasmine; Sharma, Anshul; Lee, Sulhee; Park, Young-Seo

    2018-01-01

    Lactobacillus brevis is a part of a large family of lactic acid bacteria that are present in cheese, sauerkraut, sourdough, silage, cow manure, feces, and the intestinal tract of humans and rats. It finds its use in food fermentation, and so is considered a "generally regarded as safe" organism. L. brevis strains are extensively used as probiotics and hence, there is a need for identifying and characterizing these strains. For identification and discrimination of the bacterial species at the subspecific level, repetitive element-polymerase chain reaction method is a reliable genomic fingerprinting tool. The objective of the present study was to characterize 13 strains of L. brevis isolated from various fermented foods using repetitive element-polymerase chain reaction. Repetitive element-polymerase chain reaction was performed using three primer sets, REP, Enterobacterial Repetitive Intergenic Consensus (ERIC), and (GTG) 5 , which produced different fingerprinting patterns that enable us to distinguish between the closely related strains. Fingerprinting patterns generated band range in between 150 and 5000 bp with REP, 200-7500 bp with ERIC, and 250-2000 bp with (GTG) 5 primers, respectively. The Jaccard's dissimilarity matrices were used to obtain dendrograms by the unweighted neighbor-joining method using genetic dissimilarities based on repetitive element-polymerase chain reaction fingerprinting data. Repetitive element-polymerase chain reaction proved to be a rapid and easy method that can produce reliable results in L. brevis species.

  3. Molecular typing of lung adenocarcinoma on cytological samples using a multigene next generation sequencing panel.

    Directory of Open Access Journals (Sweden)

    Aldo Scarpa

    Full Text Available Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%. In 24/36 cases (67% at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16% and 10/36 (28% cases, were mutually exclusive. Nine samples (25% showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.

  4. n-type doping through tethered functionality: a new paradigm for molecular design of solution-processed organic thermoelectrics

    Science.gov (United States)

    Russ, Boris; Robb, Maxwell J.; Popere, Bhooshan C.; Perry, Erin E.; Urban, Jeffrey J.; Chabinyc, Michael L.; Hawker, Craig J.; Segalman, Rachel A.

    2015-03-01

    A scarcity of stable n-type doping mechanisms compatible with facile processing has been a major impediment to the advancement of n-type (electron transporting) organic thermoelectric materials. We recently demonstrated that trimethylammonium functionalization with hydroxide counterions, tethered to a perylene diimide core by alkyl spacers, facilitated solution-processing and resulted in extremely high carrier concentrations (1020carriers/cm3) and best-in-class thermoelectric performance in thin films. In this presentation, we report our recent findings on the underlying mechanism enabling charge carrier generation in these self-doping materials and its influence on material thermoelectric behavior. To draw these conclusions, we complement thermoelectric characterization with insights into chemical, electronic, and structural properties from XPS, optical spectroscopy, EPR, and GIWAXS experiments. Furthermore, we show that doping through tethered functionality can be extended to other n-type small molecule systems of interest, including naphthalene diimides and diketopyrrolopyrroles. Our findings help shape promising molecular design strategies for future enhancements in n-type thermoelectric performance.

  5. A New Integrated Approach to Taxonomy: The Fusion of Molecular and Morphological Systematics with Type Material in Benthic Foraminifera.

    Science.gov (United States)

    Roberts, Angela; Austin, William; Evans, Katharine; Bird, Clare; Schweizer, Magali; Darling, Kate

    2016-01-01

    A robust and consistent taxonomy underpins the use of fossil material in palaeoenvironmental research and long-term assessment of biodiversity. This study presents a new integrated taxonomic protocol for benthic foraminifera by unequivocally reconciling the traditional taxonomic name to a specific genetic type. To implement this protocol, a fragment of the small subunit ribosomal RNA (SSU rRNA) gene is used in combination with 16 quantitative morphometric variables to fully characterise the benthic foraminiferal species concept of Elphidium williamsoni Haynes, 1973. A combination of live contemporary topotypic specimens, original type specimens and specimens of genetic outliers were utilised in this study. Through a series of multivariate statistical tests we illustrate that genetically characterised topotype specimens are morphologically congruent with both the holotype and paratype specimens of E. williamsoni Haynes, 1973. We present the first clear link between morphologically characterised type material and the unique SSU rRNA genetic type of E. williamsoni. This example provides a standard framework for the benthic foraminifera which bridges the current discontinuity between molecular and morphological lines of evidence, allowing integration with the traditional Linnaean roots of nomenclature to offer a new prospect for taxonomic stability.

  6. Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan.

    Science.gov (United States)

    Umehara, Azusa; Kawakami, Yasushi; Ooi, Hong-Kean; Uchida, Akihiko; Ohmae, Hiroshi; Sugiyama, Hiromu

    2010-10-15

    Anisakid nematodes are known to cause the zoonotic disease, anisakiasis, through the consumption of raw or undercooked fish. The parasites most frequently associated with the disease in humans are categorized as Anisakis type I, which comprise several species of the genus Anisakis. The larvae show primitive forms and lack the detailed morphological characteristics required for precise species identification. Thus, molecular characterization is necessary for determining the species of Anisakis type I larvae and acquiring important clinical and epidemiological information. In this study, we isolated Anisakis type I larvae from hairtail fish caught off the coasts of Taiwan and Japan. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was sequenced, and restriction fragment length polymorphism (RFLP) analyses using HinfI and HhaI was carried out for species identification. Most larvae isolated from hairtail caught in Taiwan were Anisakis typica (84%), while those isolated from hairtail caught in Japan were almost exclusively identified either as Anisakis simplex sensu stricto (65%) or Anisakis pegreffii (33%). This is the first report of A. typica in fish obtained from Taiwan. Our results shed the light on the epidemiology of Anisakis type I larvae, which is a potential cause of human anisakiasis in Taiwan and Japan. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Molecular Mechanisms of Latent Inflammation in Metabolic Syndrome. Possible Role of Sirtuins and Peroxisome Proliferator-Activated Receptor Type γ.

    Science.gov (United States)

    Stafeev, I S; Menshikov, M Y; Tsokolaeva, Z I; Shestakova, M V; Parfyonova, Ye V

    2015-10-01

    The problem of metabolic syndrome is one of the most important in medicine today. The main hazard of metabolic syndrome is development of latent inflammation in adipose tissue, which promotes atherosclerosis, non-alcoholic fatty liver disease, myocarditis, and a number of other illnesses. Therefore, understanding of molecular mechanisms of latent inflammation in adipose tissue is very important for treatment of metabolic syndrome. Three main components that arise during hypertrophy and hyperplasia of adipocytes underlie such inflammation: endoplasmic reticulum stress, oxidative stress, and hypoxia. Each of these components mediates activation in different ways of the key factor of inflammation - NF-κB. For metabolic syndrome therapy, it is suggested to influence a number of inflammatory signaling components by activating other cell factors to suppress development of inflammation. Such potential factors are peroxisome proliferator-activated receptors type γ that suppress transcription factor NF-κB through direct contact or via kinase of a NF-κB inhibitor (IKK), and also the antiinflammatory transcription factor AP-1. Other possible targets are type 3 NAD+-dependent histone deacetylases (sirtuins). There are mutually antagonistic relationships between NF-κB and sirtuin type 1 that prevent development of inflammation in metabolic syndrome. Moreover, sirtuin type 1 inhibits the antiinflammatory transcription factor AP-1. Study of the influence of these factors on the relationship between macrophages and adipocytes, macrophages, and adipose tissue-derived stromal cells can help to understand mechanisms of signaling and development of latent inflammation in metabolic syndrome.

  8. Molecular characterisation of ABC-type multidrug efflux systems in Bifidobacterium longum.

    Science.gov (United States)

    Moodley, Clinton; Reid, Sharon J; Abratt, Valerie R

    2015-04-01

    Administration of probiotic bacteria such as Bifidobacterium spp. can prevent antibiotic associated diarrhoea since they can survive the often harsh conditions of the gut. In Bifidobacterium longum subsp. longum(T) NCIMB 702259, two gene clusters, with homology to the ATP-binding cassette (ABC) family of efflux transporters, were identified and studied to assess their functional contribution to antibiotic resistance. Both gene clusters contained two genes encoding putative efflux transporters and a regulator gene, upstream of the structural genes. Reverse transcriptase analysis indicated that the genes in each cluster were transcribed as operons, one where all three genes, including a putative MarR-type regulator were transcribed together (BLLJ_1496/1495/1494), and the other where the two ABC-type transporter genes (BLLJ_1837/1836) were co-transcribed, but excluded the putative regulator (BLLJ_1838). Heterologous expression of the cloned BLLJ_1837/1836 transporter genes in Lactococcus lactis conferred resistance to erythromycin and tetracycline by increasing the minimum inhibitory concentration between 1.5 and 3 fold. The presence of these genes also allowed a 16% increase in the efflux of Hoechst 33342 from L. lactis cells containing the two transporter genes, BLLJ_1837-6. In B. longum, an increase in the levels of transcription of 3.3 fold was observed for BLLJ_1837 in the presence of erythromycin, as measured by multiplex quantitative PCR. In contrast to this, the expression of the genes of the BLLJ_1495/1494 operon in L. lactis did not show significant drug resistance functionality. Gel shift experiments showed that in the BLLJ_1495/1494 operon, the putative MarR-type regulator protein (BLLJ_1496) bound with high affinity to the DNA sequence upstream of the operon in which it was located but this was not erythromycin dependent. This study demonstrated the occurrence of a drug inducible, ABC-type transporter system (BLLJ_1837/1836) in B. longum as well as a

  9. Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

    Directory of Open Access Journals (Sweden)

    Bruna Baumgarten Krebs

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5-10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins.

  10. Molecular Typing of Turkish Apple Chlorotic Leaf Spot Virus Isolates Based on Partial Coat Protein Gene

    Directory of Open Access Journals (Sweden)

    C. Ulubas Serce

    2006-08-01

    Full Text Available Apple chlorotic leaf spot virus (ACLSV isolates from various hosts and geographic locations in Turkey were molecularly characterized by RFLP, nucleotide sequence analysis and the construction of a phylogenetic tree including ACLSV isolates from GenBank. Based on nucleotide sequence alignment and the phylogenetic tree, we proposed a classification of ACLSV isolates in which isolates were divided into three major groups. The first group contained mainly Far-Eastern isolates, the second group the Hungarian (eastern-European ACLSV isolates, and the third group, which contained isolates of variable characteristics, was again divided into two subgroups, subgroup I containing mixed European isolates, and subgroup II containing central European isolates. Three representative Turkish ACLSV isolates belonged to the third group; of these, one was from the mixed European cluster (subgroup I and two from the central European cluster (subgroup II. The nucleotide sequence divergence and geographic origin of the ACLSV isolates were correlated, which indicated the possible extraction of the Turkish isolates.

  11. 'Who's who' in two different flower types of Calluna vulgaris (Ericaceae: morphological and molecular analyses of flower organ identity

    Directory of Open Access Journals (Sweden)

    Krüger Katja

    2009-12-01

    Full Text Available Abstract Background The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated. Results Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types. In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2 were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics. Conclusions Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are

  12. Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid strain typing of Bacillus coagulans

    National Research Council Canada - National Science Library

    Jun Sato; Motokazu Nakayama; Ayumi Tomita; Takumi Sonoda; Motomitsu Hasumi; Takahisa Miyamoto

    2017-01-01

    In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high...

  13. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

    Directory of Open Access Journals (Sweden)

    Ming Feng Jiang

    2015-12-01

    Full Text Available Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type milk lysozyme gene (YML, was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75 which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

  14. Detection and molecular characterization of porcine type 3 orthoreoviruses circulating in South Korea.

    Science.gov (United States)

    Kwon, Hyung-Jun; Kim, Ha-Hyun; Kim, Hyun-Jeong; Park, Jun-Gyu; Son, Kyu-Yeol; Jung, Juyeon; Lee, Woo Song; Cho, Kyoung-Oh; Park, Su-Jin; Kang, Mun-Il

    2012-06-15

    Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0-98.1% and deduced amino acid identities of 96.4-98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198-204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Molecular Dependence of the Large Seebeck Effect in \\tau-type Organic Conductors

    OpenAIRE

    Aizawa, Hirohito; Kuroki, Kazuhiko; Yoshino, Harukazu; Mousdis, George A.; Papavassiliou, George C.; Murata, Keizo

    2014-01-01

    We study the Seebeck effect in the $\\tau$-type organic conductors, $\\tau$-(EDO-$S$,$S$-DMEDT-TTF)$_{2}$(AuBr$_{2}$)$_{1+y}$ and $\\tau$-(P-$S$,$S$-DMEDT-TTF)$_{2}$(AuBr$_{2}$)$_{1+y}$, where EDO-$S$,$S$-DMEDT-TTF and P-$S$,$S$-DMEDT-TTF are abbreviated as OOSS and NNSS, respectively, both experimentally and theoretically. Theoretically in particular, we perform first-principles band calculation for the two materials and construct a two-orbital model, on the basis of which we calculate the Seeb...