T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory

Science.gov (United States)

Monnier, Stéphanie; Cox, David G; Albion, Tim; Canzian, Federico

2005-01-01

Background Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow. Results We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. Conclusion We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS. PMID:16221298

T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory

Directory of Open Access Journals (Sweden)

Albion Tim

2005-10-01

Full Text Available Abstract Background Single Nucleotide Polymorphism (SNP genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS need tools to organize the data flow. Results We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. Conclusion We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS.

Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping

Directory of Open Access Journals (Sweden)

Bayoh Nabie M

2007-02-01

Full Text Available Abstract Background Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan™ single nucleotide polymorphism (SNP genotyping. Methods Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates. Results TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95% were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species, however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1% error rate for TaqMan genotyping in mistakenly identifying species hybrids. Conclusion TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

Directory of Open Access Journals (Sweden)

Valentina di Rienzo

Full Text Available In tomato, resistance to Tomato spotted wilt virus (TSWV is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke and summer (tomato crops, in the same cultivated areas of Southern Italy.

Characterizing the pathotype structure of barley powdery mildew and effectiveness of resistance genes to this pathogen in Kazakhstan.

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Rsaliyev, Aralbek; Pahratdinova, Zhazira; Rsaliyev, Shynbolat

2017-11-14

Powdery mildew of barley is a wind-borne and obligate biotrophic pathogen, which ranks among the most widespread barley pathogens worldwide. However, purposeful research towards studying the structure of the barley powdery mildew populations, of their virulence and of effectiveness of certain resistance genes against the infection was not conducted in Kazakhstan till present time. This paper is the first to describe characteristics of the pathotype structure of Blumeria graminis f.sp. hordei (Bgh) population and effectiveness of resistance genes in two regions of barley cultivation in the republic. One hundred and seven isolates of Bgh were obtained from seven populations occurring on cultivated barley at two geographically locations in Kazakhstan during 2015 and 2016. Their virulence frequency was determined on 17 differential lines Pallas. All isolates were virulent on the resistance gene Mla8 and avirulent for the resistance genes Mla9, Mla1 + MlaAl2, Mla6 + Mla14, Mla13 + MlRu3, Mla7 + MlNo3, Mla10 + MlDu2, Mla13 + MlRu3 and Mlo-5. The frequencies of isolates overcoming the genes Mla3, Mla22, Mlat Mlg + MlCP and Mla12 + MlEm2 were 0.0-33.33%, and frequencies of isolates overcoming the genes Mlra, Mlk, MlLa and Mlh ranged from 10.0 to 78.6%. Based on reactions of differential lines possessing the genes Mla22, Mlra, Mlk, Mlat, MlLa and Mlh, pathotypes were identified. In total, 23 pathotypes with virulence complexity ranging from 1 to 6 were identified. During both years in all populations of South Kazakhstan and Zhambyl regions pathotypes 24 and 64 mainly prevailed. Obtained data suggest that low similarity of populations Bgh in Kazakhstan to European, African, Australian and South-East Asian populations. The present study provides a foundation for future studies on the pathogenic variability within of Bgh populations in Kazakhstan and addresses the knowledge gap on the virulence structure of Bgh in Central Asia. Complete effectiveness of the

A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

Science.gov (United States)

Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-30

Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

Gene-gene interactions of IRF5, STAT4, IKZF1 and ETS1 in systemic lupus erythematosus.

Science.gov (United States)

Dang, J; Shan, S; Li, J; Zhao, H; Xin, Q; Liu, Y; Bian, X; Liu, Q

2014-06-01

Interferon (IFN) activation signaling and T helper 17 (Th17)-cell/B-cell regulation play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Several studies have provided convincing evidence that polymorphisms in IRF5, STAT4, IKZF1 and ETS1 from these pathways may be involved in SLE by affecting gene expression or epistasis. We analyzed the genetic interaction in known SLE susceptibility loci from the four genes in northern Han Chinese. A total of 946 northern Han Chinese participated in this study (370 unrelated SLE patients and 576 healthy controls). Subjects underwent genotyping for the single-nucleotide polymorphisms (SNPs) rs2004640 in IRF5, rs7574865 in STAT4, rs4917014 in IKZF1 and rs1128334 in ETS1 by use of a TaqMan SNP genotyping assay and direct sequencing. Gene-gene interaction analysis involved direct counting, multifactor dimensionality reduction (MDR) and linear regression analysis. SLE patients and controls differed in allele frequencies of rs7574865, rs1128334 (P < 0.001) and rs4917014 (P < 0.01). Direct counting revealed that the frequency of risk homozygote combinations was higher for SLE patients than controls (P < 0.01). Furthermore, 2-, 3- and 4-way gene-gene epistasis in SLE was confirmed by parametric methods and MDR analysis. Gene expression analysis partially supported the findings. Our study confirmed the association of the IFN pathway or Th17/B-cells and the pathogenesis of SLE, and gene-gene interaction in this pathway may increase the risk of SLE. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

Science.gov (United States)

Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

2016-01-01

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

Investigations into the biodegradation of microcystin-LR in wastewaters

International Nuclear Information System (INIS)

Ho, Lionel; Hoefel, Daniel; Palazot, Sebastien; Sawade, Emma; Newcombe, Gayle; Saint, Christopher P.; Brookes, Justin D.

2010-01-01

Microcystins are potent hepatotoxins that can be produced by cyanobacteria. These organisms can proliferate in wastewaters due to a number of factors including high concentrations of nutrients for growth. As treated wastewaters are now being considered as supplementary drinking water sources, in addition to their frequent use for irrigated agriculture, it is imperative that these wastewaters are free of toxins such as microcystins. This study investigated the potential for biodegradation of microcystin-LR (MCLR) in wastewaters through a biological sand filtration experiment and in static batch reactor experiments. MCLR was effectively removed at a range of concentrations and at various temperatures, with degradation attributed to the action of microorganisms indigenous to the wastewaters. No hepatotoxic by-products were detected following the degradation of MCLR as determined by a protein phosphatase inhibition assay. Using TaqMan polymerase chain reaction, the first gene involved in bacterial degradation of MCLR (mlrA) was detected and the responsible bacteria shown to increase with the amount of MCLR being degraded. This finding suggested that the degradation of MCLR was dependent upon the abundance of MCLR-degrading organisms present within the wastewater, and that MCLR may provide bacteria with a significant carbon source for proliferation; in turn increasing MCLR removal.

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

Science.gov (United States)

Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

2010-06-01

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

Genotyping of Hepatitis C virus isolated from hepatitis patients in Southeast of Iran by taqman realtime PCR

International Nuclear Information System (INIS)

Farivar, T.N.; Johari, P.

2011-01-01

Objectives: To check TaqMan Realtime PCR in detecting genotypes of hepatitis C virus in Iran. Methods: From July 2007 to April 2009, HCV genotyping was done on 52 patients who were referred to Research Centre for infectious Disease and Tropical Medicine, in Bou-Ali Hospital, Zahedan University of Medical Sciences. All these patients had proven hepatitis C infection. Results: Out of 52 anti HCV positive samples, 28(53.84%) had genotype 1, 2 cases (3.88 %) had genotype 2 , 12 (23.08 %) had genotype 3 and 7 (13.4 %) had genotype 4 . Mixed infection with genotypes 1 and 3 was seen in 3 cases (5.77 %). Conclusion: TaqMan probes for detecting genotyping of HCV were successful in picking genotyping of HCV infection especially those with mixed genotypes. (author)

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Science.gov (United States)

Dinoop, K P; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R P; Narayanan, P

2016-01-01

Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( Pnested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control

Science.gov (United States)

Lindenberg, Sandra; Klauck, Gisela; Pesavento, Christina; Klauck, Eberhard; Hengge, Regine

2013-01-01

C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling. PMID:23708798

Performance evaluation of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, version 2.0, for detection and quantification of hepatitis C virus RNA

NARCIS (Netherlands)

S.D. Pas (Suzan); R. Molenkamp (Richard); J. Schinkel (Janke); S. Rebers; C. Copra (Cederick); S. Seven-Deniz; D. Thamke (Diana); R.J. de Knegt (Robert); B.L. Haagmans (Bart); M. Schutten (Martin)

2013-01-01

textabstractTo evaluate the analytical performance and explore the clinical applicability of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, v2.0 (CAP/CTM v2.0), a platform comparison was performed on panels and diagnostic samples with the Roche cobas AmpliPrep/cobas TaqMan HCV test (CAP/CTM

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

Science.gov (United States)

Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

2011-01-01

Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping

Directory of Open Access Journals (Sweden)

Amanda K Riffel

2016-01-01

Full Text Available TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false positive CYP2D6*15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6*15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6*35 which is also located in exon 1. Although alternative CYP2D6*15 and *35 assays resolved the issue, we discovered a novel CYP2D6*15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6*15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696 SNP of CYP2D6*43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe

Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds.

Science.gov (United States)

Carneiro, Greice Amaral; Matić, Slavica; Ortu, Giuseppe; Garibaldi, Angelo; Spadaro, Davide; Gullino, Maria Lodovica

2017-07-01

Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by TaqMan PCR and in peripheral blood lymphocytes by competitive PCR.

Science.gov (United States)

Wadowsky, Robert M; Laus, Stella; Green, Michael; Webber, Steven A; Rowe, David

2003-11-01

Epstein-Barr virus (EBV) DNA load values were measured in samples of whole blood (n = 60) and plasma (n = 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n = 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r(2) > 0.900), whereas the plasma and PBL loads correlated poorly (r(2) = 0.512). Testing of whole blood by TaqMan PCR is an acceptable alternative to testing of PBLs by cPCR for quantifying EBV DNA load.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

OpenAIRE

Romain Barbet; Isabelle Peiffer; Antoinette Hatzfeld; Pierre Charbord; Jacques A. Hatzfeld

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs ...

Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

Science.gov (United States)

Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

2013-07-01

Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR "Double Check" Strategy

DEFF Research Database (Denmark)

Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.

2010-01-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus...... sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany......), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome...

A copula method for modeling directional dependence of genes

Directory of Open Access Journals (Sweden)

Park Changyi

2008-05-01

Full Text Available Abstract Background Genes interact with each other as basic building blocks of life, forming a complicated network. The relationship between groups of genes with different functions can be represented as gene networks. With the deposition of huge microarray data sets in public domains, study on gene networking is now possible. In recent years, there has been an increasing interest in the reconstruction of gene networks from gene expression data. Recent work includes linear models, Boolean network models, and Bayesian networks. Among them, Bayesian networks seem to be the most effective in constructing gene networks. A major problem with the Bayesian network approach is the excessive computational time. This problem is due to the interactive feature of the method that requires large search space. Since fitting a model by using the copulas does not require iterations, elicitation of the priors, and complicated calculations of posterior distributions, the need for reference to extensive search spaces can be eliminated leading to manageable computational affords. Bayesian network approach produces a discretely expression of conditional probabilities. Discreteness of the characteristics is not required in the copula approach which involves use of uniform representation of the continuous random variables. Our method is able to overcome the limitation of Bayesian network method for gene-gene interaction, i.e. information loss due to binary transformation. Results We analyzed the gene interactions for two gene data sets (one group is eight histone genes and the other group is 19 genes which include DNA polymerases, DNA helicase, type B cyclin genes, DNA primases, radiation sensitive genes, repaire related genes, replication protein A encoding gene, DNA replication initiation factor, securin gene, nucleosome assembly factor, and a subunit of the cohesin complex by adopting a measure of directional dependence based on a copula function. We have compared

Development of TaqMan probes targeting the four major celiac disease epitopes found in α-gliadin sequences of spelt (Triticum aestivum ssp. spelta) and bread wheat (Triticum aestivum ssp. aestivum).

Science.gov (United States)

Dubois, Benjamin; Bertin, Pierre; Muhovski, Yordan; Escarnot, Emmanuelle; Mingeot, Dominique

2017-01-01

Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat ( Triticum aestivum ssp. aestivum ) and spelt ( T. aestivum ssp. spelta ). Among them, the α-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed α-gliadins of spelt and bread wheat. Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed α-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed α-gliadins. The results obtained in this study provide useful tools to study the immunogenic potential of expressed α-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the

Suitable reference genes for the analysis of direct hyperplasia in mice

International Nuclear Information System (INIS)

Takagi, Soichi; Ohashi, Kazuo; Utoh, Rie; Tatsumi, Kohei; Shima, Midori; Okano, Teruo

2008-01-01

The liver is capable of undergoing a proliferative growth, known as direct hyperplasia, in which the naive liver increases in size due to stimulation with primary mitogens. To produce accurate gene expression data, housekeeping genes (HKGs) that are stably expressed need to be determined. In the present study, liver regeneration was promoted via the direct hyperplasia mode by inducing mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Gene expression levels of nine commonly used HKGs were analyzed in the liver of different timing during the regeneration. The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, we identified that PPIA and RPL4 showed the most stable expression regardless of the status of the liver regeneration. In conclusion, the present study demonstrated that the use of PPIA and RPL4 were the most optimal in providing reliable normalization of gene expression when assessing liver regeneration attributed to direct hyperplasia.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

Science.gov (United States)

Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

2013-12-27

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

Science.gov (United States)

Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

2015-12-01

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

Circumvention of normal constraints on granule protein gene expression in peripheral blood neutrophils and monocytes of patients with antineutrophil cytoplasmic autoantibody-associated glomerulonephritis.

Science.gov (United States)

Yang, Jia Jin; Pendergraft, William F; Alcorta, David A; Nachman, Patrick H; Hogan, Susan L; Thomas, Robin P; Sullivan, Pamela; Jennette, J Charles; Falk, Ronald J; Preston, Gloria A

2004-08-01

Granulopoiesis-related genes are distinctively upregulated in peripheral leukocytes of patients with antineutrophil cytoplasmic autoantibodies (ANCA)-associated glomerulonephritis. Affymetrix microarrays identified the upregulation of nine neutrophilic primary granule genes, including myeloperoxidase (MPO) and proteinase 3 (PR3), plus five secondary granule genes. Coordinate expression of granulocyte maturation marker CD35, measured by TaqMan PCR, and positive in situ staining for PR3 transcripts in polymorphic neutrophils and monocytes indicate that these genes are expressed in "mature" cells. Increased transcripts correlated with disease activity and absolute neutrophil values but not with "left shift," drug regimen, cytokine levels, hematuria, proteinuria, ANCA titer, serum creatinine, gender, or age. Upregulation of PR3 and MPO transcripts was specifically associated with ANCA disease (n = 56) as these changes were not detected in patients with ESRD (n = 25) or systemic lupus erythematosus (n = 17), as determined by TaqMan PCR. This is the first report of this phenomenon in nonneoplastic cells. The data raise the hypothesis that, in addition to the presence of anti-MPO or anti-PR3 autoantibodies, a second critical component in the cause of this disease is the reactivation of once-silenced genes leading to increased antigen availability.

RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature

Science.gov (United States)

Viollet, Coralie; Davis, David A.; Tekeste, Shewit S.; Reczko, Martin; Pezzella, Francesco; Ragoussis, Jiannis

2017-01-01

Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases. PMID:28046107

Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of Burkholderia pseudomallei and Burkholderia mallei

OpenAIRE

U'Ren, Jana M.; Van Ert, Matthew N.; Schupp, James M.; Easterday, W. Ryan; Simonson, Tatum S.; Okinaka, Richard T.; Pearson, Talima; Keim, Paul

2005-01-01

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

TaqMan探针荧光定量PCR检测花生油中掺入棕榈油的研究%Determination of palm oil adulterated in peanut oil with the TaqMan probe -based RT- PCR method

Institute of Scientific and Technical Information of China (English)

周慧; 梁宇斌; 吴苏喜; 李晓明; 裴伟; 杨涛

2011-01-01

The method of Real - time fluorescence quantitative polymerase chain reaction ( RT - PCR) with TaqMan fluorescent probe was chosen to fast detect the amount of palm oil mixed in peanut oil. MT3 - B gene of palm was selected as target gene to detect palm oil from peanut oil. The primers of MT3 - B and TaqMan probe were designed, MT3 - B gene reconstructed plasmid was built as absolute quantitative criteria for quantitative RT - PCR to establish standard curve. Peanut oil blended with 1% -40% concentration gradient palm oil was extracted DNA to test palm content by RT - PCR. The result showed that the correlation coefficient (R1) of standard curve with logarithmic linear regression analysis was 0.996. When the adulteration of palm oil in peanut oil reached 5% volume, MT3 - B gene of 17.431 copies per milli-liter of mixed oil could be detected . The method showed good sensitivity, specificity and repeatability.%根据棕榈内源基因MT3 -B设计引物和TaqMan探针,采用基因重组技术构建用于检测棕榈基因MT3 -B的重组质粒作为绝对定量标准品,建立标准曲线,对花生油中掺入棕榈油1% ～40%梯度混合油品提取DNA进行棕榈成分定量检测.结果表明,重组质粒标准品荧光定量标准曲线对数线性回归分析相关系数(R2)为0.996;花生油中掺入棕榈油达到5%时,可检出每亳升混合油品中棕榈MT3 -B基因17.431 copies,检测的重复性和特异性好.

Isolation, Identification and Phenotypic Characterization of Microcystin-Degrading Bacteria from Lake Erie

Science.gov (United States)

Krishnan, A.; Mou, X. J.

2015-12-01

Lake Erie, the smallest and warmest lake among the Laurentian Great Lakes, is known for its problem of eutrophication and frequent occurrence of harmful cyanobacterial blooms (CyanoHABs). One major harmful effect of CyanoHABs is the production of cyanotoxins, especially microcystins. Microcystins (MC) are a group of hepatotoxins and the predominant variant of them is MC-LR. Field measurements and lab experiments indicate that MC degradation in Lake Erie is mainly carried out by indigenous bacteria. However, our knowledge on taxa involved in this process is very limited. This study aimed to fill this knowledge gap using a culture-dependent approach. Water and surface sediment samples were collected from Lake Erie in 2014 and 2015 and enriched with MC-LR. Cells were plated on a number of culturing media. The obtained pure bacterial cultures were screened for MC degrading abilities by MT2 BIO-LOG assays and by growing cells in liquid media containing MC-LR as the sole carbon source. In the latter experiment, MC concentrations were measured using HPLC. Isolates showing positive MC degradation activities in the screening steps were designated MC+ bacteria and characterized based on their phenotypic properties, including colony pigmentation, elevation, opacity, margin, gram nature and motility. The taxonomic identity of MC+ bacteria was determined by 16S rRNA gene full-length DNA sequencing. The presence of mlrA, a gene encoding MC cleavage pathway, was detected by PCR. Our culturing efforts obtained 520 pure cultures; 44 of them were identified as MC+. These MC+ isolates showed diversity in taxonomic identities and differed in their morphology, gram nature, colony characteristics and motility. PCR amplification of mlrA gene yield negative results for all MC+ isolates, indicating that the primers that were used may not be ubiquitous enough to cover the heterogeneity of mlrA genes or, more likely, alternative degradative genes/pathways were employed by Lake Erie bacteria

Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

Science.gov (United States)

Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

Bone marker gene expression in calvarial bones: different bone microenvironments.

Science.gov (United States)

Al-Amer, Osama

2017-12-01

In calvarial mice, mesenchymal stem cells (MSCs) differentiate into osteoprogenitor cells and then differentiate into osteoblasts that differentiate into osteocytes, which become embedded within the bone matrix. In this case, the cells participating in bone formation include MSCs, osteoprogenitor cells, osteoblasts and osteocytes. The calvariae of C57BL/KaLwRijHsD mice consist of the following five bones: two frontal bones, two parietal bones and one interparietal bone. This study aimed to analyse some bone marker genes and bone related genes to determine whether these calvarial bones have different bone microenvironments. C57BL/KaLwRijHsD calvariae were carefully excised from five male mice that were 4-6 weeks of age. Frontal, parietal, and interparietal bones were dissected to determine the bone microenvironment in calvariae. Haematoxylin and eosin staining was used to determine the morphology of different calvarial bones under microscopy. TaqMan was used to analyse the relative expression of Runx2, OC, OSX, RANK, RANKL, OPG, N-cadherin, E-cadherin, FGF2 and FGFR1 genes in different parts of the calvariae. Histological analysis demonstrated different bone marrow (BM) areas between the different parts of the calvariae. The data show that parietal bones have the smallest BM area compared to frontal and interparietal bones. TaqMan data show a significant increase in the expression level of Runx2, OC, OSX, RANKL, OPG, FGF2 and FGFR1 genes in the parietal bones compared with the frontal and interparietal bones of calvariae. This study provides evidence that different calvarial bones, frontal, parietal and interparietal, contain different bone microenvironments.

Association of DPP4 Gene Polymorphisms with Type 2 Diabetes Mellitus in Malaysian Subjects

OpenAIRE

Ahmed, Radwan H.; Huri, Hasniza Zaman; Al-Hamodi, Zaid; Salem, Sameer D.; Al-absi, Boshra; Muniandy, Sekaran

2016-01-01

Background Genetic polymorphisms of the Dipeptidyl Peptidase 4 (DPP4) gene may play a role in the etiology of type 2 diabetes mellitus (T2DM). This study aimed to investigate the possible association of single nucleotide polymorphisms (SNPs) of the DPP4 gene in Malaysian subjects with T2DM and evaluated whether they had an effect on the serum levels of soluble dipeptidyl peptidase 4 (sDPP-IV). Method Ten DPP4 SNPs were genotyped by TaqMan genotyping assays in 314 subjects with T2DM and 235 co...

Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

OpenAIRE

Hristova, Krassimira R.; Lutenegger, Christian M.; Scow, Kate M.

2001-01-01

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribos...

Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

DEFF Research Database (Denmark)

Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan

2004-01-01

an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially...... in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.......The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate...

Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction.

Directory of Open Access Journals (Sweden)

Jeni Vuong

Full Text Available Neisseria meningitidis (Nm, Haemophilus influenzae (Hi, and Streptococcus pneumoniae (Sp are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.

Integrating high dimensional bi-directional parsing models for gene mention tagging.

Science.gov (United States)

Hsu, Chun-Nan; Chang, Yu-Ming; Kuo, Cheng-Ju; Lin, Yu-Shi; Huang, Han-Shen; Chung, I-Fang

2008-07-01

Tagging gene and gene product mentions in scientific text is an important initial step of literature mining. In this article, we describe in detail our gene mention tagger participated in BioCreative 2 challenge and analyze what contributes to its good performance. Our tagger is based on the conditional random fields model (CRF), the most prevailing method for the gene mention tagging task in BioCreative 2. Our tagger is interesting because it accomplished the highest F-scores among CRF-based methods and second over all. Moreover, we obtained our results by mostly applying open source packages, making it easy to duplicate our results. We first describe in detail how we developed our CRF-based tagger. We designed a very high dimensional feature set that includes most of information that may be relevant. We trained bi-directional CRF models with the same set of features, one applies forward parsing and the other backward, and integrated two models based on the output scores and dictionary filtering. One of the most prominent factors that contributes to the good performance of our tagger is the integration of an additional backward parsing model. However, from the definition of CRF, it appears that a CRF model is symmetric and bi-directional parsing models will produce the same results. We show that due to different feature settings, a CRF model can be asymmetric and the feature setting for our tagger in BioCreative 2 not only produces different results but also gives backward parsing models slight but constant advantage over forward parsing model. To fully explore the potential of integrating bi-directional parsing models, we applied different asymmetric feature settings to generate many bi-directional parsing models and integrate them based on the output scores. Experimental results show that this integrated model can achieve even higher F-score solely based on the training corpus for gene mention tagging. Data sets, programs and an on-line service of our gene

A 7 gene expression score predicts for radiation response in cancer cervix

International Nuclear Information System (INIS)

Rajkumar, Thangarajan; Vijayalakshmi, Neelakantan; Sabitha, Kesavan; Shirley, Sundersingh; Selvaluxmy, Ganesharaja; Bose, Mayil Vahanan; Nambaru, Lavanya

2009-01-01

Cervical cancer is the most common cancer among Indian women. The current recommendations are to treat the stage IIB, IIIA, IIIB and IVA with radical radiotherapy and weekly cisplatin based chemotherapy. However, Radiotherapy alone can help cure more than 60% of stage IIB and up to 40% of stage IIIB patients. Archival RNA samples from 15 patients who had achieved complete remission and stayed disease free for more than 36 months (No Evidence of Disease or NED group) and 10 patients who had failed radical radiotherapy (Failed group) were included in the study. The RNA were amplified, labelled and hybridized to Stanford microarray chips and analyzed using BRB Array Tools software and Significance Analysis of Microarray (SAM) analysis. 20 genes were selected for further validation using Relative Quantitation (RQ) Taqman assay in a Taqman Low-Density Array (TLDA) format. The RQ value was calculated, using each of the NED sample once as a calibrator. A scoring system was developed based on the RQ value for the genes. Using a seven gene based scoring system, it was possible to distinguish between the tumours which were likely to respond to the radiotherapy and those likely to fail. The mean score ± 2 SE (standard error of mean) was used and at a cut-off score of greater than 5.60, the sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) were 0.64, 1.0, 1.0, 0.67, respectively, for the low risk group. We have identified a 7 gene signature which could help identify patients with cervical cancer who can be treated with radiotherapy alone. However, this needs to be validated in a larger patient population

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC)

International Nuclear Information System (INIS)

Glaysher, Sharon; Modi, Paul; Rahamim, Joe; Smith, Mark E; Amer, Khalid; Addis, Bruce; Poole, Matthew; Narayanan, Ajit; Gulliford, Tim J; Andreotti, Peter E; Cree, Ian A; Yiannakis, Dennis; Gabriel, Francis G; Johnson, Penny; Polak, Marta E; Knight, Louise A; Goldthorpe, Zoe; Peregrin, Katharine; Gyi, Mya

2009-01-01

NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array™ following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue. There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer

Performance evaluation of the Cobas TaqMan MTB assay on respiratory specimens according to clinical application

Directory of Open Access Journals (Sweden)

Jong Eun Park

2017-11-01

Full Text Available Objective: To evaluate the performance of the Cobas TaqMan MTB assay (Cobas assay with respect to its clinical application. Methods: This was a retrospective analysis of 1154 results from 1034 patients for whom mycobacterial cultures and the Cobas assay were performed simultaneously. Based on the patient medical records, two categories of clinical application were defined: (1 the diagnosis of patients with a high probability of pulmonary tuberculosis according to clinical and radiological features (n = 128, and (2 the exclusion of tuberculosis in clinically indeterminate patients (n = 1026. Standard culture was used as the reference method. Results: The sensitivity of the Cobas assay for the detection of Mycobacterium tuberculosis was 70.4% (95% confidence interval (CI 49.7–85.5% for category 1, but only 25.0% (95% CI 4.5–64.4% for category 2. The specificity was ≥95.0% for both categories. The positive predictive value was 79.2% (95% CI 57.3–92.1% for category 1 and 33.3% (95% CI 6.0–75.9% for category 2, while the negative predictive value was 92.3% (95% CI 85.0–96.4% for category 1 and 99.4% (95% CI 98.7–99.8% for category 2. Conclusions: The results of this study indicate that Cobas assay results must be interpreted carefully according to the clinical purpose of the assay. Keywords: Mycobacterium tuberculosis, Pulmonary tuberculosis, Cobas TaqMan MTB assay, Korea

Effects of Transcranial Direct Current Stimulation on Expression of Immediate Early Genes (IEG’s)

Science.gov (United States)

2015-12-01

TRANSCRANIAL DIRECT CURRENT STIMULATION OF EXPRESSION OF IMMEDIATE EARLY GENES (IEG’S) Jessica...AND SUBTITLE Effects of Transcranial Direct Current Stimulation on Expression of Immediate Early Genes (IEG’s) 5a. CONTRACT NUMBER In-House 5b...community in better understanding what is occurring biologically during tDCS. 15. SUBJECT TERMS Transcranial direct current stimulation

STAT4 gene polymorphism in patients after renal allograft transplantation.

Science.gov (United States)

Dąbrowska-Żamojcin, Ewa; Dziedziejko, Violetta; Safranow, Krzysztof; Domański, Leszek; Słuczanowska-Głabowska, Sylwia; Pawlik, Andrzej

2016-01-01

STAT4 (signal transducer and activator of transcription 4) is involved in the regulation of innate and adaptive immune responses. Some studies have suggested that STAT4 may be involved in the immune response after graft transplantation. Several polymorphisms in the STAT4 gene have been identified. The most commonly studied polymorphism in the STAT4 gene is rs7574865. In our study, we examined whether this polymorphism is associated with the early and late functions of renal allografts. A total of 270 recipients of first renal transplants were included in the study. Single nucleotide polymorphisms (SNPs) within the STAT4 gene were genotyped using TaqMan genotyping assays. There were no statistically significant associations between the STAT4 gene rs7574865 polymorphism and delayed graft function, acute rejection, chronic allograft dysfunction, post-transplant diabetes mellitus, or creatinine serum concentrations after transplantation. Our results suggest a lack of association between the STAT4 rs7574865 SNP and kidney allograft function in the Polish population.

Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

Science.gov (United States)

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

Association of MBL2 Gene Polymorphism with Dental Caries in Saudi Children.

Science.gov (United States)

Alyousef, Yousef M; Borgio, J Francis; AbdulAzeez, Sayed; Al-Masoud, Naif; Al-Ali, Ali A; Al-Shwaimi, Emad; Al-Ali, Amein K

2017-01-01

The high prevalence of dental caries in children worldwide is a major oral health problem which requires early intervention. Dental caries is mainly caused by the action of acids produced by bacteria in addition to many other factors. Recent genetic studies have reported that a number of genes are associated with the susceptibility to dental caries. The majority of these genes are associated with inflammation, increased susceptibility to infection, and dentine matrix formation. Using the TaqMan assay and direct DNA sequencing, the prevalence of 6 single-nucleotide polymorphisms (SNPs) in MMP9, MBL2, MMP2, and TIMP2 genes was determined in 102 children with caries and in 100 age-matched caries-free controls. Out of the 6 SNPs tested in the 4 selected genes, only rs11003125 in the MBL2 gene was shown to be associated with a high prevalence of caries in our cohort. In addition, haplotype analysis of the 6 SNPs tested revealed that certain haplotypes, namely GT of rs11003125G and rs7501477T and GT of rs7096206G and rs7501477T, were found to be associated with a high prevalence of dental caries in our cohort, while haplotype AG of rs17576A and rs7501477G was found to have a protective effect against dental caries. In conclusion, the data indicate that rs11003125 in the MBL2 gene was shown to be associated with a high prevalence of caries in our cohort, and 2 haplotypes are also involved in the increased susceptibility to dental caries. © 2016 S. Karger AG, Basel.

Strategies used for genetically modifying bacterial genome: ite-directed mutagenesis, gene inactivation, and gene over-expression*

Science.gov (United States)

Xu, Jian-zhong; Zhang, Wei-guo

2016-01-01

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators. PMID:26834010

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC

Directory of Open Access Journals (Sweden)

Amer Khalid

2009-08-01

Full Text Available Abstract Background NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. Methods The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array™ following extraction of RNA from formalin-fixed paraffin-embedded (FFPE tissue. Results There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. Conclusion Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer.

A multianalytical approach to evaluate the association of 55 SNPs in 28 genes with obesity risk in North Indian adults.

Science.gov (United States)

Srivastava, Apurva; Mittal, Balraj; Prakash, Jai; Srivastava, Pranjal; Srivastava, Nimisha; Srivastava, Neena

2017-03-01

The aim of the study was to investigate the association of 55 SNPs in 28 genes with obesity risk in a North Indian population using a multianalytical approach. Overall, 480 subjects from the North Indian population were studied using strict inclusion/exclusion criteria. SNP Genotyping was carried out by Sequenom Mass ARRAY platform (Sequenom, San Diego, CA) and validated Taqman ® allelic discrimination (Applied Biosystems ® ). Statistical analyses were performed using SPSS software version 19.0, SNPStats, GMDR software (version 6) and GENEMANIA. Logistic regression analysis of 55 SNPs revealed significant associations (P obesity risk whereas the remaining 6 SNPs revealed no association (P > .05). The pathway-wise G-score revealed the significant role (P = .0001) of food intake-energy expenditure pathway genes. In CART analysis, the combined genotypes of FTO rs9939609 and TCF7L2 rs7903146 revealed the highest risk for BMI linked obesity. The analysis of the FTO-IRX3 locus revealed high LD and high order gene-gene interactions for BMI linked obesity. The interaction network of all of the associated genes in the present study generated by GENEMANIA revealed direct and indirect connections. In addition, the analysis with centralized obesity revealed that none of the SNPs except for FTO rs17818902 were significantly associated (P obesity risk in the North Indian population. © 2016 Wiley Periodicals, Inc.

Bystander or No Bystander for Gene Directed Enzyme Prodrug Therapy

Directory of Open Access Journals (Sweden)

Adam V. Patterson

2009-11-01

Full Text Available Gene directed enzyme prodrug therapy (GDEPT of cancer aims to improve the selectivity of chemotherapy by gene transfer, thus enabling target cells to convert nontoxic prodrugs to cytotoxic drugs. A zone of cell kill around gene-modified cells due to transfer of toxic metabolites, known as the bystander effect, leads to tumour regression. Here we discuss the implications of either striving for a strong bystander effect to overcome poor gene transfer, or avoiding the bystander effect to reduce potential systemic effects, with the aid of three successful GDEPT systems. This review concentrates on bystander effects and drug development with regard to these enzyme prodrug combinations, namely herpes simplex virus thymidine kinase (HSV-TK with ganciclovir (GCV, cytosine deaminase (CD from bacteria or yeast with 5-fluorocytodine (5-FC, and bacterial nitroreductase (NfsB with 5-(azaridin-1-yl-2,4-dinitrobenzamide (CB1954, and their respective derivatives.

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

Science.gov (United States)

Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B

2016-01-01

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV. PMID:26909355

Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.

Science.gov (United States)

Saha, Ratul; Donofrio, Robert S; Bagley, Susan T

2010-08-01

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system.

Science.gov (United States)

Baoutina, A; Coldham, T; Bains, G S; Emslie, K R

2010-08-01

As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.

Enriching the gene set analysis of genome-wide data by incorporating directionality of gene expression and combining statistical hypotheses and methods

Science.gov (United States)

Väremo, Leif; Nielsen, Jens; Nookaew, Intawat

2013-01-01

Gene set analysis (GSA) is used to elucidate genome-wide data, in particular transcriptome data. A multitude of methods have been proposed for this step of the analysis, and many of them have been compared and evaluated. Unfortunately, there is no consolidated opinion regarding what methods should be preferred, and the variety of available GSA software and implementations pose a difficulty for the end-user who wants to try out different methods. To address this, we have developed the R package Piano that collects a range of GSA methods into the same system, for the benefit of the end-user. Further on we refine the GSA workflow by using modifications of the gene-level statistics. This enables us to divide the resulting gene set P-values into three classes, describing different aspects of gene expression directionality at gene set level. We use our fully implemented workflow to investigate the impact of the individual components of GSA by using microarray and RNA-seq data. The results show that the evaluated methods are globally similar and the major separation correlates well with our defined directionality classes. As a consequence of this, we suggest to use a consensus scoring approach, based on multiple GSA runs. In combination with the directionality classes, this constitutes a more thorough basis for an enriched biological interpretation. PMID:23444143

Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

Science.gov (United States)

Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng

2017-06-01

A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developing a System for Directed Gene Introduction into Mammary Gland Via Targeted Infection of Retrovirus Receptor Transgenics

National Research Council Canada - National Science Library

Bates, Paul

1998-01-01

... (the Rous sarcoma virus receptor). Directed infection, and thus directed gene expression of cells expressing the viral receptor should provide a rapid and efficient method to test the mammary tumorigenic potential of genes in an animal model...

A TaqMan real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil

DEFF Research Database (Denmark)

Sapkota, Rumakanta; Skantar, Andrea M.; Nicolaisen, Mogens

2016-01-01

. haplaand showed no significant amplification of DNA from non-target nematodes. The assay was able to detect M. haplain a background of plant and soil DNA. A dilution series of M. haplaeggs in soil showed a high correlation ( R 2 = 0 . 95 , P ...Early detection and quantification of Meloidogyne haplain soil is essential for effective disease management. The purpose of this study was to develop a real-time PCR assay for detection of M. haplain soil. Primers and a TaqMan probe were designed for M. hapladetection. The assay detected M......-knot development in carrots by testing soils before planting. The assay could be useful for management decisions in carrot cultivation....

Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

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Adeline Bidault

2015-12-01

Full Text Available The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD in the Manila clam Venerupis (=Ruditapes philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW or extrapallial fluids (EF samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

Directory of Open Access Journals (Sweden)

Allison M Lytle

2016-01-01

Full Text Available Immune responses to coagulation factors VIII (FVIII and IX (FIX represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

Directory of Open Access Journals (Sweden)

Takao Ito

Full Text Available Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

OpenAIRE

Uozumi, N; Sakurai, K; Sasaki, T; Takekawa, S; Yamagata, H; Tsukagoshi, N; Udaka, S

1989-01-01

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other a...

Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

Directory of Open Access Journals (Sweden)

Hua-Ying Fu

2016-01-01

Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

Results of the Abbott RealTime HIV-1 assay for specimens yielding "target not detected" results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test.

Science.gov (United States)

Babady, N Esther; Germer, Jeffrey J; Yao, Joseph D C

2010-03-01

No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding "target not detected" results by CTM.

Adaptation of the short intergenic spacers between co-directional genes to the Shine-Dalgarno motif among prokaryote genomes

DEFF Research Database (Denmark)

Caro, Albert Pallejà; García-Vallvé, Santiago; Romeu, Antoni

2009-01-01

ABSTRACT: BACKGROUND: In prokaryote genomes most of the co-directional genes are in close proximity. Even the coding sequence or the stop codon of a gene can overlap with the Shine-Dalgarno (SD) sequence of the downstream co-directional gene. In this paper we analyze how the presence of SD may...... influence the stop codon usage or the spacing lengths between co-directional genes. RESULTS: The SD sequences for 530 prokaryote genomes have been predicted using computer calculations of the base-pairing free energy between translation initiation regions and the 16S rRNA 3' tail. Genomes with a large...... to the discussion of which factors affect the intergenic lengths, which cannot be totally explained by the pressure to compact the prokaryote genomes....

Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

Directory of Open Access Journals (Sweden)

López-Barragán María J

2011-11-01

Full Text Available Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

The potential of TaqMan Array Cards for detection of multiple biological agents by real-time PCR.

Directory of Open Access Journals (Sweden)

Phillip A Rachwal

Full Text Available The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels.

A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

Directory of Open Access Journals (Sweden)

Symonds Meegan L

2002-07-01

Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations.

Directory of Open Access Journals (Sweden)

Dawn N Birdsell

Full Text Available Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis, therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays

Results of the Abbott RealTime HIV-1 Assay for Specimens Yielding “Target Not Detected” Results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test▿

OpenAIRE

Babady, N. Esther; Germer, Jeffrey J.; Yao, Joseph D. C.

2009-01-01

No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding “target not detected” results by CTM.

Gene Expression Changes in Femoral Head Necrosis of Human Bone Tissue

Directory of Open Access Journals (Sweden)

Bernadett Balla

2011-01-01

Full Text Available Osteonecrosis of the femoral head (ONFH is the result of an interruption of the local circulation and the injury of vascular supply of bone. Multiple factors have been implicated in the development of the disease. However the mechanism of ischemia and necrosis in non-traumatic ONFH is not clear. The aim of our investigation was to identify genes that are differently expressed in ONFH vs. non-ONFH human bone and to describe the relationships between these genes using multivariate data analysis. Six bone tissue samples from ONFH male patients and 8 bone tissue samples from non-ONFH men were examined. The expression differences of selected 117 genes were analyzed by TaqMan probe-based quantitative real-time RT-PCR system. The significance test indicated marked differences in the expression of nine genes between ONFH and non-ONFH individuals. These altered genes code for collagen molecules, an extracellular matrix digesting metalloproteinase, a transcription factor, an adhesion molecule, and a growth factor. Canonical variates analysis demonstrated that ONFH and non-ONFH bone tissues can be distinguished by the multiple expression profile analysis of numerous genes controlled via canonical TGFB pathway as well as genes coding for extracellular matrix composing collagen type molecules. The markedly altered gene expression profile observed in the ONFH of human bone tissue may provide further insight into the pathogenetic process of osteonecrotic degeneration of bone.

Development of a rapid method for direct detection of tet(M) genes in soil from Danish farmland

DEFF Research Database (Denmark)

Agersø, Yvonne; Sengeløv, Gitte; Jensen, Lars Bogø

2004-01-01

. The tet(M) gene was directly detected in 10-80% of the samples from the various farmland soils and could be detected in all samples tested after selective enrichment. To validate the obtained results, the method was applied to garden soil samples where lower prevalence of resistance was found. Result......A method for direct detection of antibiotic resistance genes in soil samples has been developed. The tetracycline resistance gene, tet(M), was used as a model. The method was validated on Danish farmland soil that had repeatedly been treated with pig manure slurry containing resistant bacteria......: A detection limit of 10(2)-10(3) copies of the tet(M) gene per gram of soil (in a Bacillus cereus group bacterium) was achieved. tet(M) gene was detected in soil samples with the highest prevalence on farmland treated with pig manure slurry....

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide

Directory of Open Access Journals (Sweden)

Yuexia Wang

2015-09-01

Full Text Available Real-time polymerase chain reaction (PCR allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 103 CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 100 CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

Science.gov (United States)

Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

2015-09-01

Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10 3  CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10 0  CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

A novel BDNF gene promoter directs expression to skeletal muscle

Directory of Open Access Journals (Sweden)

Heinrich Gerhard

2003-06-01

Full Text Available Abstract Background Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. Results The cloning and analysis of three additional zebrafish (Danio rerio BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. Conclusion The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are

An enhanced topologically significant directed random walk in cancer classification using gene expression datasets

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Choon Sen Seah

2017-12-01

Full Text Available Microarray technology has become one of the elementary tools for researchers to study the genome of organisms. As the complexity and heterogeneity of cancer is being increasingly appreciated through genomic analysis, cancerous classification is an emerging important trend. Significant directed random walk is proposed as one of the cancerous classification approach which have higher sensitivity of risk gene prediction and higher accuracy of cancer classification. In this paper, the methodology and material used for the experiment are presented. Tuning parameter selection method and weight as parameter are applied in proposed approach. Gene expression dataset is used as the input datasets while pathway dataset is used to build a directed graph, as reference datasets, to complete the bias process in random walk approach. In addition, we demonstrate that our approach can improve sensitive predictions with higher accuracy and biological meaningful classification result. Comparison result takes place between significant directed random walk and directed random walk to show the improvement in term of sensitivity of prediction and accuracy of cancer classification.

Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

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Everett, Karin D. E.; Hornung, Linda J.; Andersen, Arthur A.

1999-01-01

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation. PMID:9986815

Direct and long-term detection of gene doping in conventional blood samples.

Science.gov (United States)

Beiter, T; Zimmermann, M; Fragasso, A; Hudemann, J; Niess, A M; Bitzer, M; Lauer, U M; Simon, P

2011-03-01

The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as 'gene doping'. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular adeno-associated virus-mediated gene transfer, one of the most likely candidate vector systems to be misused for gene doping. To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions.

Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus.

Science.gov (United States)

Liu, Jie; Ochieng, Caroline; Wiersma, Steve; Ströher, Ute; Towner, Jonathan S; Whitmer, Shannon; Nichol, Stuart T; Moore, Christopher C; Kersh, Gilbert J; Kato, Cecilia; Sexton, Christopher; Petersen, Jeannine; Massung, Robert; Hercik, Christine; Crump, John A; Kibiki, Gibson; Maro, Athanasia; Mujaga, Buliga; Gratz, Jean; Jacob, Shevin T; Banura, Patrick; Scheld, W Michael; Juma, Bonventure; Onyango, Clayton O; Montgomery, Joel M; Houpt, Eric; Fields, Barry

2016-01-01

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

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Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

2017-07-01

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order.

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Bilodeau, Guillaume J; Martin, Frank N; Coffey, Michael D; Blomquist, Cheryl L

2014-07-01

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic

Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains

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Mir A Hossain

2016-01-01

Full Text Available Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes

DEFF Research Database (Denmark)

Schepeler, Troels; Holm, Anja; Halvey, P

2012-01-01

Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array......RNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes....... MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy...

Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting genotoxins

Energy Technology Data Exchange (ETDEWEB)

Hu Ting; Gibson, David P.; Carr, Gregory J.; Torontali, Suzanne M.; Tiesman, Jay P.; Chaney, Joel G.; Aardema, Marilyn J

2004-05-18

During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK{sup +/-} mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa.

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Seong-Bin Kim

Full Text Available Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega. To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Gene expression changes as markers of early lapatinib response in a panel of breast cancer cell lines

LENUS (Irish Health Repository)

O’Neill, Fiona

2012-06-18

AbstractBackgroundLapatinib, a tyrosine kinase inhibitor of HER2 and EGFR and is approved, in combination with capecitabine, for the treatment of trastuzumab-refractory metastatic breast cancer. In order to establish a possible gene expression response to lapatinib, a panel of breast cancer cell lines with varying sensitivity to lapatinib were analysed using a combination of microarray and qPCR profiling.MethodsCo-inertia analysis (CIA), a data integration technique, was used to identify transcription factors associated with the lapatinib response on a previously published dataset of 96 microarrays. RNA was extracted from BT474, SKBR3, EFM192A, HCC1954, MDAMB453 and MDAMB231 breast cancer cell lines displaying a range of lapatinib sensitivities and HER2 expression treated with 1 μM of lapatinib for 12 hours and quantified using Taqman RT-PCR. A fold change ≥ ± 2 was considered significant.ResultsA list of 421 differentially-expressed genes and 8 transcription factors (TFs) whose potential regulatory impact was inferred in silico, were identified as associated with lapatinib response. From this group, a panel of 27 genes (including the 8 TFs) were selected for qPCR validation. 5 genes were determined to be significantly differentially expressed following the 12 hr treatment of 1 μM lapatinib across all six cell lines. Furthermore, the expression of 4 of these genes (RB1CC1, FOXO3A, NR3C1 and ERBB3) was directly correlated with the degree of sensitivity of the cell line to lapatinib and their expression was observed to “switch” from up-regulated to down-regulated when the cell lines were arranged in a lapatinib-sensitive to insensitive order. These included the novel lapatinib response-associated genes RB1CC1 and NR3C1. Additionally, Cyclin D1 (CCND1), a common regulator of the other four proteins, was also demonstrated to observe a proportional response to lapatinib exposure.ConclusionsA panel of 5 genes were determined to be differentially

Plasticity-Related Gene Expression During Eszopiclone-Induced Sleep.

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Gerashchenko, Dmitry; Pasumarthi, Ravi K; Kilduff, Thomas S

2017-07-01

Experimental evidence suggests that restorative processes depend on synaptic plasticity changes in the brain during sleep. We used the expression of plasticity-related genes to assess synaptic plasticity changes during drug-induced sleep. We first characterized sleep induced by eszopiclone in mice during baseline conditions and during the recovery from sleep deprivation. We then compared the expression of 18 genes and two miRNAs critically involved in synaptic plasticity in these mice. Gene expression was assessed in the cerebral cortex and hippocampus by the TaqMan reverse transcription polymerase chain reaction and correlated with sleep parameters. Eszopiclone reduced the latency to nonrapid eye movement (NREM) sleep and increased NREM sleep amounts. Eszopiclone had no effect on slow wave activity (SWA) during baseline conditions but reduced the SWA increase during recovery sleep (RS) after sleep deprivation. Gene expression analyses revealed three distinct patterns: (1) four genes had higher expression either in the cortex or hippocampus in the group of mice with increased amounts of wakefulness; (2) a large proportion of plasticity-related genes (7 out of 18 genes) had higher expression during RS in the cortex but not in the hippocampus; and (3) six genes and the two miRNAs showed no significant changes across conditions. Even at a relatively high dose (20 mg/kg), eszopiclone did not reduce the expression of plasticity-related genes during RS period in the cortex. These results indicate that gene expression associated with synaptic plasticity occurs in the cortex in the presence of a hypnotic medication. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Detection of growth hormone doping by gene expression profiling of peripheral blood.

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Mitchell, Christopher J; Nelson, Anne E; Cowley, Mark J; Kaplan, Warren; Stone, Glenn; Sutton, Selina K; Lau, Amie; Lee, Carol M Y; Ho, Ken K Y

2009-12-01

GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

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Chae, H S; Park, G N; Kim, S H; Jo, H J; Kim, J T; Jeoung, H Y; An, D J; Kim, N H; Shin, B W; Kang, Y I; Chang, K S

2012-08-01

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

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Kamau Edwin

2012-01-01

Full Text Available Abstract Background Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods. Methods TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD for each assay. Results Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples. Conclusion TaqMan Allelic Discrimination assay provides a good alternative tool in

Circadian clock gene aryl hydrocarbon receptor nuclear translocator-like polymorphisms are associated with seasonal affective disorder: An Indian family study.

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Rajendran, Bhagya; Janakarajan, Veeramahali Natarajan

2016-01-01

Polymorphisms in aryl hydrocarbon receptor nuclear translocator-like (ARNTL) gene, the key component of circadian clock manifests circadian rhythm abnormalities. As seasonal affective disorder (SAD) is associated with disrupted circadian rhythms, the main objective of this study was to screen an Indian family with SAD for ARNTL gene polymorphisms. In this study, 30 members of close-knit family with SAD, 30 age- and sex-matched controls of the same caste with no prior history of psychiatric illness and 30 age- and sex-matched controls belonging to 17 different castes with no prior history of psychiatric illness were genotyped for five different single nucleotide polymorphisms (SNPs) in ARNTL gene by TaqMan allele-specific genotyping assay. Statistical significance was assessed by more powerful quasi-likelihood score test-XM. Most of the family members carried the risk alleles and we observed a highly significant SNP rs2279287 (A/G) in ARNTL gene with an allelic frequency of 0.75. Polymorphisms in ARNTL gene disrupt circadian rhythms causing SAD and genetic predisposition becomes more deleterious in the presence of adverse environment.

Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis

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Ki Young Yoo

2010-03-01

Full Text Available Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs and conformation sensitive gel electrophoresis (CSGE to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%, whereas for multiplex PCR- CSGE screened sequencing, the mutations could be detected in 23 (85.2%. One patient’s mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

KAUST Repository

Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

2015-01-01

validating this direct cloning plug-and-playa approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation

Pretransplant Immune- and Apoptosis-Related Gene Expression Is Associated with Kidney Allograft Function

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Dorota Kamińska

2016-01-01

Full Text Available Renal transplant candidates present immune dysregulation, caused by chronic uremia. The aim of the study was to investigate whether pretransplant peripheral blood gene expression of immune factors affects clinical outcome of renal allograft recipients. Methods. In a prospective study, we analyzed pretransplant peripheral blood gene expression in87 renal transplant candidates with real-time PCR on custom-designed low density arrays (TaqMan. Results. Immediate posttransplant graft function (14-day GFR was influenced negatively by TGFB1 (P=0.039 and positively by IL-2 gene expression (P=0.040. Pretransplant blood mRNA expression of apoptosis-related genes (CASP3, FAS, and IL-18 and Th1-derived cytokine gene IFNG correlated positively with short- (6-month GFR CASP3: P=0.027, FAS: P=0.021, and IFNG: P=0.029 and long-term graft function (24-month GFR CASP3: P=0.003, FAS: P=0.033, IL-18: P=0.044, and IFNG: P=0.04. Conclusion. Lowered pretransplant Th1-derived cytokine and apoptosis-related gene expressions were a hallmark of subsequent worse kidney function but not of acute rejection rate. The pretransplant IFNG and CASP3 and FAS and IL-18 genes’ expression in the recipients’ peripheral blood is the possible candidate for novel biomarker of short- and long-term allograft function.

Identification of hookworm DAF-16/FOXO response elements and direct gene targets.

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Xin Gao

2010-08-01

Full Text Available The infective stage of the parasitic nematode hookworm is developmentally arrested in the environment and needs to infect a specific host to complete its life cycle. The canine hookworm (Ancylostoma caninum is an excellent model for investigating human hookworm infections. The transcription factor of A. caninum, Ac-DAF-16, which has a characteristic fork head or "winged helix" DNA binding domain (DBD, has been implicated in the resumption of hookworm development in the host. However, the precise roles of Ac-DAF-16 in hookworm parasitism and its downstream targets are unknown. In the present study, we combined molecular techniques and bioinformatics to identify a group of Ac-DAF-16 binding sites and target genes.The DNA binding domain of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DAF-16 family binding element (DBE and possible alternative Ac-DAF-16 bind motifs. The 22 genes linked to these genomic fragments were identified using bioinformatics tools and defined as candidate direct gene targets of Ac-DAF-16. Their developmental stage-specific expression patterns were examined. Also, a new putative DAF-16 binding element was identified.Our results show that Ac-DAF-16 is involved in diverse biological processes throughout hookworm development. Further investigation of these target genes will provide insights into the molecular basis by which Ac-DAF-16 regulates its downstream gene network in hookworm infection.

Direct Repression of Evening Genes by CIRCADIAN CLOCK-ASSOCIATED1 in the Arabidopsis Circadian Clock.

Science.gov (United States)

Kamioka, Mari; Takao, Saori; Suzuki, Takamasa; Taki, Kyomi; Higashiyama, Tetsuya; Kinoshita, Toshinori; Nakamichi, Norihito

2016-03-01

The circadian clock is a biological timekeeping system that provides organisms with the ability to adapt to day-night cycles. Timing of the expression of four members of the Arabidopsis thaliana PSEUDO-RESPONSE REGULATOR(PRR) family is crucial for proper clock function, and transcriptional control of PRRs remains incompletely defined. Here, we demonstrate that direct regulation of PRR5 by CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) determines the repression state of PRR5 in the morning. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses indicated that CCA1 associates with three separate regions upstream of PRR5 CCA1 and its homolog LATE ELONGATED HYPOCOTYL (LHY) suppressed PRR5 promoter activity in a transient assay. The regions bound by CCA1 in the PRR5 promoter gave rhythmic patterns with troughs in the morning, when CCA1 and LHY are at high levels. Furthermore,ChIP-seq revealed that CCA1 associates with at least 449 loci with 863 adjacent genes. Importantly, this gene set contains genes that are repressed but upregulated incca1 lhy double mutants in the morning. This study shows that direct binding by CCA1 in the morning provides strong repression of PRR5, and repression by CCA1 also temporally regulates an evening-expressed gene set that includes PRR5. © 2016 American Society of Plant Biologists. All rights reserved.

Sendai viroplexes for epidermal growth factor receptor-directed delivery of interleukin-12 and salmosin genes to cancer cells.

Science.gov (United States)

Kim, Jung Seok; Kim, Min Woo; Jeong, Hwa Yeon; Kang, Seong Jae; Park, Sang Il; Lee, Yeon Kyung; Kim, Hong Sung; Kim, Keun Sik; Park, Yong Serk

2016-07-01

The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor-directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off-target effects of gene transfection. The system consists of a well-verified cationic O,O'-dimyristyl-N-lysyl glutamate (DMKE), Sendai virus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein, referred to as cationic Sendai F/HN virosomes. To achieve tumor-specific recognition, anti-epidermal growth factor (EGF) receptor antibody was coupled to the surface of the virosomes containing interleukin-12 (IL-12) and/or salmosin genes that have potent anti-angiogenetic functions. Among the virosomal formulations, the anti-EGF receptor (EGFR) viroplexes, prepared via complexation of plasmid DNA (pDNA) with cationic DMKE lipid, exhibited more efficient gene transfection to tumor cells over-expressing EGF receptors compared to the neutrally-charged anti-EGFR virosomes encapsulating pDNA. In addition, the anti-EGFR viroplexes with IL-12 and salmosin genes exhibited the most effective therapeutic efficacy in a mouse tumor model. Especially when combined with doxorubicin, transfection of the two genes via the anti-EGFR viroplexes exhibited an enhanced inhibitory effect on tumor growth and metastasis in lungs. The results of the present study suggest that anti-EGFR viroplexes can be utilized as an effective strategy for tumor-directed gene delivery. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Prevalence of IFNL3 gene polymorphism among blood donors and its relation to genomic profile of ancestry in Brazil.

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Rizzo, Silvia Renata Cornelio Parolin; Gazito, Diana; Pott-Junior, Henrique; Latini, Flavia Roche Moreira; Castelo, Adauto

The recent development of interferon-free regimens based on direct-acting antivirals for the treatment of chronic hepatitis C virus infection has benefited many but not all patients. Some patients still experience treatment failure, possibly attributed to unknown host and viral factors, such as IFNL3 gene polymorphism. The present study assessed the prevalence of rs12979860-CC, rs12979860-CT, and rs12979860-TT genotypes of the IFNL3 gene, and its relationship with ancestry informative markers in 949 adult Brazilian healthy blood donors. Race was analyzed using ancestry informative markers as a surrogate for ancestry. IFNL3 gene was genotyped using the ABI TaqMan single nucleotide polymorphisms genotyping assays. The overall frequency of rs12979860-CC genotype was 36.9%. The contribution of African ancestry was significantly higher among donors from the northeast region in relation to southeast donors, whereas the influence of European ancestry was significantly higher in southeast donors. Donors with rs12979860-CC and rs12979860-CT genotypes had similar ancestry background. The contribution of African ancestry was higher among rs12979860-TT genotype donors in comparison to both rs12979860-CC and rs12979860-CT genotypes. The prevalence of rs12979860-CC genotype is similar to that found in the US, despite the Brazilian ancestry informative markers admixture. However, in terms of ancestry, rs12979860-CT genotype was much closer to rs12979860-CC individuals than to rs12979860-TT. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Gene expression and gene therapy imaging

International Nuclear Information System (INIS)

Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

2007-01-01

The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice

Science.gov (United States)

Zhang, Jingxu; Lu, Zuomei; Dai, Weimin; Song, Xiaoling; Peng, Yufa; Valverde, Bernal E.; Qiang, Sheng

2015-01-01

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China. PMID:26012494

Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice.

Science.gov (United States)

Zhang, Jingxu; Lu, Zuomei; Dai, Weimin; Song, Xiaoling; Peng, Yufa; Valverde, Bernal E; Qiang, Sheng

2015-05-27

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.

Direct detection of Leishmania from clinical samples.

Science.gov (United States)

Waitumbi, John N; Bast, Joshua; Nyakoe, Nancy; Magiri, Charles; Quintana, Miguel; Takhampunya, Ratree; Schuster, Anthony L; Van de Wyngaerde, Marshall T; McAvin, James C; Coleman, Russell E

2017-01-01

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

Evaluation of candidate genes associated with hepatitis A and E virus infection in Chinese Han population.

Science.gov (United States)

Gu, Maolin; Qiu, Jing; Guo, Daoxia; Xu, Yunfang; Liu, Xingxiang; Shen, Chong; Dong, Chen

2018-03-20

Recent GWAS-associated studies reported that single nucleotide polymorphisms (SNPs) in ABCB1, TGFβ1, XRCC1 genes were associated with hepatitis A virus (HAV) infection, and variants of APOA4 and APOE genes were associated with and hepatitis E virus (HEV) infection in US population. However, the associations of these loci with HAV or HEV infection in Chinese Han population remain unclear. A total of 3082 Chinese Han persons were included in this study. Anti-HAV IgG and anti-HEV IgG were detected by enzyme-linked immunosorbent assay (ELISA). Genotypes in ABCB1, TGFβ1, XRCC1, APOA4 and APOE SNPs were determined by TaqMan MGB technology. In Chinese Han population, rs1045642 C to T variation in ABCB1 was significantly associated with the decreased risk of HAV infection (P infection in our samples (P C to T variation in APOE was significantly associated with lower risk of HEV infection in males (adjusted OR infection. Additionally, Chinese Han males with rs7412 C to T variation in APOE gene are less prone to be infected by HEV.

Association of gene polymorphism of the fat mass and obesity associated gene with metabolic syndrome: a retrospective cohort study in Japanese workers.

Science.gov (United States)

Kawajiri, Tomoka; Osaki, Yoneatsu; Kishimoto, Takuji

2012-06-01

To investigate whether gene polymorphism of the fat mass and obesity associated gene (FTO) is associated with metabolic syndrome (MS), we used two MS criteria, the National Cholesterol Education Program-Adult Treatment panel III (NCEP-ATPIII) definition in 2003 and the Japanese definition in 2005. Subjects were respectively 859 and 865 Japanese workers at a company in Shimane Prefecture, Japan. They were non-MS individuals in 1998 and had regular health checkups between 1998 and 2006. The Cox proportional hazard regression was used to predict MS. Three SNPs in the FTO, rs9939609, rs1121980 and rs1558902, were genotyped by the TaqMan PCR assay and a retrospective study was performed. The three SNPs in the FTO were significantly associated with body mass index, and rs1121980 and rs1558902 were associated with fasting plasma glucose. MS defined by the NCEP-ATPIII definition was significantly associated with additive and dominant models of rs9939609 and rs1121980, and the dominant model of rs1558902, even after adjusting for confounding factors such as age, sex and lifestyle. MS defined by the Japanese definition was significantly associated with the additive model of rs1121980 and additive and dominant models of rs1558902 in multivariate analysis. These results suggested that FTO gene polymorphisms, rs9939609, rs1121980 and rs1558902, were associated with an increased risk of MS among Japanese workers.

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes.

Science.gov (United States)

Kang, Zhen; Zhang, Junli; Jin, Peng; Yang, Sen

2015-01-01

Owing to our limited understanding of the relationship between sequence and function and the interaction between intracellular pathways and regulatory systems, the rational design of enzyme-coding genes and de novo assembly of a brand-new artificial genome for a desired functionality or phenotype are difficult to achieve. As an alternative approach, directed evolution has been widely used to engineer genomes and enzyme-coding genes. In particular, significant developments toward DNA synthesis, DNA assembly (in vitro or in vivo), recombination-mediated genetic engineering, and high-throughput screening techniques in the field of synthetic biology have been matured and widely adopted, enabling rapid semi-rational genome engineering to generate variants with desired properties. In this commentary, these novel tools and their corresponding applications in the directed evolution of genomes and enzymes are discussed. Moreover, the strategies for genome engineering and rapid in vitro enzyme evolution are also proposed.

Do GnRH analogues directly affect human endometrial epithelial cell gene expression?

KAUST Repository

Zhang, Xiaomei

2010-03-04

We examined whether Gonadotrophin-releasing hormone (GnRH) analogues [leuprolide acetate (LA) and ganirelix acetate (GA)] modulate gene expression in Ishikawa cells used as surrogate for human endometrial epithelial cells in vitro. The specific aims were: (i) to study the modulatory effect of GnRH analogues by RT-PCR [in the absence and presence of E2 and P4, and cyclic adenosine monophos-phate (cAMP)] on mRNA expression of genes modulated during the window of implantation in GnRH analogues/rFSH-treated assisted reproductive technology cycles including OPTINEURIN (OPTN), CHROMATIN MODIFYING PROTEIN (CHMP1A), PROSAPOSIN (PSAP), IGFBP-5 and SORTING NEXIN 7 (SNX7), and (ii) to analyze the 5\\'-flanking regions of such genes for the presence of putative steroid-response elements [estrogen-response elements (EREs) and P4-response element (PREs)]. Ishikawa cells were cytokeratin+/vimentin2 and expressed ERa,ERb, PR and GnRH-R proteins. At 6 and 24 h, neither LA nor GA alone had an effect on gene expression. GnRH analogues alone or following E2 and/or P4 co-incubation for 24 h also had no effect on gene expression, but P4 significantly increased expression of CHMP1A.E2 + P4 treatment for 4 days, alone or followed by GA, had no effect, but E2 + P4 treatment followed by LA significantly decreased IGFBP-5 expression. The addition of 8-Br cAMP did not modify gene expression, with the exception of IGFBP-5 that was significantly increased. The GnRH analogues did not modify intracellular cAMP levels. We identified conserved EREs for OPN, CHMP1A, SNX7 and PSAP and PREs for SNX7. We conclude that GnRH analogues appear not to have major direct effects on gene expression of human endo-metrial epithelial cells in vitro. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

Science.gov (United States)

Wang, Li-Ping; Lei, Kun

2016-12-01

Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

Comparison of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test v1.0 with v2.0 in HIV-1 viral load quantification

Directory of Open Access Journals (Sweden)

Yi-Ching Tung

2015-04-01

Full Text Available Roche modified the COBAS AmpliPrep/COBAS TaqMan human immunodeficiency virus type 1 (HIV-1 test version 1.0 (CAP/CTM v1.0, resulting in the COBAS AmpliPrep/COBAS TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0. The aim of this study was to evaluate the performance of the CAP/CTM v2.0 and to compare this performance with that of the CAP/CTM v1.0. The study was conducted in a small local study group in Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. A total of 86 plasma samples from HIV-1-seropositive patients were tested using the two assays. The correlation and concordance of results between the two assays were calculated. The CAP/CTM v2.0 generated higher values than did the CAP/CTM v1.0, and five samples (5.8% yielded a difference of > 1 log10 copies/mL. In addition, our data show that CAP/CTM v1.0 and CAP/CTM v2.0 yielded relatively consistent values for 23 samples with low viral loads (< 200 copies/mL. Furthermore, when viral loads were in a medium range (2–5 log10 copies/mL, the results of the two assays were more compatible. This study shows a good correlation between CAP/CTM v1.0 and v2.0 in HIV-1 viral load measurement. Further attention must be paid to those cases in which measured viral loads present larger differences between the two assays.

Frequent down-regulation of ABC transporter genes in prostate cancer.

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Demidenko, Rita; Razanauskas, Deividas; Daniunaite, Kristina; Lazutka, Juozas Rimantas; Jankevicius, Feliksas; Jarmalaite, Sonata

2015-10-12

ATP-binding cassette (ABC) transporters are transmembrane proteins responsible for the efflux of a wide variety of substrates, including steroid metabolites, through the cellular membranes. For better characterization of the role of ABC transporters in prostate cancer (PCa) development, the profile of ABC transporter gene expression was analyzed in PCa and noncancerous prostate tissues (NPT). TaqMan Low Density Array (TLDA) human ABC transporter plates were used for the gene expression profiling in 10 PCa and 6 NPT specimens. ABCB1 transcript level was evaluated in a larger set of PCa cases (N = 78) and NPT (N = 15) by real-time PCR, the same PCa cases were assessed for the gene promoter hypermethylation by methylation-specific PCR. Expression of eight ABC transporter genes (ABCA8, ABCB1, ABCC6, ABCC9, ABCC10, ABCD2, ABCG2, and ABCG4) was significantly down-regulated in PCa as compared to NPT, and only two genes (ABCC4 and ABCG1) were up-regulated. Down-regulation of ABC transporter genes was prevalent in the TMPRSS2-ERG-negative cases. A detailed analysis of ABCB1 expression confirmed TLDA results: a reduced level of the transcript was identified in PCa in comparison to NPT (p = 0.048). Moreover, the TMPRSS2-ERG-negative PCa cases showed significantly lower expression of ABCB1 in comparison to NPT (p = 0.003) or the fusion-positive tumors (p = 0.002). Promoter methylation of ABCB1 predominantly occurred in PCa and was rarely detected in NPT (p ABC transporter genes in PCa, especially in the TMPRSS2-ERG-negative tumors.

Nonassociation of homocysteine gene polymorphisms with treatment outcome in South Indian Tamil Rheumatoid Arthritis patients.

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Muralidharan, Niveditha; Gulati, Reena; Misra, Durga Prasanna; Negi, Vir S

2018-02-01

The aim of the study was to look for any association of MTR 2756A>G and MTRR 66A>G gene polymorphisms with clinical phenotype, methotrexate (MTX) treatment response, and MTX-induced adverse events in South Indian Tamil patients with rheumatoid arthritis (RA). A total of 335 patients with RA were investigated. MTR 2756A>G gene polymorphism was analyzed by PCR-RFLP, and MTRR 66A>G SNP was analyzed by TaqMan 5' nuclease assay. The allele frequencies were compared with HapMap groups. MTR 2756G allele was found to be associated with risk of developing RA. The allele frequencies of MTR 2756A>G and MTRR 66A>G SNPs in controls differed significantly when compared with HapMap groups. Neither of the SNPs influenced the MTX treatment outcome and adverse effects. Neither of the SNPs seems to be associated with MTX treatment outcome and adverse events in South Indian Tamil patients with RA.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

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Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

2015-01-01

Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [RNA-Seq

NARCIS (Netherlands)

Muiño, J.M.; Bruijn, de S.A.; Vingron, Martin; Angenent, G.C.; Kaufmann, Kerstin

2015-01-01

Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the

Distinct actions of ancestral vinclozolin and juvenile stress on neural gene expression in the male rat

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Ross eGillette

2015-03-01

Full Text Available Exposure to the endocrine disrupting chemical vinclozolin during gestation of an F0 generation and/or chronic restraint stress during adolescence of the F3 descendants affects behavior, physiology, and gene expression in the brain. Genes related to the networks of growth factors, signaling peptides and receptors, steroid hormone receptors and enzymes, and epigenetic related factors were measured using quantitative polymerase chain reaction via Taqman low density arrays targeting 48 genes in the central amygdaloid nucleus, medial amygdaloid nucleus, medial preoptic area, lateral hypothalamus, and the ventromedial nucleus of the hypothalamus. We found that growth factors are particularly vulnerable to ancestral exposure in the central and medial amygdala; restraint stress during adolescence affected neural growth factors in the medial amygdala. Signaling peptides were affected by both ancestral exposure and stress during adolescence primarily in hypothalamic nuclei. Steroid hormone receptors and enzymes were strongly affected by restraint stress in the medial preoptic area. Epigenetic related genes were affected by stress in the ventromedial hypothalamus and by both ancestral exposure and stress during adolescence independently in the central amygdala. It is noteworthy that the lateral hypothalamus showed no effects of either manipulation. Gene expression is discussed in the context of behavioral and physiological measures previously published.

Distinct actions of ancestral vinclozolin and juvenile stress on neural gene expression in the male rat.

Science.gov (United States)

Gillette, Ross; Miller-Crews, Isaac; Skinner, Michael K; Crews, David

2015-01-01

Exposure to the endocrine disrupting chemical vinclozolin during gestation of an F0 generation and/or chronic restraint stress during adolescence of the F3 descendants affects behavior, physiology, and gene expression in the brain. Genes related to the networks of growth factors, signaling peptides, and receptors, steroid hormone receptors and enzymes, and epigenetic related factors were measured using quantitative polymerase chain reaction via Taqman low density arrays targeting 48 genes in the central amygdaloid nucleus, medial amygdaloid nucleus, medial preoptic area (mPOA), lateral hypothalamus (LH), and the ventromedial nucleus of the hypothalamus. We found that growth factors are particularly vulnerable to ancestral exposure in the central and medial amygdala; restraint stress during adolescence affected neural growth factors in the medial amygdala. Signaling peptides were affected by both ancestral exposure and stress during adolescence primarily in hypothalamic nuclei. Steroid hormone receptors and enzymes were strongly affected by restraint stress in the mPOA. Epigenetic related genes were affected by stress in the ventromedial nucleus and by both ancestral exposure and stress during adolescence independently in the central amygdala. It is noteworthy that the LH showed no effects of either manipulation. Gene expression is discussed in the context of behavioral and physiological measures previously published.

Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

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Hai Zhang

2011-07-01

Full Text Available Hypoxia-inducible factor (HIF is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation

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Zhang, Hai; Zhang, Guo; Gonzalez, Frank J.; Park, Sung-min; Cai, Dongsheng

2011-01-01

Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance. PMID:21814490

PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

Science.gov (United States)

Wimmer, Katharina; Wernstedt, Annekatrin

2014-01-01

The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

Associated factors and prevalence of erectile dysfunction in hemodialysis patients

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Marcio Rodrigues Costa

2014-01-01

Full Text Available Purpose: The proposal of this study was to determine the prevalence and the associated factors of erectile dysfunction (ED among hemodialysis (HD patients. Materials and Methods: This was a cross-sectional study based on data collected from HD male patients. Clinical, demographic and laboratory data of all patients were collected in three HD clinics from December 2010 to June 2011. Patients answered questions of erectile function domain from International Index of Erectile Function. Data were evaluated by descriptive analysis and by univariate (ULRA and multivariate logistic regression analysis (MLRA. Results: Three hundred and five patients participated of the study. The prevalence of ED was 68.19%. ED was associated with diabetes (DM, benign prostatic hyperplasia, glomerulonephritis as cause of chronic renal failure (CRF, smoking habits, lower creatinine levels (ULRA, use of calcium channel blocker (MLRA, aging, lower education level, alcohol consumption, DM (as cause of CRF and coronary insufficiency (ULRA and MLRA. Conclusions: ED was highly prevalent in the HD men. It was independently associated with aging, current use of alcohol, long alcohol use (even for those who do not drink more, lower education level, diabetes as cause of CRF, coronary insufficiency and use of channel blockers calcium.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

KAUST Repository

Li, Yongxin

2015-03-24

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning plug-and-playa approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

Science.gov (United States)

Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

2015-03-01

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

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Tsutomu Motohashi

2016-03-01

Full Text Available Neural crest cells (NC cells are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+ cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

Study of association of CD40-CD154 gene polymorphisms with disease susceptibility and cardiovascular risk in Spanish rheumatoid arthritis patients.

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Mercedes García-Bermúdez

Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease associated with increased cardiovascular (CV mortality. Since CD40-CD154 binding has direct consequences on inflammation process initiation, we aimed to replicate previous findings related to disease susceptibility in Spanish RA population. Furthermore, as the major complication in RA disease patients is the development of CV events due to accelerated atherosclerosis, and elevated levels of CD40L/CD154 are present in patients with acute myocardial infarction, we assessed the potential association of CD40 and CD154/CD40L gene variants with CV risk in Spanish RA patients.One thousand five hundred and seventy-five patients fulfilling the 1987 ACR classification criteria for RA and 1600 matched controls were genotyped for the CD40 rs1883832, rs4810485 and rs1535045 and CD154 rs3092952 and rs3092920 gene polymorphisms, using predesigned TaqMan single nucleotide polymorphism genotyping assays. Afterwards, we investigated the influence of CD40-CD154 gene variants in the development of CV events. Also, in a subgroup of 273 patients without history of CV events, we assessed the influence of these polymorphisms in the risk of subclinical atherosclerosis determined by carotid ultrasonography.Nominally significant differences in the allele frequencies for the rs1883832 CD40 gene polymorphism between RA patients and controls were found (p=0.038. Although we did not observe a significant association of CD40-CD154 gene variants with the development of CV events, an ANCOVA model adjusted for sex, age at the time of the ultrasonography assessment, follow-up time, traditional CV risk factors and anti-cyclic citrullinated peptide antibodies disclosed a significant association (p=0.0047 between CD40 rs1535045 polymorphism and carotid intima media thickness, a surrogate marker of atherosclerosis.Data from our pilot study indicate a potential association of rs1883832 CD40 gene polymorphism with susceptibility

Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [ChIP-Seq

NARCIS (Netherlands)

Muiño, J.M.; Bruijn, de S.A.; Vingron, Martin; Angenent, G.C.; Kaufmann, K.

2015-01-01

Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the

Decreased expression level of BER genes in Alzheimer's disease patients is not derivative of their DNA methylation status.

Science.gov (United States)

Sliwinska, Agnieszka; Sitarek, Przemysław; Toma, Monika; Czarny, Piotr; Synowiec, Ewelina; Krupa, Renata; Wigner, Paulina; Bialek, Katarzyna; Kwiatkowski, Dominik; Korycinska, Anna; Majsterek, Ireneusz; Szemraj, Janusz; Galecki, Piotr; Sliwinski, Tomasz

2017-10-03

Neurodegeneration in Alzheimer's disease can be caused by accumulation of oxidative DNA damage resulting from altered expression of genes involved in the base excision repair system (BER). Promoter methylation can affect the profile of BER genes expression. Decreased expression of BER genes was observed in the brains of AD patients. The aim of our study was to compare the expression and methylation profiles of six genes coding for proteins involved in BER, namely: hOGG1, APE1, MUTYH, NEIL1, PARP1 and XRCC1, in the peripheral blood cells of AD patients and healthy volunteers. The study consisted of 100 persons diagnosed with Alzheimer's disease according to DSM-IV criteria, and 110 healthy volunteers. DNA and total RNA were isolated from venous blood cells. Promoter methylation profiles were obtained by High Resolution Melting (HRM) analysis of bisulfide converted DNA samples. Real-time PCR with TaqMan probes was employed for gene expression analysis. APE1, hOGG1, MUTYH, PARP1 and NEIL1 were significantly (pgenes. The methylation status of promoters is not associated with downregulation of BER genes. Our results show that downregulation of BER genes detected in peripheral blood samples could reflect the changes occurring in the brain of patients with AD, and may be a useful biomarker of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein.

Science.gov (United States)

Yamamoto, Kenta; Sato, Yoshiki; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Sowa, Yoshihiro; Yamamoto, Toshiro; Kanamura, Narisato; Kishida, Tsunao; Mazda, Osam

2016-11-01

Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector-mediated transduction may potentially cause tumor formation from the infected cells, thus a non-viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L-Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast-like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast-specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid-driven directly converted osteoblasts (p-dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p-dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p-dOBs can be put into practical use for a novel cell-based therapy against bone diseases. J. Cell. Biochem. 117: 2538-2545, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Direct gene transfer in the Gottingen minipig CNS using stereotaxic lentiviral microinjections

DEFF Research Database (Denmark)

GLUD, AN; Hedegaard, Claus; Nielsen, Mette Slot

2010-01-01

We aim to induce direct viral mediated gene transfer in the substantia nigra (SN) of the Gottingen minipig using MRI guided stereotaxic injections of lentiviral vectors encoding enhanced green fluorescent protein (EGFP). Nine female Gottingen minipigs were injected unilaterally into the SN with 6...... per 2.5 microliters lentivirus capable of transducing cells and mediating expression of recombinant EGFP. The animals were euthanized after four (n=3) or twenty weeks (n=6). Fresh brain tissue from three animals was used for PCR. The remaining six brains were cryo- or paraffin...

Frequent down-regulation of ABC transporter genes in prostate cancer

International Nuclear Information System (INIS)

Demidenko, Rita; Razanauskas, Deividas; Daniunaite, Kristina; Lazutka, Juozas Rimantas; Jankevicius, Feliksas; Jarmalaite, Sonata

2015-01-01

ATP-binding cassette (ABC) transporters are transmembrane proteins responsible for the efflux of a wide variety of substrates, including steroid metabolites, through the cellular membranes. For better characterization of the role of ABC transporters in prostate cancer (PCa) development, the profile of ABC transporter gene expression was analyzed in PCa and noncancerous prostate tissues (NPT). TaqMan Low Density Array (TLDA) human ABC transporter plates were used for the gene expression profiling in 10 PCa and 6 NPT specimens. ABCB1 transcript level was evaluated in a larger set of PCa cases (N = 78) and NPT (N = 15) by real-time PCR, the same PCa cases were assessed for the gene promoter hypermethylation by methylation-specific PCR. Expression of eight ABC transporter genes (ABCA8, ABCB1, ABCC6, ABCC9, ABCC10, ABCD2, ABCG2, and ABCG4) was significantly down-regulated in PCa as compared to NPT, and only two genes (ABCC4 and ABCG1) were up-regulated. Down-regulation of ABC transporter genes was prevalent in the TMPRSS2-ERG-negative cases. A detailed analysis of ABCB1 expression confirmed TLDA results: a reduced level of the transcript was identified in PCa in comparison to NPT (p = 0.048). Moreover, the TMPRSS2-ERG-negative PCa cases showed significantly lower expression of ABCB1 in comparison to NPT (p = 0.003) or the fusion-positive tumors (p = 0.002). Promoter methylation of ABCB1 predominantly occurred in PCa and was rarely detected in NPT (p < 0.001). The study suggests frequent down-regulation of the ABC transporter genes in PCa, especially in the TMPRSS2-ERG-negative tumors. The online version of this article (doi:10.1186/s12885-015-1689-8) contains supplementary material, which is available to authorized users

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

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Wang Xiaoyi

2009-06-01

Full Text Available Abstract Background The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. Results We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Conclusion Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.

E112D polymorphism in the prolylcarboxypeptidase gene is associated with blood pressure response to benazepril in Chinese hypertensive patients.

Science.gov (United States)

Zhang, Yan; Hong, Xiu-mei; Xing, Hou-xun; Li, Jian-ping; Huo, Yong; Xu, Xi-ping

2009-10-20

Marked interindividual variation exists in blood pressure response to benazepril, which is considered to have genetic basis. Our objectives were to evaluate whether the E112D polymorphism in the prolylcarboxypeptidase (PRCP) gene has impact on blood pressure response to benazepril. Hypertensive patients from Huoqiu County and Yuexi County of Anhui Province received daily treatment with an oral dosage of 10 mg benazepril for 15 days. Genotypes of the E112D polymorphism in the PRCP gene were determined by TaqMan SNP genotyping assay. Multivariate linear and Logistic regressions using generalized estimating equation model were performed in a total of 1092 patients to evaluate the association of PRCP genotypes and blood pressure response to benazepril. Patients carrying ED or DD genotype had a less systolic blood pressure reduction (adjusted beta = -3.7 + or - 1.1, P benazepril treatment in hypertensive patients of Anhui Province, China.

Designing a time-effective TaqMan probe-based real-time polymerase chain reaction protocol for the identification of Yersinia enterocolitica in raw pork meat

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Milena Alicja Stachelska

2017-01-01

Full Text Available The aim of this study was to design a time-effective method comprising a short pre-enrichment step in a non-selective broth in combination with the TaqMan probe applied in the real-time polymerase chain reaction to detect Yersinia enterocolitica strains in raw pork meat. The method enabled to detect 1 colony forming unit per 25 mg of Yersinia enterocolitica in pork meat. The specificity and reliability of the method was not diminished by the company of microflora naturally present in meat. The method was found successful to detect pathogenic Yersinia enterocolitica strains in pork meat. It is advised to be used for assessing the microbial risk and for controlling the microbial quality of meat and meat products.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

Science.gov (United States)

Muhar, Matthias; Ebert, Anja; Neumann, Tobias; Umkehrer, Christian; Jude, Julian; Wieshofer, Corinna; Rescheneder, Philipp; Lipp, Jesse J; Herzog, Veronika A; Reichholf, Brian; Cisneros, David A; Hoffmann, Thomas; Schlapansky, Moritz F; Bhat, Pooja; von Haeseler, Arndt; Köcher, Thomas; Obenauf, Anna C; Popow, Johannes; Ameres, Stefan L; Zuber, Johannes

2018-05-18

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

Science.gov (United States)

Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

2002-01-01

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

Science.gov (United States)

Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Autoantibodies directed to centromere protein F in a patient with BRCA1 gene mutation

OpenAIRE

Moghaddas, Fiona; Joshua, Fredrick; Taylor, Roberta; Fritzler, Marvin J.; Toh, Ban Hock

2016-01-01

Background Autoantibodies directed to centromere protein F were first reported in 1993 and their association with malignancy has been well documented. Case We present the case of a 48-year-old Caucasian female with a BRCA1 gene mutation associated with bilateral breast cancer. Antinuclear autoantibody immunofluorescence performed for workup of possible inflammatory arthropathy showed a high titre cell cycle related nuclear speckled pattern, with subsequent confirmation by addressable laser be...

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

Science.gov (United States)

Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

2008-01-01

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Association of polymorphisms of the ADIPOQ, ADIPOR1 and ADIPOR2 genes with type 2 diabetes mellitus

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Dmitry Sergeevich Khodyrev

2015-03-01

Full Text Available The study of hereditary predisposition to multifactorial diseases is essential for diagnosis and selection of the optimal treatment. The study of polymorphisms of candidate genes whose products are involved in the pathogenesis of multifactorial diseases is of great clinical importance. Aim. The aim of this study was to investigate the association of rs2241766 and rs1501299 polymorphisms in the ADIPOQ gene, rs2275737 and rs2275738 polymorphisms in the ADIPOR1 gene and rs11061971 and rs16928751 polymorphisms in the ADIPOR2 gene with the development of type 2 diabetes mellitus (T2DM in the Russian population. Materials and methods. The study included a group of 500 patients with T2DM diagnosed based on standard diagnostic criteria (T2DM+. The control group (T2DM- was a random sample of 500 patients with no evidence of the disease and was matched to the T2DM+ group for gender, age and body mass index. The determination of alleles and genotypes was performed using real-time polymerase chain reaction with TaqMan probes. The X2 test and contingency tables were used to compare the distribution of allele and genotype frequencies. A p-value of

Genes expressed in dental enamel development are associated with molar-incisor hypomineralization.

Science.gov (United States)

Jeremias, Fabiano; Koruyucu, Mine; Küchler, Erika C; Bayram, Merve; Tuna, Elif B; Deeley, Kathleen; Pierri, Ricardo A; Souza, Juliana F; Fragelli, Camila M B; Paschoal, Marco A B; Gencay, Koray; Seymen, Figen; Caminaga, Raquel M S; dos Santos-Pinto, Lourdes; Vieira, Alexandre R

2013-10-01

Genetic disturbances during dental development influence variation of number and shape of the dentition. In this study, we tested if genetic variation in enamel formation genes is associated with molar-incisor hypomineralization (MIH), also taking into consideration caries experience. DNA samples from 163 cases with MIH and 82 unaffected controls from Turkey, and 71 cases with MIH and 89 unaffected controls from Brazil were studied. Eleven markers in five genes [ameloblastin (AMBN), amelogenin (AMELX), enamelin (ENAM), tuftelin (TUFT1), and tuftelin-interacting protein 11 (TFIP11)] were genotyped by the TaqMan method. Chi-square was used to compare allele and genotype frequencies between cases with MIH and controls. In the Brazilian data, distinct caries experience within the MIH group was also tested for association with genetic variation in enamel formation genes. The ENAM rs3796704 marker was associated with MIH in both populations (Brazil: p=0.03; OR=0.28; 95% C.I.=0.06-1.0; Turkey: p=1.22e-012; OR=17.36; 95% C.I.=5.98-56.78). Associations between TFIP11 (p=0.02), ENAM (p=0.00001), and AMELX (p=0.01) could be seen with caries independent of having MIH or genomic DNA copies of Streptococcus mutans detected by real time PCR in the Brazilian sample. Several genes involved in enamel formation appear to contribute to MIH. Copyright © 2013 Elsevier Ltd. All rights reserved.

Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms.

Science.gov (United States)

Kulesh, David A; Baker, Robert O; Loveless, Bonnie M; Norwood, David; Zwiers, Susan H; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P; Huggins, John; Jahrling, Peter B

2004-02-01

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs

Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

Science.gov (United States)

Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

2017-05-17

Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

FY 2000 Project of developing international standards for supporting new industries. Standardization of systems for detecting gene recombination products; 2000 nendo shinki sangyo shiengata kokusai hyojunka kaihatsu jigyo seika hokokusho. Idenshi kumikae seihin kenshutsu system no hyojunka

Energy Technology Data Exchange (ETDEWEB)

NONE

2001-03-01

Described herein are the FY 2000 results of the studies on the basic conditions for gene recombination products, and methods of determining content of the purchased gene recombination products in agricultural products, conducted to promote the standardization of the gene recombination product detection systems. Soybean is taken up as the gene recombination product to be studied, in which the samples containing the gene recombination product at 5, 0.5, 0.1 and 0% are prepared, and qualitatively analyzed by the DNA extraction and PCR method. No band is detected. For the quantitative examination by the real-time PCR method, the SYBRGeen, Taqman and hybridization probe methods are used to study the PCR conditions. The sample DNA extracted by the DNA Extraction kit for GMO Detection Ver.2 from the sample containing the RoundupReady{sup TM} soybean is quantitatively analyzed by the real-time PCR method for GMO content, which has produced the results for promotion of the standardization. (NEDO)

TaqMan Real-Time Polymerase Chain Reaction and ...

African Journals Online (AJOL)

ISSN: 1596-5996 (print); 1596-9827 (electronic) ... Alcohol in humans is oxidized to acetaldehyde, which in turn is oxidized to .... cool to room temperature (15 - 25 oC) for at least. 5 min, and .... logical candidate gene for alcohol dependence. A.

A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples.

Science.gov (United States)

Cascini, Fidelia; Passerotti, Stella; Martello, Simona

2012-04-10

In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug-type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the ΔΔCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

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C.P. Nunes

2000-12-01

Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

Directed partial correlation: inferring large-scale gene regulatory network through induced topology disruptions.

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Yinyin Yuan

Full Text Available Inferring regulatory relationships among many genes based on their temporal variation in transcript abundance has been a popular research topic. Due to the nature of microarray experiments, classical tools for time series analysis lose power since the number of variables far exceeds the number of the samples. In this paper, we describe some of the existing multivariate inference techniques that are applicable to hundreds of variables and show the potential challenges for small-sample, large-scale data. We propose a directed partial correlation (DPC method as an efficient and effective solution to regulatory network inference using these data. Specifically for genomic data, the proposed method is designed to deal with large-scale datasets. It combines the efficiency of partial correlation for setting up network topology by testing conditional independence, and the concept of Granger causality to assess topology change with induced interruptions. The idea is that when a transcription factor is induced artificially within a gene network, the disruption of the network by the induction signifies a genes role in transcriptional regulation. The benchmarking results using GeneNetWeaver, the simulator for the DREAM challenges, provide strong evidence of the outstanding performance of the proposed DPC method. When applied to real biological data, the inferred starch metabolism network in Arabidopsis reveals many biologically meaningful network modules worthy of further investigation. These results collectively suggest DPC is a versatile tool for genomics research. The R package DPC is available for download (http://code.google.com/p/dpcnet/.

[Gene-gene interaction on central obesity in school-aged children in China].

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Fu, L W; Zhang, M X; Wu, L J; Gao, L W; Mi, J

2017-07-10

Objective: To investigate possible effect of 6 obesity-associated SNPs in contribution to central obesity and examine whether there is an interaction in the 6 SNPs in the cause of central obesity in school-aged children in China. Methods: A total of 3 502 school-aged children who were included in Beijing Child and Adolescent Metabolic Syndrome (BCAMS) Study were selected, and based on the age and sex specific waist circumference (WC) standards in the BCAMS study, 1 196 central obese cases and 2 306 controls were identified. Genomic DNA was extracted from peripheral blood white cells using the salt fractionation method. A total of 6 single nucleotide polymorphisms ( FTO rs9939609, MC4R rs17782313, BDNF rs6265, PCSK1 rs6235, SH2B1 rs4788102, and CSK rs1378942) were genotyped by TaqMan allelic discrimination assays with the GeneAmp 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA). Logistic regression model was used to investigate the association between 6 SNPs and central obesity. Gene-gene interactions among 6 polymorphic loci were analyzed by using the Generalized Multifactor Dimensionality Reduction (GMDR) method, and then logistic regression model was constructed to confirm the best combination of loci identified in the GMDR. Results: After adjusting gender, age, Tanner stage, physical activity and family history of obesity, the FTO rs9939609-A, MC4R rs17782313-C and BDNF rs6265-G alleles were associated with central obesity under additive genetic model ( OR =1.24, 95 %CI : 1.06-1.45, P =0.008; OR =1.26, 95 %CI : 1.11-1.43, P =2.98×10(-4); OR =1.18, 95 % CI : 1.06-1.32, P =0.003). GMDR analysis showed a significant gene-gene interaction between MC4R rs17782313 and BDNF rs6265 ( P =0.001). The best two-locus combination showed the cross-validation consistency of 10/10 and testing accuracy of 0.539. This interaction showed the maximum consistency and minimum prediction error among all gene-gene interaction models evaluated. Moreover, the

Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction.

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Li, Aitao; Acevedo-Rocha, Carlos G; Sun, Zhoutong; Cox, Tony; Xu, Jia Lucy; Reetz, Manfred T

2018-02-02

Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high-fidelity solid-phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. A traditional combinatorial PCR-based SM library, produced by simultaneous randomization at several residues by using a reduced amino acid alphabet, and the respective synthetic library were constructed and compared. Statistical analysis at the DNA level with massive sequencing demonstrates that, in the synthetic approach, 97 % of the theoretically possible DNA mutants are formed, whereas the traditional SM library contained only about 50 %. Screening at the protein level also showed the superiority of the synthetic library; many highly (R,R)- and (S,S)-selective variants being discovered are not found in the traditional SM library. With the prices of synthetic genes decreasing, this approach may point the way to future directed evolution. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Association Between Stool Enteropathogen Quantity and Disease in Tanzanian Children Using TaqMan Array Cards: A Nested Case-Control Study

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Platts-Mills, James A.; Gratz, Jean; Mduma, Esto; Svensen, Erling; Amour, Caroline; Liu, Jie; Maro, Athanasia; Saidi, Queen; Swai, Ndealilia; Kumburu, Happiness; McCormick, Benjamin J. J.; Kibiki, Gibson; Houpt, Eric R.

2014-01-01

Etiologic studies of diarrhea are limited by uneven diagnostic methods and frequent asymptomatic detection of enteropathogens. Polymerase chain reaction-based stool pathogen quantification may help distinguish clinically significant infections. We performed a nested case-control study of diarrhea in infants from a community-based birth cohort in Tanzania. We tested 71 diarrheal samples and pre-diarrheal matched controls with a laboratory-developed TaqMan Array Card for 19 enteropathogens. With qualitative detection, no pathogens were significantly associated with diarrhea. When pathogen quantity was considered, rotavirus (odds ratio [OR] = 2.70 per log10 increase, P < 0.001), astrovirus (OR = 1.49, P = 0.01), and Shigella/enteroinvasive Escherichia coli (OR = 1.47, P = 0.04) were associated with diarrhea. Enterotoxigenic E. coli (0.15 SD decline in length-for-age z score after 3 months per log10 increase, P < 0.001) and Campylobacter jejuni/C. coli (0.11 SD decline, P = 0.003) in pre-diarrheal stools were associated with poor linear growth. Quantitative analysis can help refine the association between enteropathogens and disease in endemic settings. PMID:24189366

Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

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Onyango, Clayton O; Loparev, Vladimir; Lidechi, Shirley; Bhullar, Vinod; Schmid, D Scott; Radford, Kay; Lo, Michael K; Rota, Paul; Johnson, Barbara W; Munoz, Jorge; Oneko, Martina; Burton, Deron; Black, Carolyn M; Neatherlin, John; Montgomery, Joel M; Fields, Barry

2017-07-01

Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites ( Balamuthia mandrillaris and Acanthamoeba ), six bacterial pathogens ( Streptococcus pneumonia e, Haemophilus influenzae , Neisseria meningitidis , Mycoplasma pneumoniae , Mycobacterium tuberculosis , and Bartonella ), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease. Copyright © 2017 American Society for Microbiology.

Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

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Kulesh, David A.; Baker, Robert O.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P.; Huggins, John; Jahrling, Peter B.

2004-01-01

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However

Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia.

Science.gov (United States)

Fenske, Myles P; Hewett Hazelton, Kristen D; Hempton, Andrew K; Shim, Jae Sung; Yamamoto, Breanne M; Riffell, Jeffrey A; Imaizumi, Takato

2015-08-04

Flowers present a complex display of signals to attract pollinators, including the emission of floral volatiles. Volatile emission is highly regulated, and many species restrict emissions to specific times of the day. This rhythmic emission of scent is regulated by the circadian clock; however, the mechanisms have remained unknown. In Petunia hybrida, volatile emissions are dominated by products of the floral volatile benzenoid/phenylpropanoid (FVBP) metabolic pathway. Here we demonstrate that the circadian clock gene P. hybrida LATE ELONGATED HYPOCOTYL (LHY; PhLHY) regulates the daily expression patterns of the FVBP pathway genes and floral volatile production. PhLHY expression peaks in the morning, antiphasic to the expression of P. hybrida GIGANTEA (PhGI), the master scent regulator ODORANT1 (ODO1), and many other evening-expressed FVBP genes. Overexpression phenotypes of PhLHY in Arabidopsis caused an arrhythmic clock phenotype, which resembles those of LHY overexpressors. In Petunia, constitutive expression of PhLHY depressed the expression levels of PhGI, ODO1, evening-expressed FVBP pathway genes, and FVBP emission in flowers. Additionally, in the Petunia lines in which PhLHY expression was reduced, the timing of peak expression of PhGI, ODO1, and the FVBP pathway genes advanced to the morning. Moreover, PhLHY protein binds to cis-regulatory elements called evening elements that exist in promoters of ODO1 and other FVBP genes. Thus, our results imply that PhLHY directly sets the timing of floral volatile emission by restricting the expression of ODO1 and other FVBP genes to the evening in Petunia.

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens.

Science.gov (United States)

Qu, X S; Wanner, L A; Christ, B J

2011-03-01

To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Challenges to develop nitrogen-fixing cereals by direct nif-gene transfer.

Science.gov (United States)

Curatti, Leonardo; Rubio, Luis M

2014-08-01

Some regions of the developing world suffer low cereal production yields due to low fertilizer inputs, among other factors. Biological N2 fixation, catalyzed by the prokaryotic enzyme nitrogenase, is an alternative to the use of synthetic N fertilizers. The molybdenum nitrogenase is an O2-labile metalloenzyme composed of the NifDK and NifH proteins, which biosyntheses require a number of nif gene products. A challenging strategy to increase cereal crop productivity in a scenario of low N fertilization is the direct transfer of nif genes into cereals. The sensitivity of nitrogenase to O2 and the apparent complexity of nitrogenase biosynthesis are the main barriers identified so far. Expression of active NifH requires the products of nifM, nifH, and possibly nifU and nifS, whereas active NifDK requires the products of nifH, nifD, nifK, nifB, nifE, nifN, and possibly nifU, nifS, nifQ, nifV, nafY, nifW and nifZ. Plastids and mitochondria are potential subcellular locations for nitrogenase. Both could provide the ATP and electrons required for nitrogenase to function but they differ in their internal O2 levels and their ability to incorporate ammonium into amino acids. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Regulation and functions of the lms homeobox gene during development of embryonic lateral transverse muscles and direct flight muscles in Drosophila.

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Dominik Müller

Full Text Available BACKGROUND: Patterning and differentiation of developing musculatures require elaborate networks of transcriptional regulation. In Drosophila, significant progress has been made into identifying the regulators of muscle development and defining their interactive networks. One major family of transcription factors involved in these processes consists of homeodomain proteins. In flies, several members of this family serve as muscle identity genes to specify the fates of individual muscles, or groups thereof, during embryonic and/or adult muscle development. Herein, we report on the expression and function of a new Drosophila homeobox gene during both embryonic and adult muscle development. METHODOLOGY/PRINCIPAL FINDINGS: The newly described homeobox gene, termed lateral muscles scarcer (lms, which has yet uncharacterized orthologs in other invertebrates and primitive chordates but not in vertebrates, is expressed exclusively in subsets of developing muscle tissues. In embryos, lms is expressed specifically in the four lateral transverse (LT muscles and their founder cells in each hemisegment, whereas in larval wing imaginal discs, it is expressed in myoblasts that develop into direct flight muscles (DFMs, which are important for proper wing positioning. We have analyzed the regulatory inputs of various other muscle identity genes with overlapping or complementary expression patterns towards the cell type specific regulation of lms expression. Further we demonstrate that lms null mutants exhibit reduced numbers of embryonic LT muscles, and null mutant adults feature held-out-wing phenotypes. We provide a detailed description of the pattern and morphology of the direct flight muscles in the wild type and lms mutant flies by using the recently-developed ultramicroscopy and show that, in the mutants, all DFMs are present and present normal morphologies. CONCLUSIONS/SIGNIFICANCE: We have identified the homeobox gene lms as a new muscle identity gene

Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis.

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Thiesen, H-J; Steinbeck, F; Maruschke, M; Koczan, D; Ziems, B; Hakenberg, O W

2017-01-01

Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value.

Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer

International Nuclear Information System (INIS)

Zi-Bo, LI; Zhao-Jun, ZENG; Qian, CHEN; Sai-Qun, LUO; Wei-Xin, HU

2006-01-01

HSVtk/ganciclovir (GCV) gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2)-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. Recombinant adeno-associated virus-2 (rAAV) was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox) induction or without Dox induction at a vp (viral particle) number of ≥10 4 /cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at ≥10 4 vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR) analysis. The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors

Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

NARCIS (Netherlands)

Fairley, Jennifer A.; Mitchell, Louise E.; Berg, Tracy; Kenneth, Niall S.; von Schubert, Conrad; Sillje, Herman H. W.; Medema, Rene H.; Nigg, Erich A.; White, Robert J.

2012-01-01

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase Ill (pol Ill) through direct binding and phosphorylation of transcription factor Brit During interphase, Plk1 promotes

Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

Science.gov (United States)

Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens

2015-11-01

Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

Science.gov (United States)

Bouchard-Mercier, Annie; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2014-01-01

A large inter-individual variability in the plasma triglyceride (TG) response to an omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs) within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208) participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA). Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187) and ACOX1 (rs17583163) genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation. PMID:24647074

Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

Directory of Open Access Journals (Sweden)

Annie Bouchard-Mercier

2014-03-01

Full Text Available A large inter-individual variability in the plasma triglyceride (TG response to an omega-3 polyunsaturated fatty acid (n-3 PUFA supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208 participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA. Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187 and ACOX1 (rs17583163 genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation.

Gene Directed Enzyme Prodrug Therapy Using Rabbit Cytochrome P450 4B1 in Murine Colon Adenocarcinoma

Energy Technology Data Exchange (ETDEWEB)

Kim, Sung Joo; Kang, Joo Hyun; Lee, Tae Sup; Kim, Kyeong Min; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2007-07-01

The conventional cancer therapy is chemotherapy, surgical resection and/or radiotherapy. Chemotherapy using cytotoxic drug has some problems with lack of tumor selectivity resulting in toxicity to normal tissues. To enhance the tumor selectivity of cytotoxic drug, the application of suicidal gene therapy technology was designed. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a non-toxic prodrug into a cytotoxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1- tk) and cytosine deaminase (cd). Recently, a new prodrug-converting enzyme based on rabbit cytochrome P450 4B1 gene (cyp4B1) has been reported for therapy of experimental brain tumor. This enzyme activates the prodrugs such as 4-ipomeanol (4-IM) and 2- aminoanthracene (2-AA) to highly reactive furane epoxide and unsaturated dialdehyde intermediate, respectively. DNA alkylation seems to be the main mechanism of cytotoxicity of these activated drugs. In this study, we isolated cyp4B1 cDNA from rabbit lung, transduced cyp4B1 expression vector into murine colon cancer cell, and then analyzed the cytotoxic properties of cyp4b1-activated 2-AA in cyp4B1 transduced cells to verify the cyp4B1 enzyme system for gene directed enzyme prodrug therapy.

Gene Directed Enzyme Prodrug Therapy Using Rabbit Cytochrome P450 4B1 in Murine Colon Adenocarcinoma

International Nuclear Information System (INIS)

Kim, Sung Joo; Kang, Joo Hyun; Lee, Tae Sup; Kim, Kyeong Min; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo

2007-01-01

The conventional cancer therapy is chemotherapy, surgical resection and/or radiotherapy. Chemotherapy using cytotoxic drug has some problems with lack of tumor selectivity resulting in toxicity to normal tissues. To enhance the tumor selectivity of cytotoxic drug, the application of suicidal gene therapy technology was designed. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a non-toxic prodrug into a cytotoxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1- tk) and cytosine deaminase (cd). Recently, a new prodrug-converting enzyme based on rabbit cytochrome P450 4B1 gene (cyp4B1) has been reported for therapy of experimental brain tumor. This enzyme activates the prodrugs such as 4-ipomeanol (4-IM) and 2- aminoanthracene (2-AA) to highly reactive furane epoxide and unsaturated dialdehyde intermediate, respectively. DNA alkylation seems to be the main mechanism of cytotoxicity of these activated drugs. In this study, we isolated cyp4B1 cDNA from rabbit lung, transduced cyp4B1 expression vector into murine colon cancer cell, and then analyzed the cytotoxic properties of cyp4b1-activated 2-AA in cyp4B1 transduced cells to verify the cyp4B1 enzyme system for gene directed enzyme prodrug therapy

Association Study of Tumor Necrosis Factor Receptor 1 (TNFR1 Gene Polymorphisms with Schizophrenia in the Polish Population

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Renata Suchanek-Raif

2017-01-01

Full Text Available Schizophrenia is a devastating mental disorder with undetermined aetiology. Previous research has suggested that dysregulation of proinflammatory cytokines and their receptors plays a role in developing schizophrenia. We examined the association of the three single nucleotide polymorphisms (SNPs; rs4149576, rs4149577, and rs1860545 in the tumor necrosis factor receptor 1 (TNFR1 gene with the development and psychopathology of paranoid schizophrenia in the Polish Caucasian sample consisting of 388 patients and 657 control subjects. The psychopathology was assessed using a five-factor model of the Positive and Negative Syndrome Scale (PANSS. SNPs were genotyped using the TaqMan 5′-exonuclease allelic discrimination assay. The SNPs tested were not associated with a predisposition to paranoid schizophrenia in either the entire sample or after stratification according to gender. However, rs4149577 and rs1860545 SNPs were associated with the intensity of the PANSS excitement symptoms in men, which may contribute to the risk of violent behavior. Polymorphisms in the TNFR1 gene may have an impact on the symptomatology of schizophrenia in men.

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome

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Gaora Peadar Ó

2010-10-01

Full Text Available Abstract Background Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of

A common copy number variation polymorphism in the CNTNAP2 gene: sexual dimorphism in association with healthy aging and disease.

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Iakoubov, Leonid; Mossakowska, Malgorzata; Szwed, Malgorzata; Puzianowska-Kuznicka, Monika

2015-01-01

New therapeutic targets are needed to fight aging-related diseases and increase life span. A new female-specific association with diseases and limited survival past 80 years was recently reported for a copy number variation (CNV) in the CNTNAP4 gene from the neurexin superfamily. We asked whether there are CNVs that are associated with aging phenotypes within other genes from the neurexin superfamily and whether this association is sex specific. Select CNV polymorphisms were genotyped with proprietary TaqMan qPCR assays. A case/control study, in which a group of 81- to 90-year-old community-dwelling Caucasians with no chronic diseases (case) was compared to a similar control group of 65- to 75-year-olds, revealed a negative association with healthy aging for the ins allele of common esv11910 CNV in the CNTNAP2 gene (n = 388; OR = 0.29, 95% CI: 0.14-0.59, p = 0.0004 for males, and OR = 0.82, 95% CI: 0.42-1.57, p = 0.625 for females). This male-specific association was validated in a study of an independent group of 76- to 80-year-olds. To look for a corresponding positive association of the allele with aging-related diseases, two case subgroups of 81- to 90-year-olds, one composed of individuals with cognitive impairment and the other with various diseases not directly related to the nervous system, such as cardiovascular diseases, etc., were compared to a healthy control subgroup of the same age. A positive male-specific association was found for both cases (OR = 2.75, p = 0.008 for association with cognitive impairment, and OR = 3.18, p = 0.002 for other diseases combined). A new male-specific association with aging is reported for a CNV in the CNTNAP2 gene. The polymorphism might be useful for diagnosing individual genetic predispositions to healthy aging versus aging complicated by chronic diseases. © 2014 S. Karger AG, Basel.

Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii).

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Cao, Heping; Zhang, Lin; Tan, Xiaofeng; Long, Hongxu; Shockey, Jay M

2014-01-01

Triacylglycerols (TAG) are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE) are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii), whose seeds are rich in novel TAG with a wide range of industrial applications. The objectives of this study were to identify OLE genes, classify OLE proteins and analyze OLE gene expression in tung trees. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome-wide phylogenetic analysis and multiple sequence alignment demonstrated that the five tung OLE genes represented the five OLE subfamilies and all contained the "proline knot" motif (PX5SPX3P) shared among 65 OLE from 19 tree species, including the sequenced genomes of Prunus persica (peach), Populus trichocarpa (poplar), Ricinus communis (castor bean), Theobroma cacao (cacao) and Vitis vinifera (grapevine). Tung OLE1, OLE2 and OLE3 belong to the S type and OLE4 and OLE5 belong to the SM type of Arabidopsis OLE. TaqMan and SYBR Green qPCR methods were used to study the differential expression of OLE genes in tung tree tissues. Expression results demonstrated that 1) All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2) OLE mRNA levels were much higher in seeds than leaves or flowers; 3) OLE1, OLE2 and OLE3 genes were expressed in tung seeds at much higher levels than OLE4 and OLE5 genes; 4) OLE mRNA levels rapidly increased during seed development; and 5) OLE gene expression was well-coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1-3 probably play major roles in tung oil accumulation and/or oil body development. Therefore, they might be preferred targets for tung oil engineering in transgenic plants.

Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii.

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Heping Cao

Full Text Available Triacylglycerols (TAG are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii, whose seeds are rich in novel TAG with a wide range of industrial applications. The objectives of this study were to identify OLE genes, classify OLE proteins and analyze OLE gene expression in tung trees. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome-wide phylogenetic analysis and multiple sequence alignment demonstrated that the five tung OLE genes represented the five OLE subfamilies and all contained the "proline knot" motif (PX5SPX3P shared among 65 OLE from 19 tree species, including the sequenced genomes of Prunus persica (peach, Populus trichocarpa (poplar, Ricinus communis (castor bean, Theobroma cacao (cacao and Vitis vinifera (grapevine. Tung OLE1, OLE2 and OLE3 belong to the S type and OLE4 and OLE5 belong to the SM type of Arabidopsis OLE. TaqMan and SYBR Green qPCR methods were used to study the differential expression of OLE genes in tung tree tissues. Expression results demonstrated that 1 All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2 OLE mRNA levels were much higher in seeds than leaves or flowers; 3 OLE1, OLE2 and OLE3 genes were expressed in tung seeds at much higher levels than OLE4 and OLE5 genes; 4 OLE mRNA levels rapidly increased during seed development; and 5 OLE gene expression was well-coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1-3 probably play major roles in tung oil accumulation and/or oil body development. Therefore, they might be preferred targets for tung oil engineering in transgenic plants.

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.

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Doetschman, Thomas; Georgieva, Teodora

2017-03-03

Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy. Studies on its off-target activity, along with approaches to minimize this activity will be given. The advantages of gene editing versus gene targeting in embryonic stem cells, including the breadth of species and cell types to which it is applicable, will be discussed. We will also cover its use in iPSC for research and possible therapeutic purposes; and we will review its use in muscular dystrophy studies where considerable progress has been made toward dystrophin correction in mice. The CRISPR/Ca9s system is also being used for high-throughput screening of genes, gene regulatory regions, and long noncoding RNAs. In addition, the CRISPR system is being used for nongene-editing purposes such as activation and inhibition of gene expression, as well as for fluorescence tagging of chromosomal regions and individual mRNAs to track their cellular location. Finally, an approach to circumvent the inability of post-mitotic cells to support homologous recombination-based gene editing will be presented. In conclusion, applications of the CRISPR/Cas system are expanding at a breath-taking pace and are revolutionizing approaches to gain a better understanding of human diseases. © 2017 American Heart Association, Inc.

Potential role of miR-9 and miR-223 in recurrent ovarian cancer

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McGuinness Eamonn

2008-04-01

Full Text Available Abstract Background MicroRNAs (miRNAs are small, noncoding RNAs that negatively regulate gene expression by binding to target mRNAs. miRNAs have not been comprehensively studied in recurrent ovarian cancer, yet an incurable disease. Results Using real-time RT-PCR, we obtained distinct miRNA expression profiles between primary and recurrent serous papillary ovarian adenocarcinomas (n = 6 in a subset of samples previously used in a transcriptome approach. Expression levels of top dysregulated miRNA genes, miR-223 and miR-9, were examined using TaqMan PCR in independent cohorts of fresh frozen (n = 18 and FFPE serous ovarian tumours (n = 22. Concordance was observed on TaqMan analysis for miR-223 and miR-9 between the training cohort and the independent test cohorts. Target prediction analysis for the above miRNA "recurrent metastatic signature" identified genes previously validated in our transcriptome study. Common biological pathways well characterised in ovarian cancer were shared by miR-9 and miR-223 lists of predicted target genes. We provide strong evidence that miR-9 acts as a putative tumour suppressor gene in recurrent ovarian cancer. Components of the miRNA processing machinery, such as Dicer and Drosha are not responsible for miRNA deregulation in recurrent ovarian cancer, as deluded by TaqMan and immunohistochemistry. Conclusion We propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Some of the differentially deregulated miRNAs identified correlate with our previous transcriptome findings. Based on integrated transcriptome and miRNA analysis, miR-9 and miR-223 can be of potential importance as biomarkers in recurrent ovarian cancer.

Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay

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Jansen van Vuren, Petrus; Grobbelaar, Antoinette; Storm, Nadia; Conteh, Ousman; Konneh, Kelfala; Kamara, Abdul; Sanne, Ian

2015-01-01

The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks. PMID:26637383

Hypothalamus transcriptome profile suggests an anorexia-cachexia syndrome in the anx/anx mouse model.

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Mercader, Josep Maria; Lozano, Juan José; Sumoy, Lauro; Dierssen, Mara; Visa, Joana; Gratacòs, Mònica; Estivill, Xavier

2008-11-12

The anx/anx mouse displays poor appetite and lean appearance and is considered a good model for the study of anorexia nervosa. To identify new genes involved in feeding behavior and body weight regulation we performed an expression profiling in the hypothalamus of the anx/anx mice. Using commercial microarrays we detected 156 differentially expressed genes and validated 92 of those using TaqMan low-density arrays. The expression of a set of 87 candidate genes selected based on literature evidences was also quantified by TaqMan low-density arrays. Our results showed enrichment in deregulated genes involved in cell death, cell morphology, and cancer, as well as an alteration of several signaling circuits involved in energy balance including neuropeptide Y and melanocortin signaling. The expression profile along with the phenotype led us to conclude that anx/anx mice resemble the anorexia-cachexia syndrome typically observed in cancer, infection with human immunodeficiency virus or chronic diseases, rather than starvation, and that anx/anx mice could be considered a good model for the treatment and investigation of this condition.

Hypoxia-response element (HRE)-directed transcriptional regulation of the rat lysyl oxidase gene in response to cobalt and cadmium.

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Gao, Song; Zhou, Jing; Zhao, Yinzhi; Toselli, Paul; Li, Wande

2013-04-01

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

The concentration, gene expression, and spatial distribution of aggrecan in canine articular cartilage, meniscus, and anterior and posterior cruciate ligaments: a new molecular distinction between hyaline cartilage and fibrocartilage in the knee joint.

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Valiyaveettil, Manojkumar; Mort, John S; McDevitt, Cahir A

2005-01-01

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 +/- 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 +/- 4.3 nMol/g) and ligaments (ACL: 6.8 +/- 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts.

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Samuel, Stylianos; Papayiannis, Lambros C; Leroch, Michaela; Veloukas, Thomas; Hahn, Matthias; Karaoglanidis, George S

2011-08-01

Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005-2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real-time TaqMan PCR assay developed in the present study. QoI-resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse-grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI-resistant and QoI-sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. The results of the study suggest that a high risk for selection of QoI-resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real-time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre- and post-amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry.

DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1977-01-01

An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

Feasibility of using tissue microarray cores of paraffin-embedded breast cancer tissue for measurement of gene expression: a proof-of-concept study.

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Drury, Suzanne; Salter, Janine; Baehner, Frederick L; Shak, Steven; Dowsett, Mitch

2010-06-01

To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. Four matched 0.6 mm cores of invasive breast tumour and two 10 microm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan assays were performed on the 21 genes of the Oncotype DX Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. RNA yield was lower from 0.6 mm cores than from 10 microm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies.

Association analysis of calpain 10 gene variants/haplotypes with gestational diabetes mellitus among Mexican women.

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Castro-Martínez, Anna Gabriela; Sánchez-Corona, José; Vázquez-Vargas, Adriana Patricia; García-Zapién, Alejandra Guadalupe; López-Quintero, Andres; Villalpando-Velazco, Héctor Javier; Flores-Martínez, Silvia Esperanza

2018-02-28

Gestational diabetes mellitus (GDM) is a metabolically complex disease with major genetic determinants. GDM has been associated with insulin resistance and dysfunction of pancreatic beta cells, so the GDM candidate genes are those that encode proteins modulating the function and secretion of insulin, such as that for calpain 10 (CAPN10). This study aimed to assess whether single nucleotide polymorphism (SNP)-43, SNP-44, SNP-63, and the indel-19 variant, and specific haplotypes of the CAPN10 gene were associated with gestational diabetes mellitus. We studied 116 patients with gestational diabetes mellitus and 83 women with normal glucose tolerance. Measurements of anthropometric and biochemical parameters were performed. SNP-43, SNP-44, and SNP-63 were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphisms, while the indel-19 variant was detected by TaqMan qPCR assays.  The allele, genotype, and haplotype frequencies of the four variants did not differ significantly between women with gestational diabetes mellitus and controls. However, in women with gestational diabetes mellitus, glucose levels were significantly higher bearing the 3R/3R genotype than in carriers of the 3R/2R genotype of the indel-19 variant (p = 0.006). In conclusion, the 3R/3R genotype of the indel-19 variant of the CAPN-10 gene influenced increased glucose levels in these Mexican women with gestational diabetes mellitus.

Molecular Detection of Leptospiral DNA in Environmental Water on St. Kitts

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Julienne Rawlins

2014-08-01

Full Text Available Leptospirosis is an important waterborne zoonotic disease caused by pathogenic Leptospira. The pathogen is maintained in a population due to chronic colonization and shedding from renal tubules of domestic and wild animals. Humans and other animals become infected when they come in contact with urine from infected animals, either directly or through urine-contaminated surface water. In this study, we screened environmental water on the island of St. Kitts by using a TaqMan based real time quantitative polymerase chain reaction (qPCR targeting a pathogen specific leptospiral gene, lipl32. Our results indicate that around one-fifth of tested water sources have detectable leptospiral DNA.

Altered Gene Expression by Low-Dose Arsenic Exposure in Humans and Cultured Cardiomyocytes: Assessment by Real-Time PCR Arrays

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Judy Mumford

2011-06-01

Full Text Available Chronic arsenic exposure results in higher risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. The purpose of this study was to investigate the effects on expression of selected genes in the blood lymphocytes from 159 people exposed chronically to arsenic in their drinking water using a novel RT-PCR TaqMan low-density array (TLDA. We found that expression of tumor necrosis factor-α (TNF-α, which activates both inflammation and NF-κB-dependent survival pathways, was strongly associated with water and urinary arsenic levels. Expression of KCNA5, which encodes a potassium ion channel protein, was positively associated with water and toe nail arsenic levels. Expression of 2 and 11 genes were positively associated with nail and urinary arsenic, respectively. Because arsenic exposure has been reported to be associated with long QT intervals and vascular disease in humans, we also used this TLDA for analysis of gene expression in human cardiomyocytes exposed to arsenic in vitro. Expression of the ion-channel genes CACNA1, KCNH2, KCNQ1 and KCNE1 were down-regulated by 1-mM arsenic. Alteration of some common pathways, including those involved in oxidative stress, inflammatory signaling, and ion-channel function, may underlay the seemingly disparate array of arsenic-associated diseases, such as cancer, cardiovascular disease, and diabetes.

Novel Random Mutagenesis Method for Directed Evolution.

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Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

2017-01-01

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

Association analysis of nine candidate gene polymorphisms in Indian patients with type 2 diabetic retinopathy

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Govindarajan Gowthaman

2010-11-01

Full Text Available Abstract Background Diabetic retinopathy (DR is classically defined as a microvasculopathy that primarily affects the small blood vessels of the inner retina as a complication of diabetes mellitus (DM.It is a multifactorial disease with a strong genetic component. The aim of this study is to investigate the association of a set of nine candidate genes with the development of diabetic retinopathy in a South Indian cohort who have type 2 diabetes mellitus (T2DM. Methods Seven candidate genes (RAGE, PEDF, AKR1B1, EPO, HTRA1, ICAM and HFE were chosen based on reported association with DR in the literature. Two more, CFH and ARMS2, were chosen based on their roles in biological pathways previously implicated in DR. Fourteen single nucleotide polymorphisms (SNPs and one dinucleotide repeat polymorphism, previously reported to show association with DR or other related diseases, were genotyped in 345 DR and 356 diabetic patients without retinopathy (DNR. The genes which showed positive association in this screening set were tested further in additional sets of 100 DR and 90 DNR additional patients from the Aravind Eye Hospital. Those which showed association in the secondary screen were subjected to a combined analysis with the 100 DR and 100 DNR subjects previously recruited and genotyped through the Sankara Nethralaya Hospital, India. Genotypes were evaluated using a combination of direct sequencing, TaqMan SNP genotyping, RFLP analysis, and SNaPshot PCR assays. Chi-square and Fisher exact tests were used to analyze the genotype and allele frequencies. Results Among the nine loci (15 polymorphisms screened, SNP rs2070600 (G82S in the RAGE gene, showed significant association with DR (allelic P = 0.016, dominant model P = 0.012, compared to DNR. SNP rs2070600 further showed significant association with DR in the confirmation cohort (P = 0.035, dominant model P = 0.032. Combining the two cohorts gave an allelic P HTRA1, rs11200638 (G>A, showed marginal

Interaction between VEGF receptor-2 gene polymorphisms and dietary patterns on blood glucose and lipid levels in Chinese Malaysian adults.

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Yap, Roseline Wai Kuan; Shidoji, Yoshihiro; Hon, Wei Min; Masaki, Motofumi

2011-01-01

The prevalence of lifestyle-related chronic diseases is increasing and gene-diet interaction studies are limited among the Malaysian population. This study was conducted to evaluate the association and interaction effects of vascular endothelial growth factor receptor-2(VEGFR2) gene polymorphisms and dietary patterns on anthropometric and biochemical risk factors of chronic diseases in 179 Chinese Malaysian adults. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Dietary patterns were constructed from the food frequency questionnaire using factor analysis. Anthropometric measurements: body mass index (BMI), systolic and diastolic blood pressure and biomarkers: blood glucose, glycated hemoglobin A1c (HbA1c) and lipids were obtained. Two dietary patterns: 'Balanced diet' and 'Meat, rice and noodles diet' (MRND) were extracted. MRND was associated with higher BMI, blood pressure, blood glucose and lipids, while T alleles in both rs1870377 and rs2071559 were associated with higher blood lipids (p Malaysian adults. Copyright © 2012 S. Karger AG, Basel.

Identification of the interleukin 4 receptor alpha gene as a direct target for p73.

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Sasaki, Yasushi; Mita, Hiroaki; Toyota, Minoru; Ishida, Setsuko; Morimoto, Ichiro; Yamashita, Toshiharu; Tanaka, Toshihiro; Imai, Kohzoh; Nakamura, Yusuke; Tokino, Takashi

2003-12-01

p73 has a high degree of structural homology to p53 and can activate transcription of p53-responsive genes. However, analysis of p73-deficient mice revealed a marked divergence in the physiological activities of p53 family genes and distinguishes p73 from p53. Mice deficient for p73 exhibit profound defects, including hippocampal dysgenesis, chronic infection, and inflammation, as well as abnormalities in pheromone sensory pathways. p73 plays important roles in neurogenesis, sensory pathways, and homeostatic regulation. Here, we found that the interleukin 4 receptor alpha (IL-4Ralpha) gene is up-regulated by p73 but not significantly by p53 in several human cancer cell lines. IL-4Ralphatranscription is also activated in response to cisplatin, a DNA-damaging agent known to induce p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abrogates the induction of the IL-4Ralpha gene after cisplatin treatment. Furthermore, we identified a p73-binding site in the first intron of the IL-4Ralpha gene that can directly interact with the p73 protein in vivo. This p73-binding site consists of eight copies of a 10-bp consensus p53-binding motif and is a functional response element that is relatively specific for p73 among the p53 family. p73beta promoted localized nucleosomal acetylation through recruitment of coactivator p300, indicating that p73 regulates transcription of IL-4Ralpha through the unique p73-binding site. We also found that p73beta-transfected tumor cells are sensitive to IL-4-mediated apoptosis. Our data suggest that IL-4Ralpha could mediate, in part, certain immune responses and p73-dependent cell death.

Establishment of a universal and rational gene detection strategy through three-way junction-based remote transduction.

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Tang, Yidan; Lu, Baiyang; Zhu, Zhentong; Li, Bingling

2018-01-21

The polymerase chain reaction and many isothermal amplifications are able to achieve super gene amplification. Unfortunately, most commonly-used transduction methods, such as dye staining and Taqman-like probing, still suffer from shortcomings including false signals or difficult probe design, or are incompatible with multi-analysis. Here a universal and rational gene detection strategy has been established by translating isothermal amplicons to enzyme-free strand displacement circuits via three-way junction-based remote transduction. An assistant transduction probe was imported to form a partial hybrid with the target single-stranded nucleic acid. After systematic optimization the hybrid could serve as an associative trigger to activate a downstream circuit detector via a strand displacement reaction across the three-way junction. By doing so, the detection selectivity can be double-guaranteed through both amplicon-transducer recognition and the amplicon-circuit reaction. A well-optimized circuit can be immediately applied to a new target detection through simply displacing only 10-12 nt on only one component, according to the target. More importantly, this property for the first time enables multi-analysis and logic-analysis in a single reaction, sharing a single fluorescence reporter. In an applicable model, trace amounts of Cronobacter and Enterobacteria genes have been clearly distinguished from samples with no bacteria or one bacterium, with ultra-high sensitivity and selectivity.

Interleukin-21 gene polymorphism rs2221903 is associated with disease activity in patients with rheumatoid arthritis.

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Malinowski, Damian; Paradowska-Gorycka, Agnieszka; Safranow, Krzysztof; Pawlik, Andrzej

2017-08-01

Interleukin-21 (IL-21) is a cytokine which plays a significant role in the pathogenesis and disease activity of rheumatoid arthritis (RA). Genetic polymorphisms in the IL-21 gene may alter the synthesis of IL-21. The aim of this study was to examine IL-21 and IL-21R polymorphisms in patients with RA. We examined 422 patients with RA and 338 healthy controls. Single nucleotide polymorphisms (SNPs) within the IL-21 (rs6822844 G>T, rs6840978 C>T, rs2221903 T>C) and IL-21R (rs2285452 G>A) genes were genotyped using TaqMan genotyping assays. There were no statistically significant differences in the distribution of studied genotypes and alleles between RA patients and the control group. To examine whether IL-21 polymorphisms affect disease activity in RA patients, we compared the distribution of IL-21 genotypes between patients with DAS28 ≤ 2.5 (patients with remission of disease symptoms) and patients with DAS28 > 2.5 (patients with active RA). Among patients with DAS28 > 2.5, increased prevalence of rs2221903 CT and CC genotypes was observed (OR = 1.54; 95% CI: 1.04-2.28; p = 0.035). The results of this study suggest that IL-21 and IL-21R gene polymorphisms are not risk loci for RA susceptibility, whereas the IL-21 rs2221903 polymorphism is associated with disease activity.

Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

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Rupreht Ruth

2007-11-01

Full Text Available Abstract Background Hereditary hemochromatosis (HH is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. Methods Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. Results The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI 11.5 – 14.2%, 1.8% (95% CI 1.4 – 2.5% and 3.6% (95% CI 3.0 – 4.5%, respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. Conclusion The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for

Oxytocin receptor gene polymorphisms are associated with human directed social behavior in dogs (Canis familiaris.

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Anna Kis

Full Text Available The oxytocin system has a crucial role in human sociality; several results prove that polymorphisms of the oxytocin receptor gene are related to complex social behaviors in humans. Dogs' parallel evolution with humans and their adaptation to the human environment has made them a useful species to model human social interactions. Previous research indicates that dogs are eligible models for behavioral genetic research, as well. Based on these previous findings, our research investigated associations between human directed social behaviors and two newly described (-212AG, 19131AG and one known (rs8679684 single nucleotide polymorphisms (SNPs in the regulatory regions (5' and 3' UTR of the oxytocin receptor gene in German Shepherd (N = 104 and Border Collie (N = 103 dogs. Dogs' behavior traits have been estimated in a newly developed test series consisting of five episodes: Greeting by a stranger, Separation from the owner, Problem solving, Threatening approach, Hiding of the owner. Buccal samples were collected and DNA was isolated using standard protocols. SNPs in the 3' and 5' UTR regions were analyzed by polymerase chain reaction based techniques followed by subsequent electrophoresis analysis. The gene-behavior association analysis suggests that oxytocin receptor gene polymorphisms have an impact in both breeds on (i proximity seeking towards an unfamiliar person, as well as their owner, and on (ii how friendly dogs behave towards strangers, although the mediating molecular regulatory mechanisms are yet unknown. Based on these results, we conclude that similarly to humans, the social behavior of dogs towards humans is influenced by the oxytocin system.

Oxytocin receptor gene polymorphisms are associated with human directed social behavior in dogs (Canis familiaris).

Science.gov (United States)

Kis, Anna; Bence, Melinda; Lakatos, Gabriella; Pergel, Enikő; Turcsán, Borbála; Pluijmakers, Jolanda; Vas, Judit; Elek, Zsuzsanna; Brúder, Ildikó; Földi, Levente; Sasvári-Székely, Mária; Miklósi, Adám; Rónai, Zsolt; Kubinyi, Enikő

2014-01-01

The oxytocin system has a crucial role in human sociality; several results prove that polymorphisms of the oxytocin receptor gene are related to complex social behaviors in humans. Dogs' parallel evolution with humans and their adaptation to the human environment has made them a useful species to model human social interactions. Previous research indicates that dogs are eligible models for behavioral genetic research, as well. Based on these previous findings, our research investigated associations between human directed social behaviors and two newly described (-212AG, 19131AG) and one known (rs8679684) single nucleotide polymorphisms (SNPs) in the regulatory regions (5' and 3' UTR) of the oxytocin receptor gene in German Shepherd (N = 104) and Border Collie (N = 103) dogs. Dogs' behavior traits have been estimated in a newly developed test series consisting of five episodes: Greeting by a stranger, Separation from the owner, Problem solving, Threatening approach, Hiding of the owner. Buccal samples were collected and DNA was isolated using standard protocols. SNPs in the 3' and 5' UTR regions were analyzed by polymerase chain reaction based techniques followed by subsequent electrophoresis analysis. The gene-behavior association analysis suggests that oxytocin receptor gene polymorphisms have an impact in both breeds on (i) proximity seeking towards an unfamiliar person, as well as their owner, and on (ii) how friendly dogs behave towards strangers, although the mediating molecular regulatory mechanisms are yet unknown. Based on these results, we conclude that similarly to humans, the social behavior of dogs towards humans is influenced by the oxytocin system.

Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel

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Hindiyeh, Musa; Levy, Virginia; Azar, Roberto; Varsano, Noemi; Regev, Liora; Shalev, Yael; Grossman, Zehava; Mendelson, Ella

2005-01-01

The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories. PMID:15695650

Correlation of Fetuin-A gene rs1071592 and rs2593813 single nucleotide polymorphisms with polycystic ovary syndrome

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Juan YI

2016-10-01

Full Text Available Objective 　To investigate the relations of Fetuin-A gene rs1071592 and rs2593813 single nucleotide polymorphisms (SNPs with the affect ability to polycystic ovary syndrome (PCOS and its endocrine and metabolic characteristics in Chongqing Han population. Methods 　A case-control study was performed in Chinese Han subjects. The clinical data of 156 cases of normal control and 147 cases of PCOS patients were collected, and their blood glucose, lipids, sex hormone and other biochemical indexes were determined, the SNPs of rs1071592 and rs2593813 were genotyped by TaqMan SNP Genotyping Assay. Hyperinsulinemic-euglycemic clamp was performed in 147 PCOS women and 20 controls. The relative risk of developing PCOS in women with rs1071592 genotype was assessed using a binary logistic regression analysis. Results 　The distribution frequency of Fetuin-A gene homozygous rs1071592 AA genotype and A allele was significantly increased in PCOS patients than in controls (Pc0.05. Binary logistic regression analysis showed that the risk of developing PCOS was 4.93 times high in women with AA genotype of rs1071592 (OR=4.933, 95%CI 1.593-15.278, P0.05. Conclusion 　People with SNPs variants of rs1071592 in Fetuin-A gene may have an increased genetic susceptibility to PCOS. However, there won't be significant relationship between SNP of rs2593813 at Fetuin-A gene and PCOS. DOI: 10.11855/j.issn.0577-7402.2016.09.07

Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass.

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López-Mondéjar, Rubén; Antón, Anabel; Raidl, Stefan; Ros, Margarita; Pascual, José Antonio

2010-04-01

The species of the genus Trichoderma are used successfully as biocontrol agents against a wide range of phytopathogenic fungi. Among them, Trichoderma harzianum is especially effective. However, to develop more effective fungal biocontrol strategies in organic substrates and soil, tools for monitoring the control agents are required. Real-time PCR is potentially an effective tool for the quantification of fungi in environmental samples. The aim of this study consisted of the development and application of a real-time PCR-based method to the quantification of T. harzianum, and the extrapolation of these data to fungal biomass values. A set of primers and a TaqMan probe for the ITS region of the fungal genome were designed and tested, and amplification was correlated to biomass measurements obtained with optical microscopy and image analysis, of the hyphal length of the mycelium of the colony. A correlation of 0.76 between ITS copies and biomass was obtained. The extrapolation of the quantity of ITS copies, calculated based on real-time PCR data, into quantities of fungal biomass provides potentially a more accurate value of the quantity of soil fungi. Copyright 2009 Elsevier Ltd. All rights reserved.

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

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Weighardt, Florian; Barbati, Cristina; Paoletti, Claudia; Querci, Maddalena; Kay, Simon; De Beuckeleer, Marc; Van den Eede, Guy

2004-01-01

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement

Characterization of Phytophthora nicotianae isolates in southeast Spain and their detection and quantification through a real-time TaqMan PCR.

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Blaya, Josefa; Lacasa, Carmen; Lacasa, Alfredo; Martínez, Victoriano; Santísima-Trinidad, Ana B; Pascual, Jose A; Ros, Margarita

2015-04-01

The soil-borne pathogens Phytophthora nicotianae and P. capsici are the causal agents of root and stem rot of many plant species. Although P. capsici was considered the causal agent in one of the main pepper production areas of Spain to date, evidence of the presence of P. nicotianae was found. We aimed to survey the presence of P. nicotianae and study the variability in its populations in this area in order to improve the management of Tristeza disease. A new specific primer and a TaqMan probe were designed based on the internal transcribed spacer regions of ribosomal DNA to detect and quantify P. nicotianae. Both morphological and molecular analysis showed its presence and confirmed it to be the causal agent of the Phytophthora disease symptoms in the studied area. The genetic characterization among P. nicotianae populations showed a low variability of genetic diversity among the isolates. Only isolates of the A2 mating type were detected. Not only is a specific and early detection of P. nicotianae essential but also the study of genetic variability among isolates for the appropriate management of the disease, above all, in producing areas with favorable conditions for the advance of the disease. © 2014 Society of Chemical Industry.

Identification and characterization of novel mutations of the major Fanconi anemia gene FANCA in the Japanese population.

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Yagasaki, Hiroshi; Hamanoue, Satoshi; Oda, Tsukasa; Nakahata, Tatsutoshi; Asano, Shigetaka; Yamashita, Takayuki

2004-12-01

Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis, with at least 11 complementation groups. FANCA, a gene for group A, accounts for the majority of FA patients. Previous studies of FANCA mutations revealed high allelic heterogeneity, frequent occurrence of large deletions, and interpopulation differences. However, systematic mutational analysis, including gene dosage assay to detect large deletions, has not been documented for Asian populations. A newly developed TaqMan quantitative PCR-based gene dosage assay, combined with sequencing of exons and cDNA fragments, allowed for detection of 48 mutant alleles of FANCA in 27 (77%) of 35 unrelated Japanese FA families with no detectable mutations in FANCC or FANCG. We identified 29 different mutations (21 nucleotide substitutions or small deletions/insertions and eight large deletions), at least 20 of which were novel. The FANCA mutational spectrum of the Japanese was different from that of other ethnic groups so far studied. This is the largest scale of mutation analysis of FANCA in the Japanese population. Characterization of these mutations provided new information regarding the mutagenesis mechanisms and structure-function relationship of FANCA. Specifically, our data suggest that diverse mechanisms including nonhomologous recombination as well as Alu-mediated homologous recombination are involved in the generation of large deletions in FANCA. Copyright 2004 Wiley-Liss, Inc.

Vanadium NMR Chemical Shifts of (Imido)vanadium(V) Dichloride Complexes with Imidazolin-2-iminato and Imidazolidin-2-iminato Ligands: Cooperation with Quantum-Chemical Calculations and Multiple Linear Regression Analyses.

Science.gov (United States)

Yi, Jun; Yang, Wenhong; Sun, Wen-Hua; Nomura, Kotohiro; Hada, Masahiko

2017-11-30

The NMR chemical shifts of vanadium ( 51 V) in (imido)vanadium(V) dichloride complexes with imidazolin-2-iminato and imidazolidin-2-iminato ligands were calculated by the density functional theory (DFT) method with GIAO. The calculated 51 V NMR chemical shifts were analyzed by the multiple linear regression (MLR) analysis (MLRA) method with a series of calculated molecular properties. Some of calculated NMR chemical shifts were incorrect using the optimized molecular geometries of the X-ray structures. After the global minimum geometries of all of the molecules were determined, the trend of the observed chemical shifts was well reproduced by the present DFT method. The MLRA method was performed to investigate the correlation between the 51 V NMR chemical shift and the natural charge, band energy gap, and Wiberg bond index of the V═N bond. The 51 V NMR chemical shifts obtained with the present MLR model were well reproduced with a correlation coefficient of 0.97.

Social Health Insurance-Based Simultaneous Screening for 154 Mutations in 19 Deafness Genes Efficiently Identified Causative Mutations in Japanese Hearing Loss Patients.

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Kentaro Mori

Full Text Available Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500-1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, from August 2015, this genetic testing has been expanded to screen for 154 mutations in 19 deafness genes using targeted genomic enrichment with massively parallel DNA sequencing combined with the Invader assay and TaqMan genotyping. For this study we analyzed 717 unrelated Japanese hearing loss patients. The total allele frequency of 154 mutations in 19 deafness genes was 32.64% (468/1434 and the total numbers of cases associated with at least one mutation was 44.07% (316/717. Among these, we were able to diagnose 212 (30% patients, indicating that the present screening could efficiently identify causative mutations in hearing loss patients. It is noteworthy that 27 patients (3.8% had coexistent multiple mutations in different genes. Five of these 27 patients (0.7%, 5/717 overall were diagnosed with genetic hearing loss affected by concomitant with responsible mutations in more than two different genes. For patients identified with multiple mutations in different genes, it is necessary to consider that several genes might have an impact on their phenotypes.

Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

International Nuclear Information System (INIS)

Al-Salman, Fadheela; Plant, Nick

2012-01-01

The polychlorinated biphenyl group possesses high environmental persistence, leading to bioaccumulation and a number of adverse effects in mammals. Whilst coplanar PCBs elicit their toxic effects through agonism of the aryl hydrocarbon receptor; however, non-coplanar PCBs are not ligands for AhR, but may be ligands for members of the nuclear receptor family of proteins. To better understand the biological actions of non-coplanar PCBs, we have undertaken a systematic analysis of their ability to activate PXR and CAR-mediated effects. Cells were exposed to a range of non-coplanar PCBs (99, 138, 153, 180 and 194), or the coplanar PCB77: Direct activation of PXR and CAR was measured using a mammalian receptor activation assay in human liver cells, with rifampicin and CITCO used as positive controls ligands for PXR and CAR, respectively; activation of target gene expression was examined using reporter gene plasmids for CYP3A4 and MDR1 transfected into liver, intestine and lung cell lines. Several of the non-coplanar PCBs directly activated PXR and CAR, whilst the coplanar PCB77 did not. Non-coplanar PCBs were also able to activate PXR/CAR target gene expression in a substitution- and tissue-specific manner. Non-coplanar PCBs act as direct activators for the nuclear receptors PXR and CAR, and are able to elicit transcriptional activation of target genes in a substitution- and tissue-dependent manner. Chronic activation of PXR/CAR is linked to adverse effects and must be included in any risk assessment of PCBs. -- Highlights: ► Several Non-coplanar PCBs are able to directly activate both PXR and CAR in vitro. ► PCB153 is the most potent direct activator of PXR and CAR nuclear receptors. ► Non-coplanar PCB activation of CYP3A4/MDR1 reporter genes is structure-dependent. ► Non-coplanar PCB activate CYP3A4/MDR1 reporter genes in a tissue-dependent. ► PCB153 is the most potent activator of PXR/CAR target gene in all tissues.

Hypoxia-Response Element (HRE)–Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium

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Li, Wande

2013-01-01

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5′-ACGTG-3′) exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10–100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)–treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at −387/−383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation. PMID:23161664

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

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Butz, Henriett; Németh, Kinga; Czenke, Dóra; Likó, István; Czirják, Sándor; Zivkovic, Vladimir; Baghy, Kornélia; Korbonits, Márta; Kovalszky, Ilona; Igaz, Péter; Rácz, Károly; Patócs, Attila

2017-07-01

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

LENUS (Irish Health Repository)

Perry, Antoinette S

2013-04-15

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2\\/20) (SFRP1), 64.86% (48\\/74) (SFRP2), 0% (0\\/20) (SFRP4) and 60% (12\\/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6\\/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7\\/69), p < 0.0001) and BPH (11.43% (4\\/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria

International Nuclear Information System (INIS)

Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina

2015-01-01

Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3′)-IIa/nptII and aph(3′)-IIIa/nptIII – frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides – was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31–856) and 85% for nptIII (1190 copies/g soil; 13–61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0–3.3%) were positive for nptIII, none for nptII (0–0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. - Highlights: • ARM genes may act as environmental pollutants under certain conditions. • Vital criteria for rating are low endemic presence and anthropogenic ARG immission. • Agricultural soils were rarely positive for nptII with few gene copy numbers. • Most fields were nptIII positive with variable but also increased allele frequency. • NptII/III qualify as pollutants in the tested settings with low endemic abundances. - ARM genes may be considered as environmental pollutants if anthropogenic activities raise their abundance above naturally occurring background levels in exposed ecosystems.

application of multilinear regression analysis in modeling of soil

African Journals Online (AJOL)

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Accordingly [1, 3] in their work, they applied linear regression ... (MLRA) is a statistical technique that uses several explanatory ... order to check this, they adopted bivariate correlation analysis .... groups, namely A-1 through A-7, based on their relative expected ..... Multivariate Regression in Gorgan Province North of Iran” ...

Association analysis of two single-nucleotide polymorphisms of the RELN gene with autism in the South African population

KAUST Repository

Sharma, Jyoti Rajan

2013-02-01

Background: Autism (MIM209850) is a neurodevelopmental disorder characterized by a triad of impairments, namely impairment in social interaction, impaired communication skills, and restrictive and repetitive behavior. A number of family and twin studies have demonstrated that genetic factors play a pivotal role in the etiology of autistic disorder. Various reports of reduced levels of reelin protein in the brain and plasma in autistic patients highlighted the role of the reelin gene (RELN) in autism. There is no such published study on the South African (SA) population. Aims: The aim of the present study was to find the genetic association of intronic rs736707 and exonic rs362691 (single-nucleotide polymorphisms [SNPs] of the RELN gene) with autism in a SA population. Methods: Genomic DNA was isolated from cheek cell swabs from autistic (136) as well as control (208) subjects. The TaqMan ® Real-Time polymerase chain reaction and genotyping assay was utilized to determine the genotypes. Results: A significant association of SNP rs736707, but not for SNP rs362691, with autism in the SA population is observed. Conclusion: There might be a possible role of RELN in autism, especially for SA populations. The present study represents the first report on genetic association studies on the RELN gene in the SA population. © 2013, Mary Ann Liebert, Inc.

Macrophage-specific gene functions in Spi1-directed innate immunity

NARCIS (Netherlands)

Zakrzewska, Anna; Cui, Chao; Stockhammer, Oliver W.; Benard, Erica L.; Spaink, Herman P.; Meijer, Annemarie H.

2010-01-01

The Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1, the controlled gene group remains largely unknown. To identify genes dependent on Spi1, we used a microarray strategy using a knockdown approach in zebrafish embryos

Rapid approach for cloning bacterial single-genes directly from soils ...

African Journals Online (AJOL)

Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of ...

Identification of four squid species by quantitative real-time polymerase chain reaction.

Science.gov (United States)

Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

2016-02-01

Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Association and Interaction Effect of AGTR1 and AGTR2 Gene Polymorphisms with Dietary Pattern on Metabolic Risk Factors of Cardiovascular Disease in Malaysian Adults.

Science.gov (United States)

Yap, Roseline Wai Kuan; Shidoji, Yoshihiro; Yap, Wai Sum; Masaki, Motofumi

2017-08-09

Gene-diet interaction using a multifactorial approach is preferred to study the multiple risk factors of cardiovascular disease (CVD). This study examined the association and gene-diet interaction effects of the angiotensin II type 1 receptor ( AGTR1 ) gene (rs5186), and type 2 receptor ( AGTR2 ) gene (rs1403543) polymorphisms on metabolic risk factors of CVD in Malaysian adults. CVD parameters (BMI, blood pressure, glycated hemoglobin, total cholesterol (TC), triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), and TC/HDL-C ratio), and constructed dietary patterns "vegetables, fruits, and soy diet" (VFSD), and "rice, egg, and fish diet" (REFD) were obtained from previous studies. Genotyping analysis was performed by real-time PCR using Taqman probes. The subjects were 507 adults (151 Malays; 179 Chinese; and 177 Indians). Significant genetic associations were obtained on blood lipids for rs5186 in Malays and Chinese, and rs1403543 in Chinese females. The significant gene-diet interaction effects after adjusting for potential confounders were: rs5186 × VFSD on blood pressure in Malays ( p = 0.016), and in Chinese on blood lipids for rs5186 × REFD ( p = 0.009-0.023), and rs1403543 × VFSD in female subjects ( p = 0.001-0.011). Malays and Chinese showed higher risk for blood pressure and/or lipids involving rs5186 and rs1403543 SNPs together with gene-diet interactions, but not Indians.

Association study of ghrelin receptor gene polymorphisms in rheumatoid arthritis.

Science.gov (United States)

Robledo, G; Rueda, B; Gonzalez-Gay, M A; Fernández, B; Lamas, J R; Balsa, A; Pascual-Salcedo, D; García, A; Raya, E; Martín, J

2010-01-01

Ghrelin is a newly characterised growth hormone (GH) releasing peptide widely distributed that may play an important role in the regulation of metabolic balance in inflammatory diseases such as rheumatoid arthritis (RA) by decreasing the pro-inflammatory Th1 responses. In this study we investigated the possible contribution of several polymorphisms in the functional Ghrelin receptor to RA susceptibility. A screening of 3 single nucleotide polymorphisms (SNPs) was performed in a total of 950 RA patients and 990 healthy controls of Spanish Caucasian origin. Genotyping of all 3 SNPs was performed by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. We observed no statistically significant deviation between RA patients and controls for the GHSR SNPs analysed. In addition, we performed a haplotype analysis that did not reveal an association with RA susceptibility. The stratification analysis for the presence of shared epitope (SE), rheumatoid factor (RF) or antibodies anti cyclic citrullinated peptide (anti-CCP) did not detect significant association of the GHSR polymorphisms with RA. These findings suggest that the GHSR gene polymorphisms do not appear to play a major role in RA genetic predisposition in our population.

Adaptive population divergence and directional gene flow across steep elevational gradients in a climate-sensitive mammal.

Science.gov (United States)

Waterhouse, Matthew D; P Erb, Liesl; Beever, Erik A; Russello, Michael A

2018-04-25

The ecological effects of climate change have been shown in most major taxonomic groups; however, the evolutionary consequences are less well-documented. Adaptation to new climatic conditions offers a potential long-term mechanism for species to maintain viability in rapidly changing environments, but mammalian examples remain scarce. The American pika (Ochotona princeps) has been impacted by recent climate-associated extirpations and range-wide reductions in population sizes, establishing it as a sentinel mammalian species for climate change. To investigate evidence for local adaptation and reconstruct patterns of genomic diversity and gene flow across rapidly changing environments, we used a space-for-time design and restriction site-associated DNA sequencing to genotype American pikas along two steep elevational gradients at 30,966 SNPs and employed independent outlier detection methods that scanned for genotype-environment associations. We identified 338 outlier SNPs detected by two separate analyses and/or replicated in both transects, several of which were annotated to genes involved in metabolic function and oxygen transport. Additionally, we found evidence of directional gene flow primarily downslope from high-elevation populations, along with reduced gene flow at outlier loci. If this trend continues, elevational range contractions in American pikas will likely be from local extirpation rather than upward movement of low-elevation individuals; this, in turn, could limit the potential for adaptation within this landscape. These findings are of particular relevance for future conservation and management of American pikas and other elevationally-restricted, thermally-sensitive species. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Paquette, Stéphane G. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Banner, David [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Chi, Le Thi Bao [Department of Microbiology, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Carlo Urbani Centre, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Leon, Alberto J. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); Xu, Luoling; Ran, Longsi [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Huang, Stephen S.H. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Farooqui, Amber [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); and others

2014-01-05

Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

International Nuclear Information System (INIS)

Paquette, Stéphane G.; Banner, David; Chi, Le Thi Bao; Leon, Alberto J.; Xu, Luoling; Ran, Longsi; Huang, Stephen S.H.; Farooqui, Amber

2014-01-01

Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development

Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

International Nuclear Information System (INIS)

Lee, Jin Hwa; Park, Byeong Ho; Kim, Chan Sung

2003-01-01

Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x10 11 particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy

Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

Energy Technology Data Exchange (ETDEWEB)

Lee, Jin Hwa; Park, Byeong Ho; Kim, Chan Sung [Dong-A University College of Medicine, Pusan (Korea, Republic of)

2003-02-01

Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x10{sup 11} particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy.

Identification of pyrG Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Pleurotus ostreatus.

Science.gov (United States)

Zheng, Shi; Shan, Luying; Zhuang, Yongliang; Shang, Ying

2018-03-01

As a well-known edible fungus rich in nutrients, Pleurotus ostreatus has been used as an alternative to expensive wild edible fungi. Specifically, the fact that using P. ostreatus instead of other expensive wild edible fungi has damaged the rights and interests of consumers. Among the existing methods for detection of food adulteration, the amplification of endogenous reference gene is the most accurate method. However, an ideal endogenous reference gene for P. ostreatus has yet to be developed. In this study, a DNA extraction method for P. ostreatus was optimized, and pyrG was selected as a species-specific gene through sequence alignment. This gene was subsequently subjected to qualitative and quantitative Polymerase Chain Reaction (PCR) assays with 3 different P. ostreatus varieties and 7 other species. A low detection limit of 5 pg/μL was obtained by TaqMan quantitative PCR, and no pyrG amplification product was observed in the 7 other species. No allelic variation was detected in P. ostreatus varieties. These experiments confirmed that pyrG was an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus. This method was also suitable for the examination of processed P. ostreatus samples and determination of adulteration in wild mushrooms. The pyrG gene was chosen as an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus, and the detection limit was 5 pg/μL for the quantification. This method is used not only for raw materials but also for processed P. ostreatus products and other processed mushroom foods. © 2018 Institute of Food Technologists®.

Comprehensive analysis of three TYK2 gene variants in the susceptibility to Chagas disease infection and cardiomyopathy

Science.gov (United States)

Carmona, F. David; Dolade, Nuria; Vargas, Sofia; Echeverría, Luis Eduardo; González, Clara Isabel; Martin, Javier

2018-01-01

Tyrosine kinase 2 (TYK2) is a member of the Janus kinases family implicated in the signal transduction of type I interferons and several interleukins. It has been described that genetic mutations within TYK2 lead to multiple deleterious effects in the immune response. In this work, we have analyzed three functional independent variants from the frequency spectrum on the TYK2 gene (common and low-frequency variants) suggested to reduce the function of the gene in mediating cytokine signaling and the susceptibility to infections by Trypanosoma cruzi and/or the development of Chagas cardiomyopathy in the Colombian population. A total of 1,323 individuals from a Colombian endemic region for Chagas disease were enrolled in the study. They were classified as seronegative (n = 445), seropositive asymptomatic (n = 336), and chronic Chagas Cardiomyopathy subjects (n = 542). DNA samples were genotyped using TaqMan probes. Our results showed no statistically significant differences between the allelic frequencies of the three analyzed variants when seropositive and seronegative individuals were compared, therefore these variants were not associated with susceptibility to Chagas disease. Moreover, when Chagas cardiomyopathy patients were compared to asymptomatic patients, no significant associations were found. Previous reports highlighted the association of this gene in immune-related disorders under an autoimmunity context, but not predisposing patients to infectious diseases, which is consistent with our findings. Therefore, according to our results, TYK2 gene variants do not seem to play an important role in Chagas disease susceptibility and/or chronic Chagas cardiomyopathy. PMID:29304122

Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

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Mattei Marie-Geneviève

2004-07-01

Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

Polymorphisms within the neuronal cadherin (CDH2) gene are associated with obsessive-compulsive disorder (OCD) in a South African cohort.

Science.gov (United States)

McGregor, N W; Lochner, C; Stein, D J; Hemmings, S M J

2016-02-01

OCD is characterised by recurrent obsessions and compulsions that result in severe distress and increased risk for comorbidity. Recently published findings have indicated that the neuronal cadherin gene (CDH2) plays a role in the development of canine OCD, and led us to investigate the human ortholog, CDH2, in a human OCD cohort. Seven CDH2 polymorphisms were selected and genotyped in a South African Caucasian cohort of 234 OCD patients and 180 healthy controls using TaqMan assays. Polymorphisms were analysed in a single-locus and haplotypic context. Of the seven polymorphisms, two reached statistical significance for OCD under additive and codominant models of inheritance (rs1120154 and rs12605662). CDH2 SNP, rs1120154, C-allele carriers were found to be significantly associated with lower risk to develop OCD compared to TT-homozygotes (OR = 0.49; 95% CI: 0.32-0.75; p OCD compared to TT-homozygotes (OR = 0.46; 95% CI: 0.30-0.71; p OCD diagnosis (*rs8087457-rs1148374: A-T). Polymorphisms within the CDH2 gene are associated with susceptibility to OCD in a South African cohort.

Research progress in machine learning methods for gene-gene interaction detection.

Science.gov (United States)

Peng, Zhe-Ye; Tang, Zi-Jun; Xie, Min-Zhu

2018-03-20

Complex diseases are results of gene-gene and gene-environment interactions. However, the detection of high-dimensional gene-gene interactions is computationally challenging. In the last two decades, machine-learning approaches have been developed to detect gene-gene interactions with some successes. In this review, we summarize the progress in research on machine learning methods, as applied to gene-gene interaction detection. It systematically examines the principles and limitations of the current machine learning methods used in genome wide association studies (GWAS) to detect gene-gene interactions, such as neural networks (NN), random forest (RF), support vector machines (SVM) and multifactor dimensionality reduction (MDR), and provides some insights on the future research directions in the field.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

Science.gov (United States)

Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

2016-11-18

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair

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Kmiec Eric B

2007-02-01

Full Text Available Abstract Background Single-stranded oligonucleotides (ssODN are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time. Results First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours. Conclusion Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes.

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

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Corti Stefania

2011-03-01

Full Text Available Abstract Background Duchenne and Becker Muscular dystrophies (DMD/BMD are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements. Methods We selected 47 patients (41 families; 35 DMD, 6 BMD without deletions and duplications in DMD gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis. This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis. Results We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients, followed by TAG (n = 7 and TAA (n = 4. We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the DMD gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65. Conclusion The analysis of our patients' sample, carrying point mutations or complex rearrangements in DMD gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects

Relationships between genetic polymorphisms in inflammation-related factor gene and the pathogenesis of nasopharyngeal cancer.

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Qu, Yan-Li; Yu, Hong; Chen, Yan-Zhi; Zhao, Yu-Xia; Chen, Guang-Jun; Bai, Lu; Liu, Dan; Su, Hong-Xin; Wang, He-Tong

2014-09-01

Our study aims to discuss the association between inflammation-related factors such as single nucleotide polymorphisms (SNPs) with susceptibility and recurrence in nasopharyngeal carcinoma. We used Taqman real-time polymerase chain reaction (PCR) to characterize the genetic variation of five SNPs in 194 nasopharyngeal carcinoma patients and 231 healthy subjects. All statistical analysis is performed with statistical product and service solutions v13.0; odds ratio (OR) value and 95 % confidence interval (CI) were calculated. There is no relationship between TGFβ1 -869 T/C, IL-6 -634C/G, TGFβ1 -509C/T, IL1 -511C/T and nasopharyngeal carcinoma susceptibility. Both single factor and multiple factors analysis showed that IL1a -889 T/T genotype is significantly associated with nasopharyngeal carcinoma in decreasing the risk of nasopharyngeal carcinoma. A highly significant association was found between IL1a -889 T/T genotype and protective genotype as defined by various pathological types. This is more obvious in the protective genotype of the non-keratin-type squamous carcinoma undifferentiated type. We also discovered that genotype G/G and C/G + G/G of IL6 -634 gene are associated with reduced recurrence of nasopharyngeal carcinoma. IL1a -889 gene polymorphism and susceptibility is related to nasopharyngeal carcinoma and can potentially decrease the risk of nasopharyngeal carcinoma in the Han Chinese population in north China. IL1-889 TT genotype is protective genotype for nasopharyngeal carcinoma. We have provided evidence that the GG genotype of the IL6 -634 gene is associated with recurrent risk of nasopharyngeal carcinoma. The G allele is the protective gene of nasopharyngeal carcinoma recurrence.

Association of pro-ghrelin and GHS-R1A gene polymorphisms and haplotypes with heavy alcohol use and body mass.

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Landgren, Sara; Jerlhag, Elisabet; Zetterberg, Henrik; Gonzalez-Quintela, Arturo; Campos, Joaquin; Olofsson, Ulrica; Nilsson, Staffan; Blennow, Kaj; Engel, Jörgen A

2008-12-01

Ghrelin, an orexigenic peptide, acts on growth hormone secretagogue receptors (GHS-R1A), expressed in the hypothalamus as well as in important reward nodes such as the ventral tegmental area. Interestingly, ghrelin has been found to activate an important part of the reward systems, i.e., the cholinergic-dopaminergic reward link. Additionally, the rewarding and neurochemical properties of alcohol are, at least in part, mediated via this reward link. There is comorbidity between alcohol dependence and eating disorders. Thus, plasma levels of ghrelin are altered in patients with addictive behaviors such as alcohol and nicotine dependence and in binge eating disorder. This overlap prompted as to investigate the pro-ghrelin and GHS-R1A genes in a haplotype analysis of heavy alcohol-using individuals. A total of 417 Spanish individuals (abstainers, moderate, and heavy alcohol drinkers) were investigated in a haplotype analysis of the pro-ghrelin and GHS-R1A genes. Tag SNPs were chosen using HapMap data and the Tagger and Haploview softwares. These SNPs were then genotyped using TaqMan Allelic Discrimination. SNP rs2232165 of the GHS-R1A gene was associated with heavy alcohol consumption and SNP rs2948694 of the same gene as well as haplotypes of both the pro-ghrelin and the GHS-R1A genes were associated with body mass in heavy alcohol consuming individuals. The present findings are the first to disclose an association between the pro-ghrelin and GHS-R1A genes and heavy alcohol use, further strengthening the role of the ghrelin system in addictive behaviors and brain reward.

Real-time PCR quantification of human complement C4A and C4B genes

Directory of Open Access Journals (Sweden)

Fust George

2006-01-01

Full Text Available Abstract Background The fourth component of human complement (C4, an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. Results A novel quantitative real-time PCR (qPCR technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA. The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118. The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. Conclusion This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.

Role of the X-linked gene GPR174 in autoimmune Addison's disease.

Science.gov (United States)

Napier, C; Mitchell, A L; Gan, E; Wilson, I; Pearce, S H S

2015-01-01

Autoimmune endocrinopathies demonstrate a profound gender bias, but the reasons for this remain obscure. The 1000 genes on the X chromosome are likely to be implicated in this inherent susceptibility; various theories, including skewed X chromosome inactivation and fetal microchimerism, have been proposed. GPR174 is an Xq21 putative purinergic receptor that is widely expressed in lymphoid tissues. A single-nucleotide polymorphism, rs3827440, encoding Ser162Pro, has recently been associated with Graves' disease in Chinese and Polish populations, suggesting a role of this X chromosome gene in autoimmune disease. We investigated the role of rs3827440 in a UK cohort of patients with autoimmune Addison's disease (AAD). Samples from 286 AAD cases and 288 healthy controls were genotyped using TaqMan single-nucleotide polymorphism genotyping assays (C_25954273_10) on the Applied Biosystems 7900HT Fast real-time PCR system. Using a dominant (present/absent) model, the serine-encoding T allele of rs3827440 was present in 189 of 286 AAD patients (66%) compared with 132 of 288 unaffected controls (46%) [P = .010, odds ratio 1.80 (5%-95% confidence interval 1.22-2.67)]. An allele dosage model found a significant excess of the T allele in AAD patients compared with controls [P = .03, odds ratio 1.34 (5%-95% confidence interval 1.07-1.67)]. We have demonstrated a significant association of this X chromosome-encoded immunoreceptor with AAD for the first time. This X-linked gene could have a more generalized role in autoimmunity pathogenesis: G protein-coupled receptors are promising drugable targets, and further work to elucidate the functional role of GPR174 is now warranted.

Gene expression of desaturase (FADS1 and FADS2) and Elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.

Science.gov (United States)

Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Angels; López-Sabater, M Carmen

2013-01-01

It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. AE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson's rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE. Children with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1. Changes in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children.

Gene Expression of Desaturase (FADS1 and FADS2) and Elongase (ELOVL5) Enzymes in Peripheral Blood: Association with Polyunsaturated Fatty Acid Levels and Atopic Eczema in 4-Year-Old Children

Science.gov (United States)

Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Àngels; López-Sabater, M. Carmen

2013-01-01

Abstract Background It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. Methods AE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson’s rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE. Results Children with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1. Conclusions and Significance Changes in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children. PMID:24167612

Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

Science.gov (United States)

Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul

2017-05-01

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.

Association of MEP1A gene variants with insulin metabolism in central European women with polycystic ovary syndrome.

Science.gov (United States)

Lam, Uyen D P; Lerchbaum, Elisabeth; Schweighofer, Natascha; Trummer, Olivia; Eberhard, Katharina; Genser, Bernd; Pieber, Thomas R; Obermayer-Pietsch, Barbara

2014-03-10

Polycystic ovary syndrome (PCOS) shows not only hyperandrogenemia, hirsutism and fertility problems, but also metabolic disturbances including obesity, cardiovascular events and type-2 diabetes. Accumulating evidence suggests some degree of inflammation associated with prominent aspects of PCOS. We aimed to investigate the association of genetic variants 3'UTR rs17468190 (G/T) of the inflammation-associated gene MEP1A (GenBank ID: NM_005588.2) with metabolic disturbances in PCOS and healthy control women. Genetic variants rs17468190 (G/T) of MEP1A gene were analyzed in 576 PCOS women and 206 controls by using the Taqman fluorogenic 5'-exonuclease assay. This polymorphism was tested for association with anthropometric, metabolic, hormonal, and functional parameters of PCOS. There was a borderline significant difference in genotype distribution between PCOS and control women (p=0.046). In overweight/obese PCOS patients, the variants rs17468190 (G/T) in the MEP1A gene are associated with glucose and insulin metabolism. In a dominant model, the GG genotype of the MEP1A gene was more strongly associated with insulin metabolism in overweight/obese PCOS women (body mass index, BMI>25 kg/m(2)), than in GT+TT genotypes. The MEP1A GG-carriers showed a significantly increased homeostatic model assessment - insulin resistance (HOMA-IR) (p=0.003), elevation of fasting insulin (p=0.004) and stimulated insulin (30 min, pdisease modification in PCOS. It might contribute to the abnormalities of glucose metabolism and insulin sensitivity and serve as a diagnostic or therapeutic target gene for PCOS. Copyright © 2014 Elsevier B.V. All rights reserved.

Acute TNF-induced repression of cell identity genes is mediated by NFκB-directed redistribution of cofactors from super-enhancers

DEFF Research Database (Denmark)

Schmidt, Søren Fisker; Larsen, Bjørk Ditlev; Loft, Anne

2015-01-01

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) is directly involved and required for the...... specifically repressing super-enhancer-associated cell identity genes....... binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show...... that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type-specific manner. Our results propose a novel paradigm for NFκB-mediated repression, whereby NFκB selectively redistributes cofactors from high-occupancy enhancers, thereby...

Application of multilinear regression analysis in modeling of soil ...

African Journals Online (AJOL)

The application of Multi-Linear Regression Analysis (MLRA) model for predicting soil properties in Calabar South offers a technical guide and solution in foundation designs problems in the area. Forty-five soil samples were collected from fifteen different boreholes at a different depth and 270 tests were carried out for CBR, ...

Lack of association between rheumatoid arthritis and genetic variants rs10889677, rs11209026 and rs2201841 of IL-23R gene.

Science.gov (United States)

Paradowska-Gorycka, Agnieszka; Malinowski, Damian; Haladyj, Ewa; Olesinska, Marzena; Safranow, Krzysztof; Pawlik, Andrzej

2018-01-19

Rheumatoid arthritis (RA) is an autoimmune diseases, where different genetic variants in cytokine genes may play a pathogenic role. A GWAS in autoimmune diseases highlighted the IL-23R gene as a one of the susceptibility factors. We examined three candidate single nucleotide polymorphisms (SNPs) rs10889677, rs11209026 and rs2201841 of the IL-23R gene, as well as determined their possible association with RA in a Polish population. The IL-23R gene polymorphisms were genotyped for 422 RA patients and 348 healthy individuals using TaqMan SNP genotyping assay. The genotypes frequency did not deviate from HWE in each examined group. A comparison of the allele as well as genotype frequencies of the IL-23R polymorphisms under codominant, dominant and recessive genetic model revealed no significant differences between RA patients and healthy subjects. We also demonstrated that IL-23R rs2201841 and rs11209026 as well as rs11209026 and rs10889677 were in complete linkage disequilibrium (D'=1.0). Our genotype-phenotype analysis demonstrated that in carriers of rs10889677C and/or rs2201841A allele the RF, extra-articular manifestations and erosion were more frequent present than in patients with rs10889677A and/or rs2201841A allele, although this association was not significant. Present findings indicated that the autoimmune disease-associated genetic variants in IL-23R gene are not associated with RA in the Polish population. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Oral Tolerance: A New Tool for the Treatment of Gastrointestinal Inflammatory Disorders and Liver-Directed Gene Therapy

Directory of Open Access Journals (Sweden)

Yaron Ilan

1999-01-01

Full Text Available Oral tolerance is a method of downregulating an immune response by feeding antigens. The use of oral tolerance toward adenoviruses and colitis-extracted proteins for long term gene therapy and alleviation of experimental colitis, and the mechanisms of tolerance induction are presented. Adenoviruses are efficient vectors in liver-directed gene therapy; however, the antiviral immune response precludes the ability to achieve long term gene expression and prohibits the ability to reinject the recombinant virus. Oral tolerance induction via feeding of viral-extracted proteins prevented the antiadenoviral humoral and cellular immune responses, thus enabling long term gene therapy using these viruses. Moreover, pre-existing immune response to the virus was overcome by tolerance induction, enabling prolonged gene expression in a presensitized host. Inflammatory bowel diseases are immune-mediated disorders where an imbalance between proinflammatory (T helper cell type 1 and anti-inflammatory (T helper cell type 2 cytokines are thought to play a role in the pathogenesis. In the experimental colitis model, the feeding of colitis-extracted proteins downregulated the anticolon immune response. Tolerance induction toward colitis-extracted proteins ameliorated colonic inflammation as shown by decreased diarrhea and reduction of colonic ulcerations, intestinal and peritoneal adhesions, wall thickness and edema. Histological parameters for colitis were markedly improved in tolerized animals. In both models, tolerized animals developed an increase in transforming growth factor-beta, interleukin-4 and interleukin-10, and a decrease in the mRNA of interferon-gamma lymphocytes and serum levels. Adoptive transfer of tolerized lymphocytes enabled the transfer of tolerance toward adenoviruses and colon-extracted proteins. Thus, oral tolerance induces suppressor lymphocytes that mediate immune response downregulation by induction of a shift from a proinflammatory T

Direct detection of the AR-E211 G > A gene polymorphism from blood and tissue samples without DNA isolation.

Science.gov (United States)

Reptova, Silvie; Trtkova, Katerina Smesny; Kolar, Zdenek

2014-04-01

The pathogenesis of prostate cancer (CaP) involves alterations in a gene structure of the androgen receptor (AR). The single nucleotide polymorphism AR-E211 G > A localized in exon 1 of the AR gene (G1733A) was detected using direct polymerase chain reaction and restriction digestion (PCR-RFLP) method on blood and tissue samples without prior DNA isolation. We used blood samples of patients with a diagnosis of benign prostatic hyperplasia (BPH) or CaP. From monitored group of CaP patients were selected specimen in formalin-fixed paraffin-embedded tissue blocks with morphology of BPH and CaP. The main objective of our study was to develop a method based the direct PCR-RFLP analysis from blood and tissue without prior DNA isolation for faster genotyping analysis of a large number of samples. We found no statistically significant differences in allelic % of the AR-E211 G > A polymorphism between BPH and CaP patients (p ≤ 0.8462). Genotyping of the AR-E211 G > A variant in blood was not identical with tumor tissue genotyping analysis. Significant agreement between blood and tissue AR-E211 G > A polymorphism only in non-tumor tissue focus was confirmed. Although we analyzed a limited number of the tissue samples, we suppose that a presence of the minor allele A may be associated with cancer transformation-induced changes of the modified AR gene.

Expression and Sequence Variants of Inflammatory Genes; Effects on Plasma Inflammation Biomarkers Following a 6-Week Supplementation with Fish Oil.

Science.gov (United States)

Cormier, Hubert; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2016-03-15

(1) BACKGROUND: A growing body of literature suggest that polymorphisms (SNPs) from inflammation-related genes could possibly play a role in cytokine production and then interact with dietary n-3 fatty acids (FAs) to modulate inflammation. The aim of the present study was to test whether gene expression of selected inflammatory genes was altered following an n-3 PUFA supplementation and to test for gene-diet interactions modulating plasma inflammatory biomarker levels. (2) METHODS: 191 subjects completed a 6-week n-3 FA supplementation with 5 g/day of fish oil. Gene expression of TNF-α and IL6 was assessed in peripheral blood mononuclear cells (PBMCs) using the TaqMan technology. Genotyping of 20 SNPs from the TNF-LTA gene cluster, IL1β, IL6 and CRP genes was performed. (3) RESULTS: There was no significant reduction of plasma IL-6, TNF-α and C-reactive protein (CRP) levels after the 6-week fish oil supplementation. TNF-α and IL6 were slightly overexpressed in PBMCs after the supplementation (fold changes of 1.05 ± 0.38 and 1.18 ± 0.49, respectively (n = 191)), but relative quantification (RQ) within the -0.5 to 2.0 fold are considered as nonbiologically significant. In a MIXED model for repeated measures adjusted for the effects of age, sex and BMI, gene by supplementation interaction effects were observed for rs1143627, rs16944, rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF-α levels and for rs1800629 on CRP levels (p < 0.05 for all). (4) CONCLUSIONS: This study shows that a 6-week n-3 FA supplementation with 5 g/day of fish oil did not alter gene expression levels of TNF-α and IL6 in PBMCs and did not have an impact on inflammatory biomarker levels. However, gene-diet interactions were observed between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels.

Quantitative detection of Fusarium spp. and its correlation with fumonisin content in maize from South African subsistence farmers

NARCIS (Netherlands)

Waalwijk, C.; Koch, S.H.; Ncube, E.; Allwood, J.; Flett, B.; Vries, de P.M.; Kema, G.H.J.

2008-01-01

A quantitative detection tool was developed to enable the monitoring of fumonisin-producing fungi in food and feed commodities. To this end, a quantitative PCR (TaqMan) was developed that targets a conserved region in the polyketide synthase gene fum1, which is involved in the biosynthesis of

Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum

NARCIS (Netherlands)

Koek, A. G.; Bruisten, S. M.; Dierdorp, M.; van Dam, A. P.; Templeton, K.

2006-01-01

A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.

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Srivastava, Niloo; Manvati, Siddharth; Srivastava, Archita; Pal, Ranjana; Kalaiarasan, Ponnusamy; Chattopadhyay, Shilpi; Gochhait, Sailesh; Dua, Raina; Bamezai, Rameshwar N K

2011-04-04

New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

Science.gov (United States)

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

RNA-directed DNA methylation: Mechanisms and functions

KAUST Repository

Mahfouz, Magdy M.

2010-01-01

Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response

NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL1.

Science.gov (United States)

Tian, Hainan; Wang, Xianling; Guo, Hongyan; Cheng, Yuxin; Hou, Chunjiang; Chen, Jin-Gui; Wang, Shucai

2017-08-01

The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis ( Arabidopsis thaliana ). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant ( ntl8-1D ). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8 Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON ( TRY ) and TRICHOMELESS1 ( TCL1 ) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1 , in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1 . © 2017 American Society of Plant Biologists. All Rights Reserved.

Identification and validation of genes involved in cervical tumourigenesis

International Nuclear Information System (INIS)

Rajkumar, Thangarajan; Sabitha, Kesavan; Vijayalakshmi, Neelakantan; Shirley, Sundersingh; Bose, Mayil Vahanan; Gopal, Gopisetty; Selvaluxmy, Ganesharaja

2011-01-01

Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer. A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay. Our study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells. Several novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a

Cytologic atypia in the contralateral unaffected breast is related to parity and estrogen-related genes.

Science.gov (United States)

Monahan, Denise A; Wang, Jun; Lee, Oukseub; Revesz, Elizabeth; Taft, Nancy; Ivancic, David; Hansen, Nora M; Bethke, Kevin P; Zalles, C; Khan, Seema A

2016-12-01

The contralateral unaffected breast (CUB) of women with unilateral breast cancer provides a model for the study of breast tissue-based risk factors. Using random fine needle aspiration (rFNA), we have investigated hormonal and gene expression patterns related to atypia in the CUBs of newly diagnosed breast cancer patients. 83 women underwent rFNA of the CUB. Cytologic analysis was performed using the Masood Score (MS), atypia was defined as MS > 14. RNA was extracted using 80% of the sample. The expression of 20 hormone related genes was quantified using Taqman Low Density Arrays. Statistical analysis was performed using 2-tailed t tests and linear regression. Cytological atypia was more frequent in multiparous women (P = 0.0392), and was not associated with any tumor-related features in the affected breast. Masood Score was higher with shorter interval since last pregnancy (R = 0.204, P = 0.0417), higher number of births (R = 0.369, P = 0.0006), and estrogen receptor (ER) negativity of the index cancer (R = -0.203, P = 0.065). Individual cytologic features were associated with aspects of parity. Specifically, anisonucleosis was correlated with shorter interval since last pregnancy (R = 0.318, P = 0.0201), higher number of births (R = 0.382, P = 0.0004), and ER status (R = -0.314, P = 0.0038). Eight estrogen-regulated genes were increased in atypical samples (P breast cancer development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery.

Science.gov (United States)

Schnor, Noa Pereira Prada; Verlengia, Rozangela; Novais, Patrícia Fátima Sousa; Crisp, Alex Harley; Leite, Celso Vieira de Souza; Rasera-Junior, Irineu; Oliveira, Maria Rita Marques de

2017-01-01

Obesity can cause systemic arterial hypertension (SAH) and type 2 diabetes mellitus (DM2) factor that is also influenced by genetic variability. The present study aims to investigate the association between gene polymorphisms related with obesity on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass surgery. In total, 351 obese women in a Brazilian cohort completed the study. The clinical diagnosis of SAH and DM2 was monitored from medical records. Twelve gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; and rs9939609) were determined using real-time polymerase chain reaction and TaqMan assay. In the preoperative period, prevalence of SAH and DM2 was 57% and 22%, respectively. One year postoperatively, 86.8% subjects had remission of DM2 and 99.5% had control of SAH. Subjects with T allele from the serotonin receptor gene (5-HT2C, rs3813929) had five times greater chance of DM2, and the CC genotype from uncoupling protein 3 gene (UCP3, rs1800849) had three times greater chance in the preoperative period. These findings indicate that polymorphisms rs3813929 and rs1800849 from 5-HT2C and UCP3 genes were related to DM2 prevalence among the Brazilian obese women candidates for bariatric surgery.

Adaptive population divergence and directional gene flow across steep elevational gradients in a climate‐sensitive mammal

Science.gov (United States)

Waterhouse, Matthew D.; Erb, Liesl P.; Beever, Erik; Russello, Michael A.

2018-01-01

The American pika is a thermally sensitive, alpine lagomorph species. Recent climate-associated population extirpations and genetic signatures of reduced population sizes range-wide indicate the viability of this species is sensitive to climate change. To test for potential adaptive responses to climate stress, we sampled pikas along two elevational gradients (each ~470 to 1640 m) and employed three outlier detection methods, BAYESCAN, LFMM, and BAYPASS, to scan for genotype-environment associations in samples genotyped at 30,763 SNP loci. We resolved 173 loci with robust evidence of natural selection detected by either two independent analyses or replicated in both transects. A BLASTN search of these outlier loci revealed several genes associated with metabolic function and oxygen transport, indicating natural selection from thermal stress and hypoxia. We also found evidence of directional gene flow primarily downslope from large high-elevation populations and reduced gene flow at outlier loci, a pattern suggesting potential impediments to the upward elevational movement of adaptive alleles in response to contemporary climate change. Finally, we documented evidence of reduced genetic diversity associated the south-facing transect and an increase in corticosterone stress levels associated with inbreeding. This study suggests the American pika is already undergoing climate-associated natural selection at multiple genomic regions. Further analysis is needed to determine if the rate of climate adaptation in the American pika and other thermally sensitive species will be able to keep pace with rapidly changing climate conditions.

Co-relationship between california bearing ratio and index properties of jamshoro soil

International Nuclear Information System (INIS)

Iqbal, F.; Kumar, A.; Murtaza, A.

2018-01-01

Subgrade is a most important part of a pavement structure, which should have a reasonable stiffness modulus and shear strength. CBR (California Bearing Ratio) test is performed to evaluate stiffness modulus and shear strength of subgrade soils. However, CBR test is laborious and time consuming, particularly when soil is highly plastic like Jamshoro soil. In order to overcome this limitation, it may be appropriate to correlate CBR value of soil with its index properties like grain size analysis, Atterberg limits, and compaction characteristics such as MDD (Maximum Dry Density) and OMC (Optimum Moisture Content). This paper expresses the correlations between CBR value of Jamshoro soil and its index properties. SLRA (Single Linear Regression Analysis) and MLRA(Multiple Linear Regression) based Models were utilized. It is seen that MLRA gave better correlations up to R2 of about 0.984. It is observed that the Soaked CBR value can be predicted with confidence from LL (Liquid Limit), PI (Plasticity Index) and percent finer while the un-soaked CBR value can be obtained from LL, plasticity index and MDD. (author)

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

Science.gov (United States)

Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

2013-11-01

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

Associations between interleukin and interleukin receptor gene polymorphisms and risk of gout.

Science.gov (United States)

Liu, Shiguo; Zhou, Zheng; Wang, Can; Guo, Mingzhen; Chu, Nan; Li, Changgui

2015-09-24

Gout is a self-limiting, auto-inflammatory arthritis induced by the deposition of monosodium urate crystals in the synovial fluid and periarticular tissues. The aim of this study was to investigate the associations between genetic variants in the interleukin (IL) and interleukin receptor (ILR) genes IL-33, IL-1RL1, IL-23R, and signal transducer and activator of transcription 4 (STAT4) and susceptibility to gout in Chinese Han male individuals. The genetic distributions of rs3939286 in IL-33, rs13015714 in IL-1RL1, rs10889677 in IL-23R, and rs7574865 in STAT4 were detected in 1100 men with gout and 1227 ethnically matched controls, using Taqman allelic discrimination real-time polymerase chain reaction (PCR). Differences in these polymorphisms between the groups were investigated using χ(2) tests. The genotype-phenotype relationship among gout patients was tested by analysis of variance. There was a significant difference in genotypic frequencies of IL-23R rs10889677 between gout patients and controls (χ(2) = 81.386, P gout in Chinese Han male individuals. However, further studies in other ethnic groups are needed to confirm these results.

Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

DEFF Research Database (Denmark)

Angen, Øystein; Jensen, J.; Lavritsen, D. T.

2001-01-01

Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluat...

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

Directory of Open Access Journals (Sweden)

Simonas Juzėnas

Full Text Available MicroRNAs (miRNAs are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA. In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients' plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1 Gene in Primary Breast Cancer.

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Naoko Minatani

Full Text Available Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1 gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004. Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007. Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis

KAUST Repository

Zheng, Zhimin

2010-01-06

RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5′ overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5′ overhang and the partnering between RDM12 and RDR2. © 2010 Blackwell Publishing Ltd.

A Review of Land and Stream Classifications in Support of Developing a National Ordinary High Water Mark (OHWM) Classification

Science.gov (United States)

2014-08-01

LWD Large, Woody Debris MLRA Major Land Resource Area NCDWQ North Carolina Department of Water Quality NRCS Natural Resources Conservation Service...Sinuosity, bedforms (dunes, ripples, bars), banks Bedforms (bars, pools), grains, LWD ,* sinuosity, banks Grains, banks Bedforms...steps, pools), grains, LWD , banks Grains, banks Boundaries (bed and bank) Grains, LWD Dominant Sediment Sources Fluvial, bank failure

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Roeder, Martin; Vieths, Stefan; Holzhauser, Thomas

2011-01-01

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg -1 almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg -1 . We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg -1 almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg -1 . Further, between 100 and 100,000 mg kg -1 spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

Degeneration and domestication of a selfish gene in yeast: molecular evolution versus site-directed mutagenesis.

Science.gov (United States)

Koufopanou, Vassiliki; Burt, Austin

2005-07-01

VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.

Search Engine for Antimicrobial Resistance: A Cloud Compatible Pipeline and Web Interface for Rapidly Detecting Antimicrobial Resistance Genes Directly from Sequence Data.

Science.gov (United States)

Rowe, Will; Baker, Kate S; Verner-Jeffreys, David; Baker-Austin, Craig; Ryan, Jim J; Maskell, Duncan; Pearce, Gareth

2015-01-01

Antimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring. Here we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR's application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei. We have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR

Direct activation of EXPANSIN14 by LBD18 in the gene regulatory network of lateral root formation in Arabidopsis.

Science.gov (United States)

Kim, Jungmook; Lee, Han Woo

2013-02-01

Root system architecture is important for plants to adapt to a changing environment. The major determinant of the root system is lateral roots originating from the primary root. The developmental process of lateral root formation can be divided into priming, initiation, primordium development and the emergence of lateral roots, and is well characterized in Arabidopsis. The hormone auxin plays a critical role in lateral root development, and several auxin response modules involving AUXIN RESPONSE FACTORS (ARFs), transcriptional regulators of auxin-regulated genes and Aux/IAA, negative regulators of ARFs, regulate lateral root formation. The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes a unique class of transcription factors harbouring a conserved plant-specific lateral organ boundary domain and plays a role in lateral organ development of plants including lateral root formation. In our previous study, we showed that LBD18 stimulates lateral root formation in combination with LBD16 downstream of ARF7 and ARF19 during the auxin response. We have recently demonstrated that LBD18 activates expression of EXP14, a gene encoding the cell-wall loosening factor, by directly binding to the EXP14 promoter to promote lateral root emergence. Here we present the molecular function of LBD18 and its gene regulatory network during lateral root formation.

Expression and Sequence Variants of Inflammatory Genes; Effects on Plasma Inflammation Biomarkers Following a 6-Week Supplementation with Fish Oil

Science.gov (United States)

Cormier, Hubert; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2016-01-01

(1) Background: A growing body of literature suggest that polymorphisms (SNPs) from inflammation-related genes could possibly play a role in cytokine production and then interact with dietary n-3 fatty acids (FAs) to modulate inflammation. The aim of the present study was to test whether gene expression of selected inflammatory genes was altered following an n-3 PUFA supplementation and to test for gene–diet interactions modulating plasma inflammatory biomarker levels. (2) Methods: 191 subjects completed a 6-week n-3 FA supplementation with 5 g/day of fish oil. Gene expression of TNF-α and IL6 was assessed in peripheral blood mononuclear cells (PBMCs) using the TaqMan technology. Genotyping of 20 SNPs from the TNF-LTA gene cluster, IL1β, IL6 and CRP genes was performed. (3) Results: There was no significant reduction of plasma IL-6, TNF-α and C-reactive protein (CRP) levels after the 6-week fish oil supplementation. TNF-α and IL6 were slightly overexpressed in PBMCs after the supplementation (fold changes of 1.05 ± 0.38 and 1.18 ± 0.49, respectively (n = 191)), but relative quantification (RQ) within the −0.5 to 2.0 fold are considered as nonbiologically significant. In a MIXED model for repeated measures adjusted for the effects of age, sex and BMI, gene by supplementation interaction effects were observed for rs1143627, rs16944, rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF-α levels and for rs1800629 on CRP levels (p < 0.05 for all). (4) Conclusions: This study shows that a 6-week n-3 FA supplementation with 5 g/day of fish oil did not alter gene expression levels of TNF-α and IL6 in PBMCs and did not have an impact on inflammatory biomarker levels. However, gene–diet interactions were observed between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels. PMID:26999109

Expression and Sequence Variants of Inflammatory Genes; Effects on Plasma Inflammation Biomarkers Following a 6-Week Supplementation with Fish Oil

Directory of Open Access Journals (Sweden)

Hubert Cormier

2016-03-01

Full Text Available (1 Background: A growing body of literature suggest that polymorphisms (SNPs from inflammation-related genes could possibly play a role in cytokine production and then interact with dietary n-3 fatty acids (FAs to modulate inflammation. The aim of the present study was to test whether gene expression of selected inflammatory genes was altered following an n-3 PUFA supplementation and to test for gene–diet interactions modulating plasma inflammatory biomarker levels. (2 Methods: 191 subjects completed a 6-week n-3 FA supplementation with 5 g/day of fish oil. Gene expression of TNF-α and IL6 was assessed in peripheral blood mononuclear cells (PBMCs using the TaqMan technology. Genotyping of 20 SNPs from the TNF-LTA gene cluster, IL1β, IL6 and CRP genes was performed. (3 Results: There was no significant reduction of plasma IL-6, TNF-α and C-reactive protein (CRP levels after the 6-week fish oil supplementation. TNF-α and IL6 were slightly overexpressed in PBMCs after the supplementation (fold changes of 1.05 ± 0.38 and 1.18 ± 0.49, respectively (n = 191, but relative quantification (RQ within the −0.5 to 2.0 fold are considered as nonbiologically significant. In a MIXED model for repeated measures adjusted for the effects of age, sex and BMI, gene by supplementation interaction effects were observed for rs1143627, rs16944, rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF-α levels and for rs1800629 on CRP levels (p < 0.05 for all. (4 Conclusions: This study shows that a 6-week n-3 FA supplementation with 5 g/day of fish oil did not alter gene expression levels of TNF-α and IL6 in PBMCs and did not have an impact on inflammatory biomarker levels. However, gene–diet interactions were observed between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels.

Genomic signatures of local directional selection in a high gene flow marine organism, the Atlantic cod (Gadus morhua)

DEFF Research Database (Denmark)

Eg Nielsen, Einar; Hansen, Jakob Hemmer; Poulsen, Nina Aagaard

2009-01-01

-associated single nucleotide polymorphisms (SNPs) for evidence of selection in local populations of Atlantic cod (Gadus morhua L.) across the species distribution. Results: Our global genome scan analysis identified eight outlier gene loci with very high statistical support, likely to be subject to directional...... selection in local demes, or closely linked to loci under selection. Likewise, on a regional south/north transect of central and eastern Atlantic populations, seven loci displayed strongly elevated levels of genetic differentiation. Selection patterns among populations appeared to be relatively widespread...

Lack of Association Between Polymorphisms in Dopa Decarboxylase and Dopamine Receptor-1 Genes With Childhood Autism in Chinese Han Population.

Science.gov (United States)

Yu, Hong; Liu, Jun; Yang, Aiping; Yang, Guohui; Yang, Wenjun; Lei, Heyue; Quan, Jianjun; Zhang, Zengyu

2016-04-01

Genetic factors play an important role in childhood autism. This study is to determine the association of single-nucleotide polymorphisms in dopa decarboxylase (DDC) and dopamine receptor-1 (DRD1) genes with childhood autism, in a Chinese Han population. A total of 211 autistic children and 250 age- and gender-matched healthy controls were recruited. The severity of disease was determined by Children Autism Rating Scale scores. TaqMan Probe by real-time polymerase chain reaction was used to determine genotypes and allele frequencies of single-nucleotide polymorphism rs6592961 in DDC and rs251937 in DRD1. Case-control and case-only studies were respectively performed, to determine the contribution of both single-nucleotide polymorphisms to the predisposition of disease and its severity. Our results showed that there was no significant association of the genotypes and allele frequencies of both single-nucleotide polymorphisms concerning childhood autism and its severity. More studies with larger samples are needed to corroborate their predicting roles. © The Author(s) 2015.

Isolation and extreme sex-specific expression of cytochrome P450 genes in the bark beetle, Ips paraconfusus, following feeding on the phloem of host ponderosa pine, Pinus ponderosa.

Science.gov (United States)

Huber, D P W; Erickson, M L; Leutenegger, C M; Bohlmann, J; Seybold, S J

2007-06-01

We have identified cDNAs and characterized the expression of 13 novel cytochrome P450 genes of potential importance in host colonization and reproduction by the California fivespined ips, Ips paraconfusus. Twelve are of the Cyp4 family and one is of the Cyp9 family. Following feeding on host Pinus ponderosa phloem, bark beetle transcript levels of several of the Cyp4 genes increased or decreased in males only or in both sexes. In one instance (IparaCyp4A5) transcript accumulated significantly in females, but declined significantly in males. The Cyp9 gene (Cyp9T1) transcript levels in males were > 85 000 x higher at 8 h and > 25 000 x higher at 24 h after feeding compared with nonfed controls. Transcript levels in females were approximately 150 x higher at 24 h compared with nonfed controls. Cyp4G27 transcript was present constitutively regardless of sex or feeding and served as a better housekeeping gene than beta-actin or 18S rRNA for the real-time TaqMan polymerase chain reaction analysis. The expression patterns of Cyp4AY1, Cyp4BG1, and, especially, Cyp9T1 in males suggest roles for these genes in male-specific aggregation pheromone production. The differential transcript accumulation patterns of these bark beetle P450s provide insight into ecological interactions of I. paraconfusus with its host pines.

Protamine 3 expressions in crossbred bull spermatozoa may not be ...

African Journals Online (AJOL)

The mRNA expression of PRM3 gene among two groups was evaluated by real time quantitative polymerase chain reaction (PCR) using TaqMan chemistry, where peptidylprolyl isomerase A (PPIA) was used as an internal control. Our finding revealed that expression of PRM3 was down regulated in poor quality semen ...

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

Science.gov (United States)

Kim, Jung Seok; Kang, Seong Jae; Jeong, Hwa Yeon; Kim, Min Woo; Park, Sang Il; Lee, Yeon Kyung; Kim, Hong Sung; Kim, Keun Sik; Park, Yong Serk

2016-09-01

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Different contribution of BRINP3 gene in chronic periodontitis and peri-implantitis: a cross-sectional study.

Science.gov (United States)

Casado, Priscila L; Aguiar, Diego P; Costa, Lucas C; Fonseca, Marcos A; Vieira, Thays C S; Alvim-Pereira, Claudia C K; Alvim-Pereira, Fabiano; Deeley, Kathleen; Granjeiro, José M; Trevilatto, Paula C; Vieira, Alexandre R

2015-03-11

Peri-implantitis is a chronic inflammation, resulting in loss of supporting bone around implants. Chronic periodontitis is a risk indicator for implant failure. Both diseases have a common etiology regarding inflammatory destructive response. BRINP3 gene is associated with aggressive periodontitis. However, is still unclear if chronic periodontitis and peri-implantitis have the same genetic background. The aim of this work was to investigate the association between BRINP3 genetic variation (rs1342913 and rs1935881) and expression and susceptibility to both diseases. Periodontal and peri-implant examinations were performed in 215 subjects, divided into: healthy (without chronic periodontitis and peri-implantitis, n = 93); diseased (with chronic periodontitis and peri-implantitis, n = 52); chronic periodontitis only (n = 36), and peri-implantitis only (n = 34). A replication sample of 92 subjects who lost implants and 185 subjects successfully treated with implants were tested. DNA was extracted from buccal cells. Two genetic markers of BRINP3 (rs1342913 and rs1935881) were genotyped using TaqMan chemistry. Chi-square (p chronic periodontitis.

Epigenetic Alteration by DNA Methylation of ESR1, MYOD1 and hTERT Gene Promoters is Useful for Prediction of Response in Patients of Locally Advanced Invasive Cervical Carcinoma Treated by Chemoradiation.

Science.gov (United States)

Sood, S; Patel, F D; Ghosh, S; Arora, A; Dhaliwal, L K; Srinivasan, R

2015-12-01

Locally advanced invasive cervical cancer [International Federation of Gynecology and Obstetrics (FIGO) IIB/III] is treated by chemoradiation. The response to treatment is variable within a given FIGO stage. Therefore, the aim of the present study was to evaluate the gene promoter methylation profile and corresponding transcript expression of a panel of six genes to identify genes which could predict the response of patients treated by chemoradiation. In total, 100 patients with invasive cervical cancer in FIGO stage IIB/III who underwent chemoradiation treatment were evaluated. Ten patients developed systemic metastases during therapy and were excluded. On the basis of patient follow-up, 69 patients were chemoradiation-sensitive, whereas 21 were chemoradiation-resistant. Gene promoter methylation and gene expression was determined by TaqMan assay and quantitative real-time PCR, respectively, in tissue samples. The methylation frequency of ESR1, BRCA1, RASSF1A, MLH1, MYOD1 and hTERT genes ranged from 40 to 70%. Univariate and hierarchical cluster analysis revealed that gene promoter methylation of MYOD1, ESR1 and hTERT could predict for chemoradiation response. A pattern of unmethylated MYOD1, unmethylated ESR1 and methylated hTERT promoter as well as lower ESR1 transcript levels predicted for chemoradiation resistance. Methylation profiling of a panel of three genes that includes MYOD1, ESR1 and hTERT may be useful to predict the response of invasive cervical carcinoma patients treated with standard chemoradiation therapy. Copyright © 2015 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice

International Nuclear Information System (INIS)

Andres, A.C.; Schoenenberger, C.A.; Groner, B.; Henninghausen, L.; LeMeur, M.; Gelinger, P.

1987-01-01

The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras - i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors

Influence of polymorphisms within the methotrexate pathway genes on the toxicity and efficacy of methotrexate in patients with juvenile idiopathic arthritis.

Science.gov (United States)

Yanagimachi, Masakatsu; Naruto, Takuya; Hara, Takuma; Kikuchi, Masako; Hara, Ryoki; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Kaneko, Tetsuji; Morita, Satoshi; Goto, Hiroaki; Yokota, Shumpei

2011-02-01

We investigated whether several polymorphisms within the methotrexate (MTX) pathway genes were related to the toxicity and efficacy of MTX in 92 Japanese patients with articular-type juvenile idiopathic arthritis (JIA). Eight gene polymorphisms within the MTX pathway genes, namely, RFC, BCRP, MTHFR (two), FPGS, γ-glutamyl hydrolase (GGH; two) and ATIC, were genotyped using TaqMan assays. Liver dysfunction was defined as an increase in alanine transaminase to five times the normal upper limit. Non-responders to MTX were defined as patients refractory to MTX and were therefore treated with biologics. The non-TT genotype at GGH T16C was associated with a high risk of liver dysfunction (P=0.028, odds ratio=6.90, 95% confidence interval 1.38-34.5), even after adjustment for the duration of MTX treatment. A longer interval from disease onset to treatment (8.5 and 21.3 months, P=0.029) and rheumatoid factor positivity (P=0.026, odds ratio=2.87, 95% confidence interval 1.11-7.39) were associated with lower efficacy of MTX. The non-TT genotype at GGH T16C was associated with a high risk of liver dysfunction, presumably because the C allele of GGH C16T may reduce the activity of GGH. The time interval before MTX treatment and rheumatoid factor positivity were associated with the efficacy of MTX treatment. The pharmacogenetics of the MTX pathway genes affects the toxicity and efficacy of MTX in Japanese JIA patients. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.

Genome-wide identification of direct HBx genomic targets

KAUST Repository

Guerrieri, Francesca

2017-02-17

Background The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. Results ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. Conclusions Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

Development and utility of an internal threshold control (ITC real-time PCR assay for exogenous DNA detection.

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Weiyi Ni

Full Text Available Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct or negative (i.e. Ct greater than the ITC Ct. The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

American Society of Gene & Cell Therapy

Science.gov (United States)

... Gene & Cell Therapy Defined Gene therapy and cell therapy are overlapping fields of biomedical research that aim to repair the direct cause of genetic diseases. Read More Gene & Cell Therapy FAQ's Read the most common questions raised by ...

A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

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Etchells J Peter

2009-10-01

Full Text Available Abstract Background The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. Results Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. Conclusion The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.

Compositional gradients in Gramineae genes

DEFF Research Database (Denmark)

Wong, Gane Ka-Shu; Wang, Jun; Tao, Lin

2002-01-01

In this study, we describe a property of Gramineae genes, and perhaps all monocot genes, that is not observed in eudicot genes. Along the direction of transcription, beginning at the junction of the 5'-UTR and the coding region, there are gradients in GC content, codon usage, and amino-acid usage...

Gene therapy prospects--intranasal delivery of therapeutic genes.

Science.gov (United States)

Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

2012-01-01

Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

The diamine oxidase gene is associated with hypersensitivity response to non-steroidal anti-inflammatory drugs.

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José A G Agúndez

Full Text Available UNLABELLED: Non-steroidal anti-inflammatory drugs (NSAIDs are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191, which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3-2.1; Pc  = 0.0003 with a gene-dose effect (P = 0.0001. The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively. CONCLUSIONS AND IMPLICATIONS: The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.

A map of directional genetic interactions in a metazoan cell.

Science.gov (United States)

Fischer, Bernd; Sandmann, Thomas; Horn, Thomas; Billmann, Maximilian; Chaudhary, Varun; Huber, Wolfgang; Boutros, Michael

2015-03-06

Gene-gene interactions shape complex phenotypes and modify the effects of mutations during development and disease. The effects of statistical gene-gene interactions on phenotypes have been used to assign genes to functional modules. However, directional, epistatic interactions, which reflect regulatory relationships between genes, have been challenging to map at large-scale. Here, we used combinatorial RNA interference and automated single-cell phenotyping to generate a large genetic interaction map for 21 phenotypic features of Drosophila cells. We devised a method that combines genetic interactions on multiple phenotypes to reveal directional relationships. This network reconstructed the sequence of protein activities in mitosis. Moreover, it revealed that the Ras pathway interacts with the SWI/SNF chromatin-remodelling complex, an interaction that we show is conserved in human cancer cells. Our study presents a powerful approach for reconstructing directional regulatory networks and provides a resource for the interpretation of functional consequences of genetic alterations.

Direct analysis of thymic function in children with Down's syndrome

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Meschiari Liviana

2005-02-01

Full Text Available Abstract Background Down's syndrome (DS is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE in children with DS. Methods We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC. Results In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found. Conclusions The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.

Regional Supplement to the Corps of Engineers Wetland Delineation Manual: Western Mountains, Valleys, and Coast Region (Version 2.0)

Science.gov (United States)

2010-05-01

monophylla or P. edulis), junipers (Juniperus), cottonwoods (e.g., Populus fremontii), willows (Salix), or hardwoods (e.g., Quercus , Platanus...pine in association with incense cedar and California black oak ( Quercus kelloggii) on the western slopes and Jeffrey pine on the eastern slopes...corkbark fir (Abies lasiocarpa var. arizonica ), limber pine (Pinus flexilis), and bristlecone pine (P. aristata) (Bailey 1995). Black Hills (MLRA

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African Journals Online (AJOL)

NWUuser

2013-09-12

Sep 12, 2013 ... Afr J Mar Sci 503; Gen N 434 in GG 36413 of 25 April 2013 1-2; Hauck and Sowman Waves of. Change .... definition of the term in the MLRA emphasises the element of consumptive use as ... In other words, fishing activities are ... fishers that is the primary focus of this contribution and it is therefore useful to.

Toxicogenomics-based identification of mechanisms for direct immunotoxicity.

Science.gov (United States)

Shao, Jia; Katika, Madhumohan R; Schmeits, Peter C J; Hendriksen, Peter J M; van Loveren, Henk; Peijnenburg, Ad A C M; Volger, Oscar L

2013-10-01

Compounds with direct immunotoxic properties, including metals, mycotoxins, agricultural pesticides, and industrial chemicals, form potential human health risks due to exposure through food, drinking water, and the environment. Insights into the mechanisms of action are currently lacking for the majority of these direct immunotoxicants. Therefore, the present work aimed to gain insights into the molecular mechanisms underlying direct immunotoxicity. To this end, we assessed in vitro the effects of 31 test compounds on the transcriptome of the human Jurkat T-cell line. These compounds included direct immunotoxicants, immunosuppressive drugs with different mode of actions, and nonimmunotoxic control chemicals. Pathway analysis of the microarray data allowed us to identify canonical pathways and Gene Ontology processes that were transcriptionally regulated in common by immunotoxicants (1) with structural similarities, such as tributyltin chloride and tributyltin oxide that activated the retinoic acid/X receptor signaling pathway and (2) without structural similarities, such as As2O3, dibutyltin chloride, diazinon, MeHg, ochratoxin A (OTA), S9-treated OTA, S9-treated cyclophosphamide, and S9-treated benzo[a]pyrene, which activated unfolded protein response, and FTY720, lindane, and propanil, which activated the cholesterol biosynthesis pathway. In addition, processes uniquely affected by individual immunotoxicants were identified, such as the induction of Notch receptor signaling and the downregulation of acute-phase response genes by OTA. These findings were validated by quantitative real-time PCR analysis of genes involved in these processes. Our study indicated that diverse modes of action are involved in direct immunotoxicity and that a set of pathways or genes, rather than one single gene, can be used to screen compounds for direct immunotoxicity.

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

Science.gov (United States)

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

Science.gov (United States)

The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

Association analysis of the IL-1 gene cluster polymorphisms with aggressive and chronic periodontitis in the Algerian population.

Science.gov (United States)

Boukortt, Kawther Nourelhouda; Saidi-Ouahrani, Nadjia; Boukerzaza, Boubaker; Ouhaibi-Djellouli, Hadjira; Hachmaoui, Khalida; Benaissa, Fatima Zohra; Taleb, Leila; Drabla-Ouahrani, Hayet; Deba, Tahria; Ouledhamou, Sid Ahmed; Mehtar, Nadhera; Boudjema, Abdellah

2015-10-01

There is strong evidence that genetic as well as environmental factors affect the development of periodontitis. Various studies suggest that genetic polymorphisms of the interleukin-1 (IL-1) genes are associated with an increased risk of developing the pathogenesis. The aim of the present study was to investigate the possible relationship between two polymorphisms of IL-1 gene cluster IL-1B (C+3954T) (rs1143634) and IL-1A (C-889T) (rs1800587) SNPs and the aggressive and chronic periodontitis risk in a case control study in Algerian population. 279 subjects were recruited and received a periodontal examination: 128 healthy controls and 151 cases. From cases, 91 patients were having a chronic disease whereas 60 subjects with aggressive form. All these subjects were genotyped for IL-1A (C-889T) and IL-1B (C+3954T) polymorphisms using TaqMan real time PCR technology. Frequencies of IL-1 alleles, genotypes and the haplotypes were also examined. Significant differences were found in the carriage rate of both minor alleles of the IL-1A (C-889T) and IL-1B (C+3954T) polymorphisms of aggressive periodontitis cases compared with healthy controls (OR [95%CI]=1.61 [1.03-2.49], p=0.03), (OR [95%CI]=1.69 [1.09-2.63], p=0.01), respectively. The result did not reach significance with the chronic form. The studied polymorphisms of the IL-1 genes appear to be associated with susceptibility to aggressive periodontitis (AgP) in the Algerian population. Copyright © 2015 Elsevier Ltd. All rights reserved.

IRF5, PTPN22, CD28, IL2RA, KIF5A, BLK and TNFAIP3 genes polymorphisms and lupus susceptibility in a cohort from the Egypt Delta; relation to other ethnic groups.

Science.gov (United States)

Elghzaly, Ashraf A; Metwally, Shereen S; El-Chennawi, Farha A; Elgayaar, Maha A; Mosaad, Youssef M; El-Toraby, Ehab E; Hegab, Mohsen M; Ibrahim, Saleh M

2015-07-01

To replicate a single nucleotide polymorphism (SNP) of known genes for lupus (IRF5 rs10488631, PTPN22 rs2476601, BLK rs2736340 and TNFAIP3 rs5029939) and other autoimmune diseases (CD28 rs1980422, IL2RA rs2104286 and KIF5A rs1678542) on a newly studied Egyptian cohort to investigate the genetic disparity with different studied ethnic groups in relation to lupus susceptibility. 170 Egyptian patients from Egypt Delta with SLE and 241 matched healthy controls were genotyped by Taqman real time PCR for the selected SNPs. The results revealed significant association with IRF5 (p<0.0001) and PTPN22 (p=0.008) and insignificant association with KIF5A, CD28, IL2RA, BLK and TNFAIP3 genes. This study may provide an additional evidence for the association between IRF5 and PTPN22 and lupus susceptibility and may exclude it for CD28, IL2RA, and KIF5A. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Effect of methotrexate combined with ginger, silymarin or propolis on ...

African Journals Online (AJOL)

The present study was performed to evaluate the effect of three natural antioxidants on the adverse effect of methotrexate (MTX) in normal liver cells. TaqMan RT-PCR technology was used to estimate the mRNA expression levels for three genes after rats injection with a single dose of 20 mg/kg b.w MTX or the same MTX ...

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

International Nuclear Information System (INIS)

Obermayr, Eva; Heinze, Georg; Tong, Dan; Zeillinger, Robert; Sanchez-Cabo, Fatima; Tea, Muy-Kheng M; Singer, Christian F; Krainer, Michael; Fischer, Michael B; Sehouli, Jalid; Reinthaller, Alexander; Horvat, Reinhard

2010-01-01

The presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients. Gene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan ® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection. Six genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients. The

FunGene: the functional gene pipeline and repository.

Science.gov (United States)

Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

2013-01-01

Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

FunGene: the Functional Gene Pipeline and Repository

Directory of Open Access Journals (Sweden)

Jordan A. Fish

2013-10-01

Full Text Available Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer.While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/ offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

SOX9 governs differentiation stage-specific gene expression in growth plate chondrocytes via direct concomitant transactivation and repression.

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Victor Y L Leung

2011-11-01

Full Text Available Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9-GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

Science.gov (United States)

Gouran, Hossein; Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

The hOGG1 Ser326Cys Gene Polymorphism and Breast Cancer Risk in Saudi Population.

Science.gov (United States)

Alanazi, Mohammed; Pathan, Akbar Ali Khan; Shaik, Jilani P; Alhadheq, Abdullah; Khan, Zahid; Khan, Wajahatullah; Al Naeem, Abdulrahman; Parine, Narasimha Reddy

2017-07-01

The purpose of this study was to test the association between human 8-oxoguanine glycosylase 1 (hOGG1) gene polymorphisms and susceptibility to breast cancer in Saudi population. We have also aimed to screen the hOGG1 Ser326Cys polymorphism effect on structural and functional properties of the hOGG1 protein using in silico tools. We have analyzed four SNPs of hOGG1 gene among Saudi breast cancer patients along with healthy controls. Genotypes were screened using TaqMan SNP genotype analysis method. Experimental data was analyzed using Chi-square, t test and logistic regression analysis using SPSS software (v.16). In silco analysis was conducted using discovery studio and HOPE program. Genotypic analysis showed that hOGG1 rs1052133 (Ser326Cys) is significantly associated with breast cancer samples in Saudi population, however rs293795 (T >C), rs2072668 (C>G) and rs2075747 (G >A) did not show any association with breast cancer. The hOGG1 SNP rs1052133 (Ser326Cys) minor allele T showed a significant association with breast cancer samples (OR = 1.78, χ2 = 7.86, p = 0.02024). In silico structural analysis was carried out to compare the wild type (Ser326) and mutant (Cys326) protein structures. The structural prediction studies revealed that Ser326Cys variant may destabilize the protein structure and it may disturb the hOGG1 function. Taken together this is the first In silico study report to confirm Ser326Cys variant effect on structural and functional properties of hOGG1 gene and Ser326Cys role in breast cancer susceptibility in Saudi population.

Direct Detection and Differentiation of Pathogenic Leptospira Species Using a Multi-Gene Targeted Real Time PCR Approach

Science.gov (United States)

Ferreira, Ana Sofia; Costa, Pedro; Rocha, Teresa; Amaro, Ana; Vieira, Maria Luísa; Ahmed, Ahmed; Thompson, Gertrude; Hartskeerl, Rudy A.; Inácio, João

2014-01-01

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis. PMID:25398140

GeneTopics - interpretation of gene sets via literature-driven topic models

Science.gov (United States)

2013-01-01

Background Annotation of a set of genes is often accomplished through comparison to a library of labelled gene sets such as biological processes or canonical pathways. However, this approach might fail if the employed libraries are not up to date with the latest research, don't capture relevant biological themes or are curated at a different level of granularity than is required to appropriately analyze the input gene set. At the same time, the vast biomedical literature offers an unstructured repository of the latest research findings that can be tapped to provide thematic sub-groupings for any input gene set. Methods Our proposed method relies on a gene-specific text corpus and extracts commonalities between documents in an unsupervised manner using a topic model approach. We automatically determine the number of topics summarizing the corpus and calculate a gene relevancy score for each topic allowing us to eliminate non-specific topics. As a result we obtain a set of literature topics in which each topic is associated with a subset of the input genes providing directly interpretable keywords and corresponding documents for literature research. Results We validate our method based on labelled gene sets from the KEGG metabolic pathway collection and the genetic association database (GAD) and show that the approach is able to detect topics consistent with the labelled annotation. Furthermore, we discuss the results on three different types of experimentally derived gene sets, (1) differentially expressed genes from a cardiac hypertrophy experiment in mice, (2) altered transcript abundance in human pancreatic beta cells, and (3) genes implicated by GWA studies to be associated with metabolite levels in a healthy population. In all three cases, we are able to replicate findings from the original papers in a quick and semi-automated manner. Conclusions Our approach provides a novel way of automatically generating meaningful annotations for gene sets that are directly

Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

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Benjamin Mayne

2016-10-01

Full Text Available The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analysed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes, followed by the heart (375 genes, kidney (224 genes, colon (218 genes and thyroid (163 genes. More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

Science.gov (United States)

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Impacts of CA9 gene polymorphisms on urothelial cell carcinoma susceptibility and clinicopathologic characteristics in Taiwan.

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Shian-Shiang Wang

Full Text Available Carbonic anhydrase 9 (CA9 is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study explored the effect of CA9 gene polymorphisms on the susceptibility of developing urothelial cell carcinoma (UCC and the clinicopathological status.A total of 442 participants, including 221 healthy people and 221 patients with UCC, were recruited for this study. Four single-nucleotide polymorphisms (SNPs of the CA9 gene were assessed by a real-time PCR with the TaqMan assay. After adjusting for other co-variants, the individuals carrying at least one A allele at CA9 rs1048638 had a 2.303-fold risk of developing UCC than did wild-type (CC carriers. Furthermore, UCC patients who carried at least one A allele at rs1048638 had a higher invasive stage risk (p< 0.05 than did patients carrying the wild-type allele. Moreover, among the UCC patients with smoker, people with at least one A allele of CA9 polymorphisms (rs1048638 had a 4.75-fold (95% CI = 1.204-18.746 increased risk of invasive cancer.The rs1048638 polymorphic genotypes of CA9 might contribute to the prediction of susceptibility to and pathological development of UCC. This is the first study to provide insight into risk factors associated with CA9 variants in carcinogenesis of UCC in Taiwan.

Site-directed mutagenesis of the foot-and-mouth disease virus RNA-polymerase gene

International Nuclear Information System (INIS)

Brindeiro, R.M.; Soares, M.A.; Vianna, A.L.M.; Pontes, O.H.A. de; Pacheco, A.B.F.; Almeida, D.F. de; Tanuri, A.

1991-01-01

The foot-and-mouth disease virus RNA-polymerase gene was mutagenised in its active site. Pst I digestion of the polymerase gene (cDNA) generated a 790 bp fragment containing the critical sequence. This fragment was subcloned in M13mp8 for mutagenesis method. The polymerase gene was then reconstructed and subcloned in pUC19. These mutants will be used to study the enzyme structure and activity and to develop intracellular immunization assays in eukaryotic cells. (author)

Human γ-globin genes silenced independently of other genes in the β-globin locus.

NARCIS (Netherlands)

N.O. Dillon (Niall); F.G. Grosveld (Frank)

1991-01-01

textabstractErythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5'

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

Energy Technology Data Exchange (ETDEWEB)

Roeder, Martin; Vieths, Stefan [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany); Holzhauser, Thomas, E-mail: holth@pei.de [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany)

2011-01-24

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg{sup -1} almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg{sup -1}. We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg{sup -1} almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg{sup -1}. Further, between 100 and 100,000 mg kg{sup -1} spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a

Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies.

Science.gov (United States)

Passot, Christophe; Azzopardi, Nicolas; Renault, Sylvaine; Baroukh, Nadine; Arnoult, Christophe; Ohresser, Marc; Boisdron-Celle, Michèle; Gamelin, Erick; Watier, Hervé; Paintaud, Gilles; Gouilleux-Gruart, Valérie

2013-01-01

The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman (®) technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs' distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

Science.gov (United States)

Zarrinfar, Hossein; Makimura, Koichi; Satoh, Kazuo; Khodadadi, Hossein; Mirhendi, Hossein

2013-05-01

Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group. © 2013 Wiley Periodicals, Inc.

TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease.

Science.gov (United States)

Bohuski, Elizabeth; Lorch, Jeffrey M; Griffin, Kathryn M; Blehert, David S

2015-04-15

Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease. We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

Analysis of variants in DNA damage signalling genes in bladder cancer

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Bishop D Timothy

2008-07-01

Full Text Available Abstract Background Chemicals from occupational exposure and components of cigarette smoke can cause DNA damage in bladder urothelium. Failure to repair DNA damage by DNA repair proteins may result in mutations leading to genetic instability and the development of bladder cancer. Immunohistochemistry studies have shown DNA damage signal activation in precancerous bladder lesions which is lost on progression, suggesting that the damage signalling mechanism acts as a brake to further tumorigenesis. Single nucleotide polymorphisms (SNPs in DSB signalling genes may alter protein function. We hypothesized that SNPs in DSB signalling genes may modulate predisposition to bladder cancer and influence the effects of environmental exposures. Methods We recruited 771 cases and 800 controls (573 hospital-based and 227 population-based from a previous case-control study and interviewed them regarding their smoking habits and occupational history. DNA was extracted from a peripheral blood sample and genotyping of 24 SNPs in MRE11, NBS1, RAD50, H2AX and ATM was undertaken using an allelic discrimination method (Taqman. Results Smoking and occupational dye exposure were strongly associated with bladder cancer risk. Using logistic regression adjusting for age, sex, smoking and occupational dye exposure, there was a marginal increase in risk of bladder cancer for an MRE11 3'UTR SNP (rs2155209, adjusted odds ratio 1.54 95% CI (1.13–2.08, p = 0.01 for individuals homozygous for the rare allele compared to those carrying the common homozygous or heterozygous genotype. However, in the hospital-based controls, the genotype distribution for this SNP deviated from Hardy-Weinberg equilibrium. None of the other SNPs showed an association with bladder cancer and we did not find any significant interaction between any of these polymorphisms and exposure to smoking or dye exposure. Conclusion Apart from a possible effect for one MRE11 3'UTR SNP, our study does not support

CYP19A1 gene polymorphism and colorectal cancer etiology in Saudi population: case–control study

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Al-Mukaynizi FB

2017-09-01

Full Text Available Fatimah Basil Al-Mukaynizi,1 Mohammed Alanazi,1 Sooad Al-Daihan,1 Narasimha Reddy Parine,1 Majid Almadi,2 Abdulrahman Aljebreen,2 Nahla Azzam,2 Othman Alharbi,2 Maha Arafah,3 Arjumand Warsy4 1Department of Biochemistry, College of Science, 2Department of Internal Medicine, 3Department of Pathology, College of Medicine, 4Central Laboratory, Female Center for Scientific & Medical Colleges, King Saud University, Riyadh, Saudi Arabia Background: Considerable interest is directed toward the enzyme aromatase (CYP19A1 and the development of cancer, due to CYP19A1’s role in estrogen biosynthesis. Several cancers display excessive intra-tumor accumulation of estrogens, and aromatase inhibitors are used for treatment. The CYP19A1 gene exhibits polymorphism and mutations that can alter its expression or aromatase activity and influence estrogen production. We designed this study to investigate the link between CYP19A1 polymorphism and susceptibility to colorectal cancer (CRC development in Saudis. Patients and methods: Blood samples from 100 CRC patients and 100 healthy controls were drawn for DNA extractions. Three polymorphic sites, rs4774585, rs936308, and rs4775936, were genotyped using Taqman genotyping by real-time polymerase chain reaction. Allelic and genotype frequencies were calculated and compared in the two groups. Results: All single nucleotide polymorphisms (SNPs were polymorphic in Saudis, and comparison of allele frequencies showed several differences when compared to other populations. None of the SNPs were associated with the risk of CRC development in Saudis (P>0.05. Some gender and location (colon or rectal differences were observed. Discussion: The results of this study highlighted the genetic heterogeneity existing between populations in the prevalence of different SNPs and their relation to disease state. It showed that, although rs4774585, rs936308, and rs4775936 are involved in CRC development in several populations, their role

From gene engineering to gene modulation and manipulation: can we prevent or detect gene doping in sports?

Science.gov (United States)

Fischetto, Giuseppe; Bermon, Stéphane

2013-10-01

During the last 2 decades, progress in deciphering the human gene map as well as the discovery of specific defective genes encoding particular proteins in some serious human diseases have resulted in attempts to treat sick patients with gene therapy. There has been considerable focus on human recombinant proteins which were gene-engineered and produced in vitro (insulin, growth hormone, insulin-like growth factor-1, erythropoietin). Unfortunately, these substances and methods also became improper tools for unscrupulous athletes. Biomedical research has focused on the possible direct insertion of gene material into the body, in order to replace some defective genes in vivo and/or to promote long-lasting endogenous synthesis of deficient proteins. Theoretically, diabetes, anaemia, muscular dystrophies, immune deficiency, cardiovascular diseases and numerous other illnesses could benefit from such innovative biomedical research, though much work remains to be done. Considering recent findings linking specific genotypes and physical performance, it is tempting to submit the young athletic population to genetic screening or, alternatively, to artificial gene expression modulation. Much research is already being conducted in order to achieve a safe transfer of genetic material to humans. This is of critical importance since uncontrolled production of the specifically coded protein, with serious secondary adverse effects (polycythaemia, acute cardiovascular problems, cancer, etc.), could occur. Other unpredictable reactions (immunogenicity of vectors or DNA-vector complex, autoimmune anaemia, production of wild genetic material) also remain possible at the individual level. Some new substances (myostatin blockers or anti-myostatin antibodies), although not gene material, might represent a useful and well-tolerated treatment to prevent progression of muscular dystrophies. Similarly, other molecules, in the roles of gene or metabolic activators [5-aminoimidazole-4

How does the extracellular matrix direct gene expression

Energy Technology Data Exchange (ETDEWEB)

Bissell, M J; Hall, H G; Parry, G

1982-01-01

Based on the existing literature, a model is presented that postulates a ''dynamic reciprocity'' between the extracellular matrix (ECM) on the one hand and the cytoskeleton and the nuclear matrix on the other hand. The ECM is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with mRNA and the interaction of the chromatin with the nuclear matrix. This, in turn, would affect the ECM, which would affect the cell.

A unique nuclear receptor direct repeat 17 (DR17) is present within the upstream region of Schistosoma mansoni female-specific p14 gene

International Nuclear Information System (INIS)

Fantappie, Marcelo Rosado; Furtado, Daniel Rodrigues; Rumjanek, Franklin David; LoVerde, Philip T.

2008-01-01

The eggs produced by sexually mature female Schistosma mansoni are responsible for the pathogenesis of the disease. The eggshell precursor gene p14 is expressed only in the vitelline cells of sexually mature female worms in response to a yet unidentified male stimulus. Herein, we report the identification of a novel nuclear receptor response element in the upstream region of the p14 gene. This element contains the canonical hexameric DNA core motif, 5'-PuGGTCA, composed of an atypically spaced direct repeat (DR17). Schistosome nuclear receptors SmRXR1 and SmNR1 specifically bound to the p14-DR17 element as a heterodimer. SmRXR1, but not SmNR1, bound to the motif as a monomer. Introduction of mutations in the TCA core sequence completely abolished the binding by SmRXR1/SmNR1 heterodimer. This finding supports our hypothesis that the expression of Schistosoma mansonip14 gene is regulated through the nuclear receptor signaling pathway

Apoptosis Gene Information System--AGIS.

Science.gov (United States)

Sakharkar, Kishore R; Clement, Marie V; Chow, Vincent T K; Pervaiz, Shazib

2006-05-01

Genes implicated in apoptosis have great relevance to biology, medicine and oncology. Here, we describe a unique resource, Apoptosis Gene Information System (AGIS) that provides data for over 2400 genes involved directly or indirectly, in apoptotic pathways of more than 350 different organisms. The organization of this information system is based on the principle of one-gene, one record. AGIS will be updated on a six monthly basis as new information becomes available. AGIS can be accessed at: http://www.cellfate.org/AGIS/.

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis.

Science.gov (United States)

Okada, Morihiro; Miller, Thomas C; Fu, Liezhen; Shi, Yun-Bo

2015-09-01

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis.

Study of the association between 3111T/C polymorphism of the CLOCK gene and the presence of overweight in schoolchildren.

Science.gov (United States)

Giovaninni, Nayara P; Fuly, Jeanne T; Moraes, Leonardo I; Coutinho, Thais N; Trarbach, Ericka B; Jorge, Alexander A de L; Costalonga, Everlayny F

2014-01-01

To evaluate the association between 3111T/C polymorphism of the CLOCK gene and the presence of obesity and sleep duration in children aged 6-13 years. In adults, this genetic variant has been associated with duration of sleep, ghrelin levels, weight, and eating habits. Although short sleep duration has been linked to obesity in children, no study has aimed to identify the possible molecular mechanisms of this association to date. Weight, height, and circumferences were transformed into Z-scores for age and gender. Genotyping was performed using TaqMan methodology. A questionnaire regarding hours of sleep was provided to parents. The appropriate statistical tests were performed. This study evaluated 370 children (45% males, 55% females, mean age 8.5 ± 1.5 years). The prevalence of overweight was 18%. The duration of sleep was, on average, 9.7hours, and was inversely related to age (ppolymorphism was not significantly associated with overweight or sleep duration in children in this city. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay.

Science.gov (United States)

Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

2011-01-24

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(®) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(®) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

Subgenomic Diversity Patterns Caused by Directional Selection in Bread Wheat Gene Pools

Directory of Open Access Journals (Sweden)

Kai Voss-Fels

2015-07-01

Full Text Available Genetic diversity represents the fundamental key to breeding success, providing the basis for breeders to select varieties with constantly improving yield performance. On the other hand, strong selection during domestication and breeding have eliminated considerable genetic diversity in the breeding pools of major crops, causing erosion of genetic potential for adaptation to emerging challenges like climate change. High-throughput genomic technologies can address this dilemma by providing detailed knowledge to characterize and replenish genetic diversity in breeding programs. In hexaploid bread wheat ( L., the staple food for 35% of the world’s population, bottlenecks during allopolyploidisation followed by strong artificial selection have considerably narrowed diversity to the extent that yields in many regions appear to be unexpectedly stagnating. In this study, we used a 90,000 single nucleotide polymorphism (SNP wheat genotyping array to assay high-frequency, polymorphic SNP markers in 460 accessions representing different phenological diversity groups from Asian, Australian, European, and North American bread wheat breeding materials. Detailed analysis of subgroup diversity at the chromosome and subgenome scale revealed highly distinct patterns of conserved linkage disequilibrium between different gene pools. The data enable identification of genome regions in most need of rejuvenation with novel diversity and provide a high-resolution molecular basis for genomic-assisted introgression of new variation into chromosome segments surrounding directionally selected metaloci conferring important adaptation and quality traits.

Efficient in vivo gene transfer to xenotransplanted human skin by lentivirus-mediated, but not by AAV-directed, gene delivery

DEFF Research Database (Denmark)

Jakobsen, Maria Vad; Askou, Anne Louise; Dokkedahl, Karin Stenderup

skin graft, and firefly luciferase expression was observed primarily in neighboring tissue beneath or surrounding the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin...... graft only. The study demonstrates limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo....

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

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L. Gavrilovic

2009-12-01

Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Correlation of HAMP gene polymorphisms and expression with the susceptibility and length of hospital stays in Taiwanese children with Kawasaki disease

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Lu, Hsing-Fang; Wong, Henry Sung-Ching; Yu, Hong-Ren; Kuo, Hsing-Chun; Huang, Fu-Chen; Lo, Mao-Hung; Hsieh, Kai-Sheng; Chen, Su-Fen; Chang, Wei-Chiao; Kuo, Ho-Chang

2017-01-01

Kawasaki disease (KD) is a form of systemic vasculitis. Regarding its pathogenesis, HAMP gene encoding hepcidin, which is significant for iron metabolism, has a vital function. In this study, we recruited a total of 381 KD patients for genotyping. Data from 997 subjects (500 subjects from cohort 1; 497 subjects from cohort 2) were used for analysis. Using TaqMan allelic discrimination, we determined five tag SNPs (rs916145, rs10421768, rs3817623, rs7251432, and rs2293689). Treatment outcome data related to such clinical phenotypes as coronary artery lesions (CAL), coronary artery aneurysms (CAA), and intravenous immunoglobulin (IVIG) effects were also collected. Furthermore, we measured plasma hepcidin levels with an enzyme-linked immunosorbent assay. We found that HAMP gene polymorphism (rs7251432, and rs2293689) was significantly correlated with KD risk and that plasma hepcidin levels both before and after IVIG treatment had a significantly positive correlation with length of hospital stays (R = 0.217, p = 0.046 and R = 0.381, p < 0.0001, respectively). In contrast, plasma hepcidin levels has a negative correlation with KD patients’ albumin levels (R = −0.27, p < 0.001) prior to IVIG treatment. This study's findings indicate that HAMP might have a role in the disease susceptibility, as well as its expressions correlated length of hospital stays, and albumin levels in Taiwanese children with KD. PMID:28881695

Nano-scale gene delivery systems; current technology, obstacles, and future directions.

Science.gov (United States)

Garcia-Guerra, Antonio; Dunwell, Thomas L; Trigueros, Sonia

2018-01-07

Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review. Copyright© Bentham Science Publishers; For

Gene-Environment Interactions in Genome-Wide Association Studies: Current Approaches and New Directions

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Winham, Stacey J.; Biernacka, Joanna M.

2013-01-01

Background: Complex psychiatric traits have long been thought to be the result of a combination of genetic and environmental factors, and gene-environment interactions are thought to play a crucial role in behavioral phenotypes and the susceptibility and progression of psychiatric disorders. Candidate gene studies to investigate hypothesized…

Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

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Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

2017-05-01

Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

Diagnostic efficacy of a real time-PCR assay for Chlamydia trachomatis infection in infertile women in north India

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Benu Dhawan

2014-01-01

Full Text Available Background & objectives: Little is known about the prevalence of Chlamydia trachomatis infection in Indian women with infertility. To improve the diagnosis of C. trachomatis infection in developing countries, there is an urgent need to establish cost-effective molecular test with high sensitivity and specificity. This study was conducted to determine the diagnostic utility of a real time-PCR assay for detention of C. trachomatis infection in infertile women attending an infertility clinic in north India. The in house real time-PCR assay was also compared with a commercial real-time PCR based detection system. Methods: Endocervical swabs, collected from 200 infertile women were tested for C. trachomatis by three different PCR assays viz. in-house real time-PCR targeting the cryptic plasmid using published primers, along with omp1 gene and cryptic plasmid based conventional PCR assays. Specimens were also subjected to direct fluorescence assay (DFA and enzyme immunoassay (EIA Performance of in-house real time-PCR was compared with that of COBAS Taqman C. trachomatis Test, version 2.0 on all in-house real time-PCR positive sample and 30 consecutive negative samples. Results: C. trachomatis infection was found in 13.5 per cent (27/200 infertile women by in-house real time-PCR, 11.5 per cent (23/200 by cryptic plasmid and/or omp1 gene based conventional PCR, 9 per cent (18/200 by DFA and 6.5 per cent (7/200 by EIA. The in-house real time-PCR exhibited a sensitivity and specificity of 100 per cent, considering COBAS Taqman CT Test as the gold standard. The negative and positive predictive values of the in-house real time-PCR were 100 per cent. The in-house real time-PCR could detect as low as 10 copies of C. trachomatis DNA per reaction. Interpretation & conclusions: In-house real time-PCR targeting the cryptic plasmid of C. trachomatis exhibited an excellent sensitivity and specificity similar to that of COBAS Taqman CT Test, v2.0 for detection of C

A Single Nucleotide Polymorphism (rs4236480 in TRPV5 Calcium Channel Gene Is Associated with Stone Multiplicity in Calcium Nephrolithiasis Patients

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Anas Khaleel

2015-01-01

Full Text Available Nephrolithiasis is characterized by calcification of stones in the kidneys from an unknown cause. Animal models demonstrated the functional roles of the transient receptor potential vanilloid member 5 (TRPV5 gene in calcium renal reabsorption and hypercalciuria. Therefore, TRPV5 was suggested to be involved in calcium homeostasis. However, whether genetic polymorphisms of TRPV5 are associated with kidney stone multiplicity or recurrence is unclear. In this study, 365 Taiwanese kidney-stone patients were recruited. Both biochemical data and DNA samples were collected. Genotyping was performed by a TaqMan allelic discrimination assay. We found that a TRPV5 polymorphism (rs4236480 was observed to be associated with stone multiplicity of calcium nephrolithiasis, as the risk of stone multiplicity was higher in patients with the TT+CT genotype than in patients with the CC genotype (p=0.0271. In summary, despite the complexity of nephrolithiasis and the potential association of numerous calcium homeostatic absorption/reabsorption factors, TRPV5 plays an important role in the pathogenesis of calcium nephrolithiasis.

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus.

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Zsuzsanna Elek

Full Text Available The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ2-test in combination with correction for multiple testing. For functional analysis, the entire 3' UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus

Science.gov (United States)

Elek, Zsuzsanna; Németh, Nóra; Nagy, Géza; Németh, Helga; Somogyi, Anikó; Hosszufalusi, Nóra; Sasvári-Székely, Mária; Rónai, Zsolt

2015-01-01

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ 2-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function. PMID:26426397

Development of chimeric gene promoters responsive to hypoxia and ionizing radiation

International Nuclear Information System (INIS)

Zheng Aiqing; Yu Jinming

2004-01-01

The authors describe two systems that make use of gene-directed enzyme prodrug therapy, regulated by radiation or hypoxic-responsive promoters. The use of treatment-, condition- or tumor-specific promoters to control gene-directed enzyme prodrug therapy is one such method for targeting gene expression to the tumor. The development of such strategies that achieve tumor targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy

Genetic variations in the vitamin-D receptor (VDR) gene in preeclampsia patients in the Chinese Han population.

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Zhan, Ying; Liu, Mengchun; You, Yuelan; Zhang, Yan; Wang, Jingli; Wang, Xunfeng; Liu, Shiguo; Liu, Xuemei

2015-07-01

Previous studies have indicated that vitamin D deficiency is linked to a risk of preeclampsia (PE). The aim of our study was to investigate the association between genetic variations in the vitamin-D receptor (VDR) gene and the susceptibility to PE in the Chinese Han population. We examined the genotypes VDR rs2228570, rs11568820 and rs1544410 in 402 PE patients and 554 normal pregnant women in the third trimester by TaqMan allelic discrimination real-time polymerase chain reaction. The clinical data of the individuals were collected to enable genotype-phenotype analysis. A significant statistical difference in the genotypic frequencies of rs2228570 between cases and controls was found (χ(2)=13.750, P=0.001). The G allele was the risk factor for the risk of PE (χ(2)=9.456, P=0.002, OR=1.137, 95% CI 1.111-1.610). There was no difference in the genotypic and allelic distributions of rs11568820 and rs1544410 between the two groups (P> 0.05). Our results provide evidence for a possible link between VDR and the development of PE in the Chinese Han population.

Targeted in vitro and in vivo gene transfer into T Lymphocytes: potential of direct inhibition of allo-immune activation

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Mehra Mandeep R

2006-11-01

Full Text Available Abstract Background Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-α and IFN-γ mRNA expression. Results Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2 resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines. Conclusion These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation.

The association of elastin gene variants with two angiographic subtypes of polypoidal choroidal vasculopathy.

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Suiho Yanagisawa

Full Text Available To compare the association of elastin (ELN gene variants between two different angiographic phenotypes of polypoidal choroidal vasculopathy (PCV.We included 411 treatment-naïve PCV patients and 350 controls in the present study. PCV was classified into two phenotypes (152 Type 1 and 259 Type 2 according to the presence or absence of feeding vessels found in indocyanine-green angiography. Single nucleotide polymorphisms (SNPs in the ELN region including rs868005, rs884843, rs2301995, rs13239907 and rs2856728 were genotyped using TaqMan Genotyping Assays.In the allelic association analyses, rs868005 showed the strongest association with Type 2 PCV (allelic odds ratio 1.56; p = 7.4x10(-6, while no SNP was significantly associated with Type 1 PCV. Genotype association analyses revealed the significant association of rs868005 with Type 2 PCV in log additive model and predominant model (odds ratio 1.75; p = 1.5x10(-6 and odds ratio 1.60; p = 0.0044, respectively, but not with Type 1 PCV. These findings were further corroborated by another control group in the literature.There may be significantly different associations in genetic variants of elastin between two angiographic phenotypes of PCV.

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

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Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

2008-12-01

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.

Science.gov (United States)

Choi, Goro; Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Seo, Ji Hyun; Choi, Jong Seob; Kim, Do Hyun; Seo, Tae Seok

2016-06-21

In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

International Nuclear Information System (INIS)

Kang, Yu; Zhang, Xiaoyan; Jiang, Wei; Wu, Chaoqun; Chen, Chunmei; Zheng, Yufang; Gu, Jianren; Xu, Congjian

2009-01-01

Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors. A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for gene therapy in ovarian cancer

Investigation of the ZNF804A gene polymorphism with genetic risk for bipolar disorder in attention deficit hyperactivity disorder

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Xu Xiaohui

2013-01-01

Full Text Available Abstract Background Genome-wide association studies (GWAS have been conducted on many psychiatric disorders. Evidence from large GWAS indicates that the single nucleotide polymorphism (SNP rs1344706 in the zinc-finger protein 804A gene (ZNF804A is associated with psychotic disorders including bipolar disorder and schizophrenia. One study also found significant association between rs1344706 and the executive control network of attention. In this study we examine the role of the rs1344706 polymorphism that previously showed association with BD and is known to alter expression of the gene in two clinical family-based ADHD samples from the UK and Taiwan. Findings To investigate the association between rs1344706 and ADHD, two family samples of ADHD probands from the United Kingdom (n = 180 and Taiwan (n = 212 were genotyped using TaqMan SNP genotyping assays and analysed using within-family transmission disequilibrium test. No significant associations were found between rs1344706 polymorphism and ADHD in either of the samples from Taiwan (P = 0.91 and UK (P = 0.41. Even combining the two datasets together the A allele of rs1344706 SNP was still not significantly over-transmitted to affected probands (P = 0.50. Furthermore, there was no evidence of association with the specific symptoms subgroups of inattention or hyperactivity-impulsivity. Conclusions In this study we used family-based ADHD data in the UK and Taiwanese population to test for an association between rs1344706 SNP in the ZNF804A gene and ADHD. Results showed no significant association of rs1344706 with ADHD in UK and Taiwanese samples.

Genomic signatures of local directional selection in a high gene flow marine organism; the Atlantic cod (Gadus morhua

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Mittelholzer Christian

2009-12-01

Full Text Available Abstract Background Marine fishes have been shown to display low levels of genetic structuring and associated high levels of gene flow, suggesting shallow evolutionary trajectories and, possibly, limited or lacking adaptive divergence among local populations. We investigated variation in 98 gene-associated single nucleotide polymorphisms (SNPs for evidence of selection in local populations of Atlantic cod (Gadus morhua L. across the species distribution. Results Our global genome scan analysis identified eight outlier gene loci with very high statistical support, likely to be subject to directional selection in local demes, or closely linked to loci under selection. Likewise, on a regional south/north transect of central and eastern Atlantic populations, seven loci displayed strongly elevated levels of genetic differentiation. Selection patterns among populations appeared to be relatively widespread and complex, i.e. outlier loci were generally not only associated with one of a few divergent local populations. Even on a limited geographical scale between the proximate North Sea and Baltic Sea populations four loci displayed evidence of adaptive evolution. Temporal genome scan analysis applied to DNA from archived otoliths from a Faeroese population demonstrated stability of the intra-population variation over 24 years. An exploratory landscape genetic analysis was used to elucidate potential effects of the most likely environmental factors responsible for the signatures of local adaptation. We found that genetic variation at several of the outlier loci was better correlated with temperature and/or salinity conditions at spawning grounds at spawning time than with geographic distance per se. Conclusion These findings illustrate that adaptive population divergence may indeed be prevalent despite seemingly high levels of gene flow, as found in most marine fishes. Thus, results have important implications for our understanding of the interplay of

The metabolic regulator CodY links L. monocytogenes metabolism to virulence by directly activating the virulence regulatory gene, prfA

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Lobel, Lior; Sigal, Nadejda; Borovok, Ilya; Belitsky, Boris R.; Sonenshein, Abraham L.; Herskovits, Anat A.

2015-01-01

Summary Metabolic adaptations are critical to the ability of bacterial pathogens to grow within host cells and are normally preceded by sensing of host-specific metabolic signals, which in turn can influence the pathogen's virulence state. Previously, we reported that the intracellular bacterial pathogen Listeria monocytogenes responds to low availability of branched-chain amino acids (BCAA) within mammalian cells by up-regulating both BCAA biosynthesis and virulence genes. The induction of virulence genes required the BCAA-responsive transcription regulator, CodY, but the molecular mechanism governing this mode of regulation was unclear. In this report, we demonstrate that CodY directly binds the coding sequence of the L. monocytogenes master virulence activator gene, prfA, 15 nt downstream of its start codon, and that this binding results in up-regulation of prfA transcription specifically under low concentrations of BCAA. Mutating this site abolished CodY binding and reduced prfA transcription in macrophages, and attenuated bacterial virulence in mice. Notably, the mutated binding site did not alter prfA transcription or PrfA activity under other conditions that are known to activate PrfA, such as during growth in the presence of glucose-1-phosphate. This study highlights the tight crosstalk between L. monocytogenes metabolism and virulence' while revealing novel features of CodY-mediated regulation. PMID:25430920

A first insight into the occurrence and expression of functional amoA and accA genes of autotrophic and ammonia-oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea

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Yakimov, Michail M.; Cono, Violetta La; Denaro, Renata

2009-05-01

The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000 m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan ® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000 m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000 m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

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Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

2006-07-01

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Direct sequencing of FAH gene in Pakistani tyrosinemia type 1 families reveals a novel mutation.

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Ijaz, Sadaqat; Zahoor, Muhammad Yasir; Imran, Muhammad; Afzal, Sibtain; Bhinder, Munir A; Ullah, Ihsan; Cheema, Huma Arshad; Ramzan, Khushnooda; Shehzad, Wasim

2016-03-01

Hereditary tyrosinemia type 1 (HT1) is a rare inborn error of tyrosine catabolism with a worldwide prevalence of one out of 100,000 live births. HT1 is clinically characterized by hepatic and renal dysfunction resulting from the deficiency of fumarylacetoacetate hydrolase (FAH) enzyme, caused by recessive mutations in the FAH gene. We present here the first report on identification of FAH mutations in HT1 patients from Pakistan with a novel one. Three Pakistani families, each having one child affected with HT1, were enrolled over a period of 1.5 years. Two of the affected children had died as they were presented late with acute form. All regions of the FAH gene spanning exons and splicing sites were amplified by polymerase chain reaction (PCR) and mutation analysis was carried out by direct sequencing. Results of sequencing were confirmed by restriction fragment length polymorphism (PCR-RFLP) analysis. Three different FAH mutations, one in each family, were found to co-segregate with the disease phenotype. Two of these FAH mutations have been known (c.192G>T and c.1062+5G>A [IVS12+5G>A]), while c.67T>C (p.Ser23Pro) was a novel mutation. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. Most of the HT1 patients die before they present to hospitals in Pakistan, as is indicated by enrollment of only three families in 1.5 years. Most of those with late clinical presentation do not survive due to delayed diagnosis followed by untimely treatment. This tragic condition advocates the establishment of expanded newborn screening program for HT1 within Pakistan.

Evolution of genetic architecture under directional selection.

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Hansen, Thomas F; Alvarez-Castro, José M; Carter, Ashley J R; Hermisson, Joachim; Wagner, Günter P

2006-08-01

We investigate the multilinear epistatic model under mutation-limited directional selection. We confirm previous results that only directional epistasis, in which genes on average reinforce or diminish each other's effects, contribute to the initial evolution of mutational effects. Thus, either canalization or decanalization can occur under directional selection, depending on whether positive or negative epistasis is prevalent. We then focus on the evolution of the epistatic coefficients themselves. In the absence of higher-order epistasis, positive pairwise epistasis will tend to weaken relative to additive effects, while negative pairwise epistasis will tend to become strengthened. Positive third-order epistasis will counteract these effects, while negative third-order epistasis will reinforce them. More generally, gene interactions of all orders have an inherent tendency for negative changes under directional selection, which can only be modified by higher-order directional epistasis. We identify three types of nonadditive quasi-equilibrium architectures that, although not strictly stable, can be maintained for an extended time: (1) nondirectional epistatic architectures; (2) canalized architectures with strong epistasis; and (3) near-additive architectures in which additive effects keep increasing relative to epistasis.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test.

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Kim, Hanah; Hur, Mina; Bae, Eunsin; Lee, Kyung-A; Lee, Woo-In

2018-02-19

Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0-1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

Gene Ontology and KEGG Enrichment Analyses of Genes Related to Age-Related Macular Degeneration

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Jian Zhang

2014-01-01

Full Text Available Identifying disease genes is one of the most important topics in biomedicine and may facilitate studies on the mechanisms underlying disease. Age-related macular degeneration (AMD is a serious eye disease; it typically affects older adults and results in a loss of vision due to retina damage. In this study, we attempt to develop an effective method for distinguishing AMD-related genes. Gene ontology and KEGG enrichment analyses of known AMD-related genes were performed, and a classification system was established. In detail, each gene was encoded into a vector by extracting enrichment scores of the gene set, including it and its direct neighbors in STRING, and gene ontology terms or KEGG pathways. Then certain feature-selection methods, including minimum redundancy maximum relevance and incremental feature selection, were adopted to extract key features for the classification system. As a result, 720 GO terms and 11 KEGG pathways were deemed the most important factors for predicting AMD-related genes.

Evaluation of candidate stromal epithelial cross-talk genes identifies association between risk of serous ovarian cancer and TERT, a cancer susceptibility "hot-spot".

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Sharon E Johnatty

2010-07-01

Full Text Available We hypothesized that variants in genes expressed as a consequence of interactions between ovarian cancer cells and the host micro-environment could contribute to cancer susceptibility. We therefore used a two-stage approach to evaluate common single nucleotide polymorphisms (SNPs in 173 genes involved in stromal epithelial interactions in the Ovarian Cancer Association Consortium (OCAC. In the discovery stage, cases with epithelial ovarian cancer (n=675 and controls (n=1,162 were genotyped at 1,536 SNPs using an Illumina GoldenGate assay. Based on Positive Predictive Value estimates, three SNPs-PODXL rs1013368, ITGA6 rs13027811, and MMP3 rs522616-were selected for replication using TaqMan genotyping in up to 3,059 serous invasive cases and 8,905 controls from 16 OCAC case-control studies. An additional 18 SNPs with Pper-alleleor=0.5. However genotypes at TERT rs7726159 were associated with ovarian cancer risk in the smaller, five-study replication study (Pper-allele=0.03. Combined analysis of the discovery and replication sets for this TERT SNP showed an increased risk of serous ovarian cancer among non-Hispanic whites [adj. ORper-allele 1.14 (1.04-1.24 p=0.003]. Our study adds to the growing evidence that, like the 8q24 locus, the telomerase reverse transcriptase locus at 5p15.33, is a general cancer susceptibility locus.

Visual gene developer: a fully programmable bioinformatics software for synthetic gene optimization

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McDonald Karen

2011-08-01

Full Text Available Abstract Background Direct gene synthesis is becoming more popular owing to decreases in gene synthesis pricing. Compared with using natural genes, gene synthesis provides a good opportunity to optimize gene sequence for specific applications. In order to facilitate gene optimization, we have developed a stand-alone software called Visual Gene Developer. Results The software not only provides general functions for gene analysis and optimization along with an interactive user-friendly interface, but also includes unique features such as programming capability, dedicated mRNA secondary structure prediction, artificial neural network modeling, network & multi-threaded computing, and user-accessible programming modules. The software allows a user to analyze and optimize a sequence using main menu functions or specialized module windows. Alternatively, gene optimization can be initiated by designing a gene construct and configuring an optimization strategy. A user can choose several predefined or user-defined algorithms to design a complicated strategy. The software provides expandable functionality as platform software supporting module development using popular script languages such as VBScript and JScript in the software programming environment. Conclusion Visual Gene Developer is useful for both researchers who want to quickly analyze and optimize genes, and those who are interested in developing and testing new algorithms in bioinformatics. The software is available for free download at http://www.visualgenedeveloper.net.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor.

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Bahadur, Urvashi; Ganjam, Goutham K; Vasudevan, Nandini; Kondaiah, Paturu

2005-02-28

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-alpha) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ERalpha antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the

A BAYESIAN NONPARAMETRIC MIXTURE MODEL FOR SELECTING GENES AND GENE SUBNETWORKS.

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Zhao, Yize; Kang, Jian; Yu, Tianwei

2014-06-01

It is very challenging to select informative features from tens of thousands of measured features in high-throughput data analysis. Recently, several parametric/regression models have been developed utilizing the gene network information to select genes or pathways strongly associated with a clinical/biological outcome. Alternatively, in this paper, we propose a nonparametric Bayesian model for gene selection incorporating network information. In addition to identifying genes that have a strong association with a clinical outcome, our model can select genes with particular expressional behavior, in which case the regression models are not directly applicable. We show that our proposed model is equivalent to an infinity mixture model for which we develop a posterior computation algorithm based on Markov chain Monte Carlo (MCMC) methods. We also propose two fast computing algorithms that approximate the posterior simulation with good accuracy but relatively low computational cost. We illustrate our methods on simulation studies and the analysis of Spellman yeast cell cycle microarray data.

Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants

International Nuclear Information System (INIS)

Kelly, R.; Miller, S.M.; Kurtz, M.B.; Kirsch, D.R.

1987-01-01

A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms

[Association of the genetic variations of bone morphogenetic protein 7 gene with diabetes and insulin resistance in Xinjiang Uygur population].

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Yan, Zhi-tao; Li, Nan-fang; Guo, Yan-ying; Yao, Xiao-guang; Wang, Hong-mei; Hu, Jun-li

2011-06-01

To investigate the association between the genetic variations of the functional region in bone morphogenetic protein gene (BMP7) with type 2 diabetes mellitus in Chinese Uygur individuals. A case-control study was conducted based on epidemiological investigation. A total of 717 Uygur subjects (276 males and 441 females) were selected and divided into two groups: diabetes mellitus group (n = 502, 191 males and 311 females) and control group (n = 215, 85 males and 130 females). All exons, flanking introns and the promoter regions of (BMP7) gene were sequenced in 48 Uygur diabetics. Representative variations were selected according to the minor allele frequency (MAF) and linkage disequilibrium and genotyped using the TaqMan polymerase chain reaction method in 717 Uygur individuals, a relatively isolated general population in a relatively homogeneous environment and a case-control study was conducted to test the association between the genetic variations of (BMP7) gene and type 2 diabetes mellitus. Five novel and 8 known variations in the (BMP7) gene were identified. All genotype distributions were tested for deviations from Hardy-Weinberg equilibrium (P> 0.05). There was significant difference of genotype distribution of rs6025422 between type 2 diabetes mellitus and control groups in the male population (P 0.05), but there was no difference in total and female population (P> 0.05). And the means of fasting blood glucose (FBG), fasting insulin and HOMA-index significantly decreased in individuals with AA, AG and GG genotypes of rs6025422 in male population (Ppopulation (P> 0.05). The logistic regression analysis showed that GG genotype of rs6025422 variation might be a protective factor for diabetes in male (OR= 0.637, 95% confidence interval 0.439-0.923, P< 0.05). The present study suggests that the rs6025422 polymorphism in (BMP7) gene may be associated with diabetes mellitus and insulin resistance in Uygur men.

Modulation of cortisol responses to the DEX/CRH test by polymorphisms of the interleukin-1beta gene in healthy adults

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Ota Miho

2011-07-01

Full Text Available Abstract Background Recently, hypothalamus-pituitary-adrenal (HPA axis function assessed with the combined dexamethasone (DEX/corticotropin releasing hormone (CRH test has been shown to be associated with response to antidepressant treatment. A polymorphism (rs16944 in the interleukin-1beta (IL-1β gene has also been reported to be associated with the medication response in depression. These findings prompted us to examine the possible association between IL-1β gene polymorphisms and HPA axis function assessed with the DEX/CRH test. Methods DEX/CRH test was performed in 179 healthy volunteers (45 males: mean age 40.5 ± 15.8 years; 134 females: mean age 47.1 ± 13.2 years. Five tagging single nucleotide polymorphisms (SNPs of IL-1β gene (rs2853550, rs1143634, rs1143633, rs1143630, rs16944 were selected at an r2 threshold of 0.80 with a minor allele frequency > 0.1. Genotyping was performed by the TaqMan allelic discrimination assay. A two-way factorial analysis of variance (ANOVA was performed with the DEX/CRH test results as the dependent variable and genotype and gender as independent variables. To account for multiple testing, P values Results The cortisol levels after DEX administration (DST-Cortisol showed significant associations with the genotypes of rs16944 (P = 0.00049 and rs1143633 (P = 0.0060, with no significant gender effect or genotype × gender interaction. On the other hand, cortisol levels after CRH administration (DEX/CRH-Cortisol were affected by gender but were not significantly influenced by the genotype of the examined SNPs, with no significant genotype × gender interaction. Conclusions Our results suggest that genetic variations in the IL-1β gene contribute to the HPA axis alteration assessed by DST-Cortisol in healthy subjects. On the other hand, no significant associations of the IL-1β gene polymorphisms with the DEX/CRH-Cortisol were observed. Confirmation of our findings in futures studies may add new insight into

Modulation of cortisol responses to the DEX/CRH test by polymorphisms of the interleukin-1beta gene in healthy adults.

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Sasayama, Daimei; Hori, Hiroaki; Iijima, Yoshimi; Teraishi, Toshiya; Hattori, Kotaro; Ota, Miho; Fujii, Takashi; Higuchi, Teruhiko; Amano, Naoji; Kunugi, Hiroshi

2011-07-05

Recently, hypothalamus-pituitary-adrenal (HPA) axis function assessed with the combined dexamethasone (DEX)/corticotropin releasing hormone (CRH) test has been shown to be associated with response to antidepressant treatment. A polymorphism (rs16944) in the interleukin-1beta (IL-1β) gene has also been reported to be associated with the medication response in depression. These findings prompted us to examine the possible association between IL-1β gene polymorphisms and HPA axis function assessed with the DEX/CRH test. DEX/CRH test was performed in 179 healthy volunteers (45 males: mean age 40.5 ± 15.8 years; 134 females: mean age 47.1 ± 13.2 years). Five tagging single nucleotide polymorphisms (SNPs) of IL-1β gene (rs2853550, rs1143634, rs1143633, rs1143630, rs16944) were selected at an r2 threshold of 0.80 with a minor allele frequency > 0.1. Genotyping was performed by the TaqMan allelic discrimination assay. A two-way factorial analysis of variance (ANOVA) was performed with the DEX/CRH test results as the dependent variable and genotype and gender as independent variables. To account for multiple testing, P values rs16944 (P = 0.00049) and rs1143633 (P = 0.0060), with no significant gender effect or genotype × gender interaction. On the other hand, cortisol levels after CRH administration (DEX/CRH-Cortisol) were affected by gender but were not significantly influenced by the genotype of the examined SNPs, with no significant genotype × gender interaction. Our results suggest that genetic variations in the IL-1β gene contribute to the HPA axis alteration assessed by DST-Cortisol in healthy subjects. On the other hand, no significant associations of the IL-1β gene polymorphisms with the DEX/CRH-Cortisol were observed. Confirmation of our findings in futures studies may add new insight into the communication between the immune system and the HPA axis.

Inferring Gene Regulatory Networks Using Conditional Regulation Pattern to Guide Candidate Genes.

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Fei Xiao

Full Text Available Combining path consistency (PC algorithms with conditional mutual information (CMI are widely used in reconstruction of gene regulatory networks. CMI has many advantages over Pearson correlation coefficient in measuring non-linear dependence to infer gene regulatory networks. It can also discriminate the direct regulations from indirect ones. However, it is still a challenge to select the conditional genes in an optimal way, which affects the performance and computation complexity of the PC algorithm. In this study, we develop a novel conditional mutual information-based algorithm, namely RPNI (Regulation Pattern based Network Inference, to infer gene regulatory networks. For conditional gene selection, we define the co-regulation pattern, indirect-regulation pattern and mixture-regulation pattern as three candidate patterns to guide the selection of candidate genes. To demonstrate the potential of our algorithm, we apply it to gene expression data from DREAM challenge. Experimental results show that RPNI outperforms existing conditional mutual information-based methods in both accuracy and time complexity for different sizes of gene samples. Furthermore, the robustness of our algorithm is demonstrated by noisy interference analysis using different types of noise.

Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

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Martin, Guiomar; Soy, Judit; Monte, Elena

2016-01-01

Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.

The evolution of milk casein genes from tooth genes before the origin of mammals.

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Kawasaki, Kazuhiko; Lafont, Anne-Gaelle; Sire, Jean-Yves

2011-07-01

Caseins are among cardinal proteins that evolved in the lineage leading to mammals. In milk, caseins and calcium phosphate (CaP) form a huge complex called casein micelle. By forming the micelle, milk maintains high CaP concentrations, which help altricial mammalian neonates to grow bone and teeth. Two types of caseins are known. Ca-sensitive caseins (α(s)- and β-caseins) bind Ca but precipitate at high Ca concentrations, whereas Ca-insensitive casein (κ-casein) does not usually interact with Ca but instead stabilizes the micelle. Thus, it is thought that these two types of caseins are both necessary for stable micelle formation. Both types of caseins show high substitution rates, which make it difficult to elucidate the evolution of caseins. Yet, recent studies have revealed that all casein genes belong to the secretory calcium-binding phosphoprotein (SCPP) gene family that arose by gene duplication. In the present study, we investigated exon-intron structures and phylogenetic distributions of casein and other SCPP genes, particularly the odontogenic ameloblast-associated (ODAM) gene, the SCPP-Pro-Gln-rich 1 (SCPPPQ1) gene, and the follicular dendritic cell secreted peptide (FDCSP) gene. The results suggest that contemporary Ca-sensitive casein genes arose from a putative common ancestor, which we refer to as CSN1/2. The six putative exons comprising CSN1/2 are all found in SCPPPQ1, although ODAM also shares four of these exons. By contrast, the five exons of the Ca-insensitive casein gene are all reminiscent of FDCSP. The phylogenetic distribution of these genes suggests that both SCPPPQ1 and FDCSP arose from ODAM. We thus argue that all casein genes evolved from ODAM via two different pathways; Ca-sensitive casein genes likely originated directly from SCPPPQ1, whereas the Ca-insensitive casein genes directly differentiated from FDCSP. Further, expression of ODAM, SCPPPQ1, and FDCSP was detected in dental tissues, supporting the idea that both types of caseins

Learning gene networks under SNP perturbations using eQTL datasets.

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Lingxue Zhang

2014-02-01

Full Text Available The standard approach for identifying gene networks is based on experimental perturbations of gene regulatory systems such as gene knock-out experiments, followed by a genome-wide profiling of differential gene expressions. However, this approach is significantly limited in that it is not possible to perturb more than one or two genes simultaneously to discover complex gene interactions or to distinguish between direct and indirect downstream regulations of the differentially-expressed genes. As an alternative, genetical genomics study has been proposed to treat naturally-occurring genetic variants as potential perturbants of gene regulatory system and to recover gene networks via analysis of population gene-expression and genotype data. Despite many advantages of genetical genomics data analysis, the computational challenge that the effects of multifactorial genetic perturbations should be decoded simultaneously from data has prevented a widespread application of genetical genomics analysis. In this article, we propose a statistical framework for learning gene networks that overcomes the limitations of experimental perturbation methods and addresses the challenges of genetical genomics analysis. We introduce a new statistical model, called a sparse conditional Gaussian graphical model, and describe an efficient learning algorithm that simultaneously decodes the perturbations of gene regulatory system by a large number of SNPs to identify a gene network along with expression quantitative trait loci (eQTLs that perturb this network. While our statistical model captures direct genetic perturbations of gene network, by performing inference on the probabilistic graphical model, we obtain detailed characterizations of how the direct SNP perturbation effects propagate through the gene network to perturb other genes indirectly. We demonstrate our statistical method using HapMap-simulated and yeast eQTL datasets. In particular, the yeast gene network

Association of interleukin-1 gene variations with moderate to severe chronic periodontitis in multiple ethnicities

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Wu, X; Offenbacher, S; Lόpez, N J; Chen, D; Wang, H-Y; Rogus, J; Zhou, J; Beck, J; Jiang, S; Bao, X; Wilkins, L; Doucette-Stamm, L; Kornman, K

2015-01-01

Background and Objective Genetic markers associated with disease are often non-functional and generally tag one or more functional “causative” variants in linkage disequilibrium. Markers may not show tight linkage to the causative variants across multiple ethnicities due to evolutionary divergence, and therefore may not be informative across different population groups. Validated markers of disease suggest causative variants exist in the gene and, if the causative variants can be identified, it is reasonable to hypothesize that such variants will be informative across diverse populations. The aim of this study was to test that hypothesis using functional Interleukin-1 (IL-1) gene variations across multiple ethnic populations to replace the non-functional markers originally associated with chronic adult periodontitis in Caucasians. Material and Methods Adult chronic periodontitis cases and controls from four ethnic groups (Caucasians, African Americans, Hispanics and Asians) were recruited in the USA, Chile and China. Genotypes of IL1B gene single nucleotide polymorphisms (SNPs), including three functional SNPs (rs16944, rs1143623, rs4848306) in the promoter and one intronic SNP (rs1143633), were determined using a single base extension method or TaqMan 5′ nuclease assay. Logistic regression and other statistical analyses were used to examine the association between moderate to severe periodontitis and IL1B gene variations, including SNPs, haplotypes and composite genotypes. Genotype patterns associated with disease in the discovery study were then evaluated in independent validation studies. Results Significant associations were identified in the discovery study, consisting of Caucasians and African Americans, between moderate to severe adult chronic periodontitis and functional variations in the IL1B gene, including a pattern of four IL1B SNPs (OR = 1.87, p < 0.0001). The association between the disease and this IL1B composite genotype pattern was validated

Analysis of the influence of the T393C polymorphism of the GNAS gene on the clinical expression of primary hyperparathyroidism.

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Piedra, María; Berja, Ana; Ramos, Laura; García-Unzueta, María Teresa; Morán, Jesús Manuel; Ruiz, David; Amado, José Antonio

2017-12-01

The receptor of parathyroid hormone and parathyroid hormone-related-protein (PTH/PTHrp) is located in the cell membrane of target tissues - kidney and osteoblasts. It is a G protein-coupled-receptor whose G s α subunit is encoded by the GNAS gene. Our aim was to study whether the single nucleotide polymorphism (SNP) T393C of the GNAS gene is associated with renal stones, bone mineral density (BMD), or bone remodelling markers in primary hyperparathyroidism (PHPT). An analysis was made of clinical and biochemical parameters and densitometric values in three areas and their relationship with the T393C SNP of the GNAS gene in 261 patients with primary hyperparathyroidism and in 328 healthy controls. Genotyping was performed using the Custom Taqman ® SNP Genotyping assay. The genotype frequencies of GNAS T/C 393 were similar in the control and PHPT groups. No association was found between genotypes and clinical expression of PHPT (renal stones and bone fractures). A nonstatistically significant trend was seen to lower BMD in the lumbar spine, femoral neck, and total hip in both PHPT and control C homozygote subjects. Genetic susceptibility to PHPT related to the GNAS T393C polymorphism or a major influence in its development and clinical expression were found. A C allele-related susceptibility to lower BMD in trabecular bone in both PHPT and control subjects is not sufficient to suggest a more severe clinical expression of PHPT. This trend may be considered as a basis for further studies with larger sample sizes and complementary functional evaluation. Copyright © 2017 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

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Mizoshiri, N; Kishida, T; Yamamoto, K; Shirai, T; Terauchi, R; Tsuchida, S; Mori, Y; Ejima, A; Sato, Y; Arai, Y; Fujiwara, H; Yamamoto, T; Kanamura, N; Mazda, O; Kubo, T

2015-11-27

Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Artificial neural network inference (ANNI: a study on gene-gene interaction for biomarkers in childhood sarcomas.

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Dong Ling Tong

Full Text Available OBJECTIVE: To model the potential interaction between previously identified biomarkers in children sarcomas using artificial neural network inference (ANNI. METHOD: To concisely demonstrate the biological interactions between correlated genes in an interaction network map, only 2 types of sarcomas in the children small round blue cell tumors (SRBCTs dataset are discussed in this paper. A backpropagation neural network was used to model the potential interaction between genes. The prediction weights and signal directions were used to model the strengths of the interaction signals and the direction of the interaction link between genes. The ANN model was validated using Monte Carlo cross-validation to minimize the risk of over-fitting and to optimize generalization ability of the model. RESULTS: Strong connection links on certain genes (TNNT1 and FNDC5 in rhabdomyosarcoma (RMS; FCGRT and OLFM1 in Ewing's sarcoma (EWS suggested their potency as central hubs in the interconnection of genes with different functionalities. The results showed that the RMS patients in this dataset are likely to be congenital and at low risk of cardiomyopathy development. The EWS patients are likely to be complicated by EWS-FLI fusion and deficiency in various signaling pathways, including Wnt, Fas/Rho and intracellular oxygen. CONCLUSIONS: The ANN network inference approach and the examination of identified genes in the published literature within the context of the disease highlights the substantial influence of certain genes in sarcomas.

Expression of Five Neuroblastoma Genes in Bone Marrow or Blood of Patients with Relapsed/Refractory Neuroblastoma Provides a New Biomarker for Disease and Prognosis.

Science.gov (United States)

Marachelian, Araz; Villablanca, Judith G; Liu, Cathy W; Liu, Betty; Goodarzian, Fariba; Lai, Hollie A; Shimada, Hiroyuki; Tran, Hung C; Parra, Jaime A; Gallego, Richard; Bedrossian, Nora; Young, Sabrina; Czarnecki, Scarlett; Kennedy, Rebekah; Weiss, Brian D; Goldsmith, Kelly; Granger, Meaghan; Matthay, Katherine K; Groshen, Susan; Asgharzadeh, Shahab; Sposto, Richard; Seeger, Robert C

2017-09-15

Purpose: We determined whether quantifying neuroblastoma-associated mRNAs (NB-mRNAs) in bone marrow and blood improves assessment of disease and prediction of disease progression in patients with relapsed/refractory neuroblastoma. Experimental Design: mRNA for CHGA, DCX, DDC, PHOX2B, and TH was quantified in bone marrow and blood from 101 patients concurrently with clinical disease evaluations. Correlation between NB-mRNA (delta cycle threshold, Δ C t , for the geometric mean of genes from the TaqMan Low Density Array NB5 assay) and morphologically defined tumor cell percentage in bone marrow, 123 I-meta-iodobenzylguanidine (MIBG) Curie score, and CT/MRI-defined tumor longest diameter was determined. Time-dependent covariate Cox regression was used to analyze the relationship between Δ C t and progression-free survival (PFS). Results: NB-mRNA was detectable in 83% of bone marrow (185/223) and 63% (89/142) of blood specimens, and their Δ C t values were correlated (Spearman r = 0.67, P neuroblastoma. Clin Cancer Res; 23(18); 5374-83. ©2017 AACR . ©2017 American Association for Cancer Research.

Gene mining a marama bean expressed sequence tags (ESTs ...

African Journals Online (AJOL)

The authors reported the identification of genes associated with embryonic development and microsatellite sequences. The future direction will entail characterization of these genes using gene over-expression and mutant assays. Key words: Namibia, simple sequence repeats (SSR), data mining, homology searches, ...

Association of hypoxia inducible factor-1 alpha gene polymorphism with both type 1 and type 2 diabetes in a Caucasian (Hungarian sample

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Panczel Pal

2009-08-01

Full Text Available Abstract Background Hypoxia inducible factor-1 alpha (HIF-1α is a transcription factor that plays an important role in neo-vascularisation, embryonic pancreas beta-cell mass development, and beta cell protection. Recently a non synonymous single nucleotide polymorphism (g.C45035T SNP, rs11549465 of HIF-1α gene, resulting in the p.P582S amino acid change has been shown to be associated with type 2 diabetes (T2DM in a Japanese population. Our aim was to replicate these findings on a Caucasian (Hungarian population, as well as to study whether this genetic effect is restricted to T2DM or can be expanded to diabetes in general. Methods A large Caucasian sample (N = 890 was recruited including 370 T2DM, 166 T1DM and 354 healthy subjects. Genotyping was validated by two independent methods: a restriction fragment analysis (RFLP and a real time PCR using TaqMan probes. An overestimation of heterozygotes by RFLP was observed as a consequence of a nearby SNP (rs34005929. Therefore genotyping results of the justified TaqMan system were accepted. The measured genotype distribution corresponded to Hardy-Weinberg equilibrium (P = 0.740 Results As the TT genotype was extremely rare in the population (0.6% in clinical sample and 2.5% in controls, the genotypes were grouped as T absent (CC and T present (CT and TT. Genotype-wise analysis showed a significant increase of T present group in controls (24.0% as compared to patients (16.8%, P = 0.008. This genetic effect was demonstrated in the separated samples of type 1 (15.1%, P = 0.020, and also in type 2 (17.6%, P = 0.032 diabetes. Allele-wise analysis gave identical results showing a higher frequency of the T allele in the control sample (13.3% than in the clinical sample (8.7%, P = 0.002 with similar results in type 1 (7.8%, P = 0.010 and type 2 (9.1%, P = 0.011 diabetes. The odds ratio for diabetes (either type 1 or 2 was 1.56 in the presence of the C allele. Conclusion We confirmed the protective effect

ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

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Jeron Andreas

2012-12-01

Full Text Available Abstract Background The transcription factor (TF forkhead box P3 (FOXP3 is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs. It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

The bystander effect of cancer gene therapy

International Nuclear Information System (INIS)

Lumniczky, K.; Safrany, G.

2008-01-01

Cancer gene therapy is a new, promising therapeutic agent. In the clinic, it should be used in combination with existing modalities, such as tumour irradiation. First, we summarise the most important fields of cancer gene therapy: gene directed enzyme pro-drug therapy; the activation of an anti-tumour immune attack; restoration of the wild type p53 status; the application of new, replication competent and oncolytic viral vectors; tumour specific, as well as radiation- and hypoxia-induced gene expression. Special emphasizes are put on the combined effect of these modalities with local tumour irradiation. Using the available vector systems, only a small portion of the cancer cells will contain the therapeutic genes under therapeutic situations. Bystander cell killing might contribute to the success of various gene therapy protocols. We summarise the evidences that lethal bystander effects may occur during cancer gene therapy. Bystander effects are especially important in the gene directed enzyme pro-drug therapy. There, bystander cell killing might have different routes: cell communication through gap junction intercellular contacts; release of toxic metabolites into the neighbourhood or to larger distances; phagocytosis of apoptotic bodies; and the activation of the immune system. Bystander cell killing can be enhanced by the introduction of gap junction proteins into the cells, by further activating the immune system with immune-stimulatory molecules, or by introducing genes into the cells that help the transfer of cytotoxic genes and / or metabolites into the bystander cells. In conclusion, there should be additional improvements in cancer gene therapy for the more efficient clinical application. (orig.)

Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes

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Allan Andrew C

2008-07-01

Full Text Available Abstract Background Transcription factors (TFs co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1 leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA

Regulation of T cell activation by HIV-1 accessory proteins: Vpr acts via distinct mechanisms to cooperate with Nef in NFAT-directed gene expression and to promote transactivation by CREB

International Nuclear Information System (INIS)

Lahti, Anna L.; Manninen, Aki; Saksela, Kalle

2003-01-01

Nef and Vpr are lentiviral accessory proteins that have been implicated in regulation of cellular gene expression. We noticed that Vpr can potentiate Nef-induced activation of nuclear factor of activated T cells (NFAT)-dependent transcription. Unlike Nef, which stimulated calcium signaling to activate NFAT, Vpr functioned farther downstream. Similar to the positive effects of Vpr on most of the transcriptional test systems that we used, potentiation of NFAT-directed gene expression was relatively modest in magnitude (two- to threefold) and depended on the cell cycle-arresting capacity of Vpr. By contrast, we found that Vpr could cause more than fivefold upregulation of cyclic AMP response element (CRE)-directed transcription via a mechanism that did not require Vpr-induced G2/M arrest. This effect, however, was only evident under suboptimal conditions known to lead to serine phosphorylation of the CRE binding factor (CREB) but not to CREB-dependent gene expression. This suggested that Vpr may act by stabilizing interactions with CREB and its transcriptional cofactor CREB binding protein (CBP). Indeed, this effect could be blocked by cotransfection of the adenoviral CBP inhibitor E1A. These results provide additional evidence for cell cycle-independent regulation of gene expression by Vpr and implicate CREB as a potentially important target for Vpr action in HIV-infected host cells

Gene × Smoking Interactions on Human Brain Gene Expression: Finding Common Mechanisms in Adolescents and Adults

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Wolock, Samuel L.; Yates, Andrew; Petrill, Stephen A.; Bohland, Jason W.; Blair, Clancy; Li, Ning; Machiraju, Raghu; Huang, Kun; Bartlett, Christopher W.

2013-01-01

Background: Numerous studies have examined gene × environment interactions (G × E) in cognitive and behavioral domains. However, these studies have been limited in that they have not been able to directly assess differential patterns of gene expression in the human brain. Here, we assessed G × E interactions using two publically available datasets…

The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

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Tydén, E; Löfgren, M; Hakhverdyan, M; Tjälve, H; Larsson, P

2013-08-01

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury. © 2012 John Wiley & Sons Ltd.

Using the gene ontology to scan multilevel gene sets for associations in genome wide association studies.

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Schaid, Daniel J; Sinnwell, Jason P; Jenkins, Gregory D; McDonnell, Shannon K; Ingle, James N; Kubo, Michiaki; Goss, Paul E; Costantino, Joseph P; Wickerham, D Lawrence; Weinshilboum, Richard M

2012-01-01

Gene-set analyses have been widely used in gene expression studies, and some of the developed methods have been extended to genome wide association studies (GWAS). Yet, complications due to linkage disequilibrium (LD) among single nucleotide polymorphisms (SNPs), and variable numbers of SNPs per gene and genes per gene-set, have plagued current approaches, often leading to ad hoc "fixes." To overcome some of the current limitations, we developed a general approach to scan GWAS SNP data for both gene-level and gene-set analyses, building on score statistics for generalized linear models, and taking advantage of the directed acyclic graph structure of the gene ontology when creating gene-sets. However, other types of gene-set structures can be used, such as the popular Kyoto Encyclopedia of Genes and Genomes (KEGG). Our approach combines SNPs into genes, and genes into gene-sets, but assures that positive and negative effects of genes on a trait do not cancel. To control for multiple testing of many gene-sets, we use an efficient computational strategy that accounts for LD and provides accurate step-down adjusted P-values for each gene-set. Application of our methods to two different GWAS provide guidance on the potential strengths and weaknesses of our proposed gene-set analyses. © 2011 Wiley Periodicals, Inc.

Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data

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Tintle Nathan L

2012-08-01

Full Text Available Abstract Background Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. Results We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Conclusions Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

Deconstructing the pluripotency gene regulatory network

KAUST Repository

Li, Mo

2018-04-04

Pluripotent stem cells can be isolated from embryos or derived by reprogramming. Pluripotency is stabilized by an interconnected network of pluripotency genes that cooperatively regulate gene expression. Here we describe the molecular principles of pluripotency gene function and highlight post-transcriptional controls, particularly those induced by RNA-binding proteins and alternative splicing, as an important regulatory layer of pluripotency. We also discuss heterogeneity in pluripotency regulation, alternative pluripotency states and future directions of pluripotent stem cell research.

Deconstructing the pluripotency gene regulatory network

KAUST Repository

Li, Mo; Belmonte, Juan Carlos Izpisua

2018-01-01

Pluripotent stem cells can be isolated from embryos or derived by reprogramming. Pluripotency is stabilized by an interconnected network of pluripotency genes that cooperatively regulate gene expression. Here we describe the molecular principles of pluripotency gene function and highlight post-transcriptional controls, particularly those induced by RNA-binding proteins and alternative splicing, as an important regulatory layer of pluripotency. We also discuss heterogeneity in pluripotency regulation, alternative pluripotency states and future directions of pluripotent stem cell research.

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification.

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Schaub, Christoph; Nagaso, Hideyuki; Jin, Hong; Frasch, Manfred

2012-03-01

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

Improved animal models for testing gene therapy for atherosclerosis.

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Du, Liang; Zhang, Jingwan; De Meyer, Guido R Y; Flynn, Rowan; Dichek, David A

2014-04-01

Gene therapy delivered to the blood vessel wall could augment current therapies for atherosclerosis, including systemic drug therapy and stenting. However, identification of clinically useful vectors and effective therapeutic transgenes remains at the preclinical stage. Identification of effective vectors and transgenes would be accelerated by availability of animal models that allow practical and expeditious testing of vessel-wall-directed gene therapy. Such models would include humanlike lesions that develop rapidly in vessels that are amenable to efficient gene delivery. Moreover, because human atherosclerosis develops in normal vessels, gene therapy that prevents atherosclerosis is most logically tested in relatively normal arteries. Similarly, gene therapy that causes atherosclerosis regression requires gene delivery to an existing lesion. Here we report development of three new rabbit models for testing vessel-wall-directed gene therapy that either prevents or reverses atherosclerosis. Carotid artery intimal lesions in these new models develop within 2-7 months after initiation of a high-fat diet and are 20-80 times larger than lesions in a model we described previously. Individual models allow generation of lesions that are relatively rich in either macrophages or smooth muscle cells, permitting testing of gene therapy strategies targeted at either cell type. Two of the models include gene delivery to essentially normal arteries and will be useful for identifying strategies that prevent lesion development. The third model generates lesions rapidly in vector-naïve animals and can be used for testing gene therapy that promotes lesion regression. These models are optimized for testing helper-dependent adenovirus (HDAd)-mediated gene therapy; however, they could be easily adapted for testing of other vectors or of different types of molecular therapies, delivered directly to the blood vessel wall. Our data also supports the promise of HDAd to deliver long

The rs7517847 polymorphism in the IL-23R gene is associated with gout in a Chinese Han male population.

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Liu, Shiguo; He, Hongmei; Yu, Renchao; Han, Lin; Wang, Can; Cui, Ying; Li, Changgui

2015-05-01

Gout is a polygenic auto-inflammatory disease, in which inflammation plays an important role in disease pathogenesis. The cytokine interleukin (IL)-23 promotes inflammation and helps to guide inflammatory cells, while studies have shown that the IL-23R gene is associated with susceptibility to several immune-related diseases. This study aimed to determine whether the IL-23R rs7517847 (G/T) polymorphism is associated with gout in a Chinese Han male population. We recruited 400 patients with gout and 582 gout-free controls. After obtaining blood samples for DNA extraction, genotyping of the rs7517847 polymorphism was performed by fluorescence-based quantitative PCR using TaqMan probes. An association analysis was carried out using the χ(2) test. A genotype-phenotype analysis was also conducted. Both genotypic and allelic frequencies of rs7517847 differed significantly between gout patients and controls (χ(2) = 6.792, df = 2, P = 0.034 by genotype; χ(2) = 4.202, df = 1, P = 0.04 by allele). IL-23R may be associated with gout in a Chinese Han male population, although our findings should be confirmed using larger sample sizes and other independent populations.

Ghrelin gene polymorphism as a genetic biomarker for prediction of therapy induced clearance in Egyptian chronic HCV patients.

Science.gov (United States)

Hamdy, Marwa; Kassim, Samar Kamal; Khairy, Eman; Maher, Mohsen; Mansour, Khaled Amr; Albreedy, Ashraf M

2018-04-05

Ghrelin (GHRL) has important implications for liver disease. It has anti-inflammatory effects, regulates cell proliferation, modulates the fibrogenic response and protects liver tissue. Genetic variations in the GHRL gene may play a crucial role in the development of chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Therefore, we examined the association of GHRL gene polymorphisms (rs26312 and rs27647), and its serum level to virologic responses to combined sofosbuvir and Simeprevir therapy for a course of 12 successive weeks in Egyptian chronic hepatitis C (CHC) patients. Human genomic and clinical data were collected from 100 Egyptian participants in this study, 90 HCV patients who received sofosbuvir and Simeprevir and 10 non-HCV healthy subjects. Genotyping of GHRL rs26312 and rs27647, were determined with the TaqMan qRT-PCR allele detection assay. The serum GHRL concentrations were determined using enzyme-linked immunosorbent assay (ELISA). GHRL polymorphisms (rs26312 and rs27647) genotype distributions and allele frequencies did not differ between HCV patients and normal healthy subjects or between patient groups when compared according to the therapeutic response. In addition, we found significant lower serum GHRL levels in CHC patients compared with the healthy controls. However, there was no significant association of the GHRL rs26312 and rs27647 polymorphisms with GHRL levels in CHC patients. We conclude that GHRL SNPs (rs26312 and rs27647) do not affect response to combined sofosbuvir and Simeprevir treatment in chronic Egyptian HCV patients. Copyright © 2018 Elsevier B.V. All rights reserved.

A Multiple Interaction Analysis Reveals ADRB3 as a Potential Candidate for Gallbladder Cancer Predisposition via a Complex Interaction with Other Candidate Gene Variations

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Rajani Rai

2015-11-01

Full Text Available Gallbladder cancer is the most common and a highly aggressive biliary tract malignancy with a dismal outcome. The pathogenesis of the disease is multifactorial, comprising the combined effect of multiple genetic variations of mild consequence along with numerous dietary and environmental risk factors. Previously, we demonstrated the association of several candidate gene variations with GBC risk. In this study, we aimed to identify the combination of gene variants and their possible interactions contributing towards genetic susceptibility of GBC. Here, we performed Multifactor-Dimensionality Reduction (MDR and Classification and Regression Tree Analysis (CRT to investigate the gene–gene interactions and the combined effect of 14 SNPs in nine genes (DR4 (rs20576, rs6557634; FAS (rs2234767; FASL (rs763110; DCC (rs2229080, rs4078288, rs7504990, rs714; PSCA (rs2294008, rs2978974; ADRA2A (rs1801253; ADRB1 (rs1800544; ADRB3 (rs4994; CYP17 (rs2486758 involved in various signaling pathways. Genotyping was accomplished by PCR-RFLP or Taqman allelic discrimination assays. SPSS software version 16.0 and MDR software version 2.0 were used for all the statistical analysis. Single locus investigation demonstrated significant association of DR4 (rs20576, rs6557634, DCC (rs714, rs2229080, rs4078288 and ADRB3 (rs4994 polymorphisms with GBC risk. MDR analysis revealed ADRB3 (rs4994 to be crucial candidate in GBC susceptibility that may act either alone (p < 0.0001, CVC = 10/10 or in combination with DCC (rs714 and rs2229080, p < 0.0001, CVC = 9/10. Our CRT results are in agreement with the above findings. Further, in-silico results of studied SNPs advocated their role in splicing, transcriptional and/or protein coding regulation. Overall, our result suggested complex interactions amongst the studied SNPs and ADRB3 rs4994 as candidate influencing GBC susceptibility.

Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples.

Science.gov (United States)

Schalasta, Gunnar; Börner, Anna; Speicher, Andrea; Enders, Martin

2018-03-28

Proper management of patients with chronic hepatitis B virus (HBV) infection requires monitoring of plasma or serum HBV DNA levels using a highly sensitive nucleic acid amplification test. Because commercially available assays differ in performance, we compared herein the performance of the Hologic Aptima HBV Quant assay (Aptima) to that of the Roche Cobas TaqMan HBV test for use with the high pure system (HPS/CTM). Assay performance was assessed using HBV reference panels as well as plasma and serum samples from chronically HBV-infected patients. Method correlation, analytical sensitivity, precision/reproducibility, linearity, bias and influence of genotype were evaluated. Data analysis was performed using linear regression, Deming correlation analysis and Bland-Altman analysis. Agreement between the assays for the two reference panels was good, with a difference in assay values vs. target 0.98). The two assays had similar bias and precision across the different genotypes tested at low viral loads (25-1000 IU/mL). Aptima has a performance comparable with that of HPS/CTM, making it suitable for use for HBV infection monitoring. Aptima runs on a fully automated platform (the Panther system) and therefore offers a significantly improved workflow compared with HPS/CTM.

Discovering genes underlying QTL

Energy Technology Data Exchange (ETDEWEB)

Vanavichit, Apichart [Kasetsart University, Kamphaengsaen, Nakorn Pathom (Thailand)

2002-02-01

A map-based approach has allowed scientists to discover few genes at a time. In addition, the reproductive barrier between cultivated rice and wild relatives has prevented us from utilizing the germ plasm by a map-based approach. Most genetic traits important to agriculture or human diseases are manifested as observable, quantitative phenotypes called Quantitative Trait Loci (QTL). In many instances, the complexity of the phenotype/genotype interaction and the general lack of clearly identifiable gene products render the direct molecular cloning approach ineffective, thus additional strategies like genome mapping are required to identify the QTL in question. Genome mapping requires no prior knowledge of the gene function, but utilizes statistical methods to identify the most likely gene location. To completely characterize genes of interest, the initially mapped region of a gene location will have to be narrowed down to a size that is suitable for cloning and sequencing. Strategies for gene identification within the critical region have to be applied after the sequencing of a potentially large clone or set of clones that contains this gene(s). Tremendous success of positional cloning has been shown for cloning many genes responsible for human diseases, including cystic fibrosis and muscular dystrophy as well as plant disease resistance genes. Genome and QTL mapping, positional cloning: the pre-genomics era, comparative approaches to gene identification, and positional cloning: the genomics era are discussed in the report. (M. Suetake)

A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

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Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

2016-04-01

The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

hebp3, a novel member of the heme-binding protein gene family, is expressed in the medaka meninges with higher abundance in females due to a direct stimulating action of ovarian estrogens.

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Nakasone, Kiyoshi; Nagahama, Yoshitaka; Okubo, Kataaki

2013-02-01

The brains of teleost fish exhibit remarkable sexual plasticity throughout their life span. To dissect the molecular basis for the development and reversal of sex differences in the teleost brain, we screened for genes differentially expressed between sexes in the brain of medaka (Oryzias latipes). One of the genes identified in the screen as being preferentially expressed in females was found to be a new member of the heme-binding protein gene family that includes hebp1 and hebp2 and was designated here as hebp3. The medaka hebp3 is expressed in the meninges with higher abundance in females, whereas there is no expression within the brain parenchyma. This female-biased expression of hebp3 is not attributable to the direct action of sex chromosome genes but results from the transient and reversible action of estrogens derived from the ovary. Moreover, estrogens directly activate the transcription of hebp3 via a palindromic estrogen-responsive element in the hebp3 promoter. Taken together, our findings demonstrate that hebp3 is a novel transcriptional target of estrogens, with female-biased expression in the meninges. The definite but reversible sexual dimorphism of the meningeal hebp3 expression may contribute to the development and reversal of sex differences in the teleost brain.

Assessment of the potential for gene flow from transgenic maize (Zea mays L.) to eastern gamagrass (Tripsacum dactyloides L.).

Science.gov (United States)

Lee, Moon-Sub; Anderson, Eric K; Stojšin, Duška; McPherson, Marc A; Baltazar, Baltazar; Horak, Michael J; de la Fuente, Juan Manuel; Wu, Kunsheng; Crowley, James H; Rayburn, A Lane; Lee, D K

2017-08-01

Eastern gamagrass (Tripsacum dactyloides L.) belongs to the same tribe of the Poaceae family as maize (Zea mays L.) and grows naturally in the same region where maize is commercially produced in the USA. Although no evidence exists of gene flow from maize to eastern gamagrass in nature, experimental crosses between the two species were produced using specific techniques. As part of environmental risk assessment, the possibility of transgene flow from maize to eastern gamagrass populations in nature was evaluated with the objectives: (1) to assess the seeds of eastern gamagrass populations naturally growing near commercial maize fields for the presence of a transgenic glyphosate-tolerance gene (cp4 epsps) that would indicate cross-pollination between the two species, and (2) to evaluate the possibility of interspecific hybridization between transgenic maize used as male parent and eastern gamagrass used as female parent. A total of 46,643 seeds from 54 eastern gamagrass populations collected in proximity of maize fields in Illinois, USA were planted in a field in 2014 and 2015. Emerged seedlings were treated with glyphosate herbicide and assessed for survival. An additional 48,000 seeds from the same 54 eastern gamagrass populations were tested for the presence of the cp4 epsps transgene markers using TaqMan ® PCR method. The results from these trials showed that no seedlings survived the herbicide treatment and no seed indicated presence of the herbicide tolerant cp4 epsps transgene, even though these eastern gamagrass populations were exposed to glyphosate-tolerant maize pollen for years. Furthermore, no interspecific hybrid seeds were produced from 135 hand-pollination attempts involving 1529 eastern gamagrass spikelets exposed to maize pollen. Together, these results indicate that there is no evidence of gene flow from maize to eastern gamagrass in natural habitats. The outcome of this study should be taken in consideration when assessing for environmental

An intronic polymorphism of IRF4 gene influences gene transcription in vitro and shows a risk association with childhood acute lymphoblastic leukemia in males.

Science.gov (United States)

Do, Thuy N; Ucisik-Akkaya, Esma; Davis, Charronne F; Morrison, Brittany A; Dorak, M Tevfik

2010-02-01

The interferon regulatory factor (IRF) family of DNA-binding proteins regulates expression of interferon-inducible genes with roles in the immune response and carcinogenesis. IRF4 is involved in the differentiation of B and T cells and is overexpressed in B-cell malignancies as a result of c-REL (NF-kappaB) hyperactivation. IRF4 polymorphisms are associated with susceptibility to chronic lymphoid leukemia (CLL) and non-Hodgkin lymphoma (NHL). We examined 13 IRF4 SNPs in 114 cases of childhood acute lymphoblastic leukemia (ALL) and 388 newborn controls from Wales (U.K.) using TaqMan assays. IRF4 intron 4 SNP rs12203592 showed a male-specific risk association (OR=4.4, 95% CI=1.5 to 12.6, P=0.007). Functional consequences of the C>T substitution at this SNP were assessed by cell-based reporter assays using three different cell lines. We found a repressive effect of the rs12203592 wildtype allele C on IRF4 promoter activity (Pcell line tested. Thus, homozygosity for the rs12203592 variant allele would result in increased IRF4 expression. This increase would be compounded by high levels of NF-kappaB activity in males due to the absence of estrogen. IRF4 differs from other IRFs in its anti-interferon activity which interferes with immune surveillance. We propose that a detailed study of IRF4 can provide information on the mechanism of the sex effect and the role of immune surveillance in childhood ALL development. Copyright 2009 Elsevier B.V. All rights reserved.

Estimating directional epistasis

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Le Rouzic, Arnaud

2014-01-01

Epistasis, i.e., the fact that gene effects depend on the genetic background, is a direct consequence of the complexity of genetic architectures. Despite this, most of the models used in evolutionary and quantitative genetics pay scant attention to genetic interactions. For instance, the traditional decomposition of genetic effects models epistasis as noise around the evolutionarily-relevant additive effects. Such an approach is only valid if it is assumed that there is no general pattern among interactions—a highly speculative scenario. Systematic interactions generate directional epistasis, which has major evolutionary consequences. In spite of its importance, directional epistasis is rarely measured or reported by quantitative geneticists, not only because its relevance is generally ignored, but also due to the lack of simple, operational, and accessible methods for its estimation. This paper describes conceptual and statistical tools that can be used to estimate directional epistasis from various kinds of data, including QTL mapping results, phenotype measurements in mutants, and artificial selection responses. As an illustration, I measured directional epistasis from a real-life example. I then discuss the interpretation of the estimates, showing how they can be used to draw meaningful biological inferences. PMID:25071828

Association of CFH and CFB Gene Polymorphisms with Retinopathy in Type 2 Diabetic Patients

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Jun Wang

2013-01-01

Full Text Available Objectives. The complement system is a key component of innate immunity and has been implicated in the pathogenesis of diabetic retinopathy (DR. This study aimed at investigating whether polymorphisms of two genes in the complement pathway, complement factor H (CFH and complement factor B (CFB, are associated with DR. Methods. 552 well-defined subjects with type 2 diabetes, consisting of 277 DR patients and 275 diabetic controls, were recruited. Four Tag-SNPs rs1048709, rs537160, rs4151657, and rs2072633 in CFB and rs800292 (I62V in CFH were examined using TaqMan Genotyping Assays. Results. There were significant increases in the frequencies of A allele and AA genotype for rs1048709 in DR patients compared with diabetic controls (Pcorr=0.035, OR=1.42; Pcorr=0.02, OR=2.27, resp.: meanwhile, significant decreases in the frequencies of A allele and AA genotype for rs800292 were observed in DR patients compared with diabetic controls (Pcorr=0.04, OR=0.72; Pcorr=0.015, OR=0.51, resp.. Joint effect of these two loci was also identified. Moreover, rs800292/AA genotype was found to be related with delayed progression to DR. Conclusions. CFH-rs800292 and CFB-rs1048709 are associated with the presence of DR, which strengthens the concept that complement system plays an important role in the pathogenesis of DR.

Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches.

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Fernandes, Telmo J R; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

2018-04-15

Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001-50%) than the EvaGreen (0.05-50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+

International Nuclear Information System (INIS)

Doloff, Joshua C; Su, Ting; Waxman, David J

2010-01-01

Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37). To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase. The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 'factory' cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing. The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products

Intratumor genetic heterogeneity of breast carcinomas as determined by fine needle aspiration and TaqMan low density array

DEFF Research Database (Denmark)

Lyng, Maria B.; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

2007-01-01

BACKGROUND: Gene expression profiling is thought to be an important tool in determining treatment strategies for breast cancer patients. Tissues for such analysis may at a preoperative stage be obtained, by fine needle aspiration (FNA) allowing initiation of neoadjuvant treatment. To evaluate the...... of breast carcinomas, as sampled by FNA, does not prohibit generation of useful gene profiles for treatment decision making. However, sampling and analysis strategies should take heterogeneity within a tumor, and varying heterogeneity amongst the single genes, into account...

Tracking microorganisms and gene in the environment

International Nuclear Information System (INIS)

Atlas, R.M.; Sayler, G.S.

1988-01-01

Studies have been conducted to determine the sensitivities and limitations of various methods for determining the fate of genetically engineered microorganisms (GEMs) and their genes in the environment. Selective viable plate count procedures can be designed to detect the introduced organisms with high sensitivity; but they are restricted by potential mutations affecting the expression of the selective characteristic in the introduced organism, the occurrence of the particular selective characteristic in the indigenous organisms, and the need to culture the organism. The accuracy of this approach is greatly improved by colony hybridization procedures that use a specific gene probe to detect the introduced genes, but this approach is still only as sensitive as the plating procedure. Direct extraction of DNA from environmental samples, coupled with dot blot hybridization with radiolabeled probe DNA or solution hybridization, gives a high degree of both sensitivity and precision. This approach does not require culturing of the organism; and even if an introduced gene moves into a new organism or if the introduced organism is viable but nonculturable, the gene probe methods will detect the persistence of the introduced genes in the environment. Efficient direct DNA extraction methods have been developed and tested following in vitro experimental additions of GEMs to sediment and water samples

Association of the STAT4 gene with increased susceptibility for some immune-mediated diseases.

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Martínez, A; Varadé, J; Márquez, A; Cénit, M C; Espino, L; Perdigones, N; Santiago, J L; Fernández-Arquero, M; de la Calle, H; Arroyo, R; Mendoza, J L; Fernández-Gutiérrez, B; de la Concha, E G; Urcelay, E

2008-09-01

The STAT4 gene encodes a transcription factor involved in the signaling pathways of several cytokines, including interleukin-12 (IL-12), the type I interferons, and IL-23. Recently, the association of a STAT4 haplotype marked by rs7574865 with rheumatoid arthritis (RA) and systemic lupus erythematosus was reported. The aim of this study was to investigate the role of this STAT4 tagging polymorphism in other immune-mediated diseases. The study group comprised 2,776 consecutively recruited Spanish individuals: 575 with RA, 440 with multiple sclerosis, 700 with inflammatory bowel disease, 311 with type 1 diabetes, and 723 ethnically matched healthy control subjects. The STAT4 polymorphism rs7574865 was genotyped using a predesigned TaqMan assay. Allele and genotype frequencies in patients and control subjects were compared by chi-square test. The association of STAT4 polymorphism rs7574865 with RA was validated in patients of Spanish origin (for T versus G, P = 1.2 x 10(-6), odds ratio [OR] 1.59, 95% confidence interval [95% CI] 1.31-1.92), and the association was described for the first time in both clinical forms of inflammatory bowel disease, Crohn's disease and ulcerative colitis (for T versus G, P = 0.006, OR 1.29, 95% CI 1.07-1.55), and in type 1 diabetes mellitus (for T versus G, P = 0.008, OR 1.36, 95% CI 1.07-1.71). In contrast, the genotypic distribution of this polymorphism showed no difference between patients with multiple sclerosis and healthy control subjects (for T versus G, P = 0.83, OR 1.02, 95% CI 0.82-1.28). The STAT4 gene is emerging as a novel common risk factor for diverse complex diseases.

Associations between interleukin and interleukin receptor gene polymorphisms and risk of gout

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Liu, Shiguo; Zhou, Zheng; Wang, Can; Guo, Mingzhen; Chu, Nan; Li, Changgui

2015-01-01

Gout is a self-limiting, auto-inflammatory arthritis induced by the deposition of monosodium urate crystals in the synovial fluid and periarticular tissues. The aim of this study was to investigate the associations between genetic variants in the interleukin (IL) and interleukin receptor (ILR) genes IL-33, IL-1RL1, IL-23R, and signal transducer and activator of transcription 4 (STAT4) and susceptibility to gout in Chinese Han male individuals. The genetic distributions of rs3939286 in IL-33, rs13015714 in IL-1RL1, rs10889677 in IL-23R, and rs7574865 in STAT4 were detected in 1100 men with gout and 1227 ethnically matched controls, using Taqman allelic discrimination real-time polymerase chain reaction (PCR). Differences in these polymorphisms between the groups were investigated using χ2 tests. The genotype-phenotype relationship among gout patients was tested by analysis of variance. There was a significant difference in genotypic frequencies of IL-23R rs10889677 between gout patients and controls (χ2 = 81.386, P < 0.001). However, there were no significant differences in distributions of the other polymorphisms between the groups. Our results revealed that the rs10889677 variant in IL-23R may be involved in the development of gout in Chinese Han male individuals. However, further studies in other ethnic groups are needed to confirm these results. PMID:26399911

STAT4 single nucleotide gene polymorphisms and susceptibility to endometriosis-related infertility.

Science.gov (United States)

Zamani, Mohammad Reza; Salmaninejad, Arash; Akbari Asbagh, Firouzeh; Masoud, Ahmad; Rezaei, Nima

2016-08-01

Endometriosis is a multifactorial benign gynecologic disorder, characterized by the ectopic growth of misplaced endometrial cells with complex genetic inheritance and changing of some immune based factors and also shares some autoimmune characteristics. However, it is not clear yet that how and when these immunological factors affect the initiation or progression of the disease. It has been shown that STAT4 is a predisposing gene in the development of some autoimmune diseases. The study group comprised 114 patients with endometriosis and 92 sex-, age-, and ethnicity-matched healthy controls of Iranian ancestry. Four SNPs (rs7574865, rs7601754, rs7582694 and rs11889341) were genotyped using the MGB TaqMan. A significant association in rs7582694 between C allele (P=0.002, OR=1.986, 95% CI: 1.262-3.126) and endometriosis was found in our study, while the G allele (P=0.002, OR=0.0503, 95% CI: 0.319-0.792) was significantly decreased in the patients population. The GC genotype (P=0.004, OR=2.234, 95% CI: 1.301-4.150) was also significantly overrepresented in the patients with endometriosis, while the frequency of GG genotype was significantly lower in the patient group, compared to the controls (P=0.007, OR=0.457, 95% CI: 0.256-0.813). Our results for the first time showed a significant association between rs7582694 alleles and genotypes and susceptibility to endometriosis in a population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

AAV Vectorization of DSB-mediated Gene Editing Technologies.

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Moser, Rachel J; Hirsch, Matthew L

2016-01-01

Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

Bioreactor Study Employing Bacteria with Enhanced Activity toward Cyanobacterial Toxins Microcystins

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Dariusz Dziga

2014-08-01

Full Text Available An important aim of white (grey biotechnology is bioremediation, where microbes are employed to remove unwanted chemicals. Microcystins (MCs and other cyanobacterial toxins are not industrial or agricultural pollutants; however, their occurrence as a consequence of human activity and water reservoir eutrophication is regarded as anthropogenic. Microbial degradation of microcystins is suggested as an alternative to chemical and physical methods of their elimination. This paper describes a possible technique of the practical application of the biodegradation process. The idea relies on the utilization of bacteria with a significantly enhanced MC-degradation ability (in comparison with wild strains. The cells of an Escherichia coli laboratory strain expressing microcystinase (MlrA responsible for the detoxification of MCs were immobilized in alginate beads. The degradation potency of the tested bioreactors was monitored by HPLC detection of linear microcystin LR (MC-LR as the MlrA degradation product. An open system based on a column filled with alginate-entrapped cells was shown to operate more efficiently than a closed system (alginate beads shaken in a glass container. The maximal degradation rate calculated per one liter of carrier was 219.9 µg h−1 of degraded MC-LR. A comparison of the efficiency of the described system with other biological and chemo-physical proposals suggests that this new idea presents several advantages and is worth investigating in future studies.

Small-scale Fisheries Governance and Understanding the Snoek (Thyrsites atun Supply Chain in the Ocean View Fishing Community, Western Cape, South Africa

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Moenieba Isaacs

2013-12-01

Full Text Available Postapartheid fisheries reform in South Africa, through the Marine Living Resources Act (MLRA 18 of 1998, used individual transferable quotas (ITQs to broaden resource access through allocating quotas to new entrants, even though the system has been created to reduce capacity through a reduction in the number of active fishers. The formal action space created through fisheries reform in South Africa left many artisanal fishers to operate in the informal action spaces, selling Thyrsites atun (snoek to poor communities to sustain their livelihoods. Artisanal fishers were not recognized by MLRA of 1998 and through class action case brought against the ITQ system, and in out of court settlement with the claimants in 2007, 1000 interim relief permits will be allocated to artisanal fishes and the development of a new small-scale fisheries policy for South Africa. In this case study of a fishing community in Ocean View, Cape Town I examine a snoek fishery that operates differently, through a community supply chain and informal markets, than that of the high value ITQ regulated species, yet plays a significant role in the livelihoods of artisanal fishers and in the food security of poor households. The findings of this case study show the failures of existing policy frameworks and the implications for the implementation of the new small-scale fisheries policy in South Africa.

Genetic variants in ATM, H2AFX and MRE11 genes and susceptibility to breast cancer in the polish population.

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Podralska, Marta; Ziółkowska-Suchanek, Iwona; Żurawek, Magdalena; Dzikiewicz-Krawczyk, Agnieszka; Słomski, Ryszard; Nowak, Jerzy; Stembalska, Agnieszka; Pesz, Karolina; Mosor, Maria

2018-04-20

DNA damage repair is a complex process, which can trigger the development of cancer if disturbed. In this study, we hypothesize a role of variants in the ATM, H2AFX and MRE11 genes in determining breast cancer (BC) susceptibility. We examined the whole sequence of the ATM kinase domain and estimated the frequency of founder mutations in the ATM gene (c.5932G > T, c.6095G > A, and c.7630-2A > C) and single nucleotide polymorphisms (SNPs) in H2AFX (rs643788, rs8551, rs7759, and rs2509049) and MRE11 (rs1061956 and rs2155209) among 315 breast cancer patients and 515 controls. The analysis was performed using high-resolution melting for new variants and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for recurrent ATM mutations. H2AFX and MRE11 polymorphisms were analyzed using TaqMan assays. The cumulative genetic risk scores (CGRS) were calculated using unweighted and weighted approaches. We identified four mutations (c.6067G > A, c.8314G > A, c.8187A > T, and c.6095G > A) in the ATM gene in three BC cases and two control subjects. We observed a statistically significant association of H2AFX variants with BC. Risk alleles (the G of rs7759 and the T of rs8551 and rs2509049) were observed more frequently in BC cases compared to the control group, with P values, odds ratios (OR) and 95% confidence intervals (CIs) of 0.0018, 1.47 (1.19 to 1.82); 0.018, 1.33 (1.09 to 1.64); and 0.024, 1.3 (1.06 to 1.59), respectively. Haplotype-based tests identified a significant association of the H2AFX CACT haplotype with BC (P ATM gene to the development of breast cancer needs further detailed study.

No Effect of the Transforming Growth Factor {beta}1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

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Reuther, Sebastian; Metzke, Elisabeth [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Bonin, Michael [Department of Medical Genetics, University of Tuebingen (Germany); Petersen, Cordula [Clinic of Radiotherapy and Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Dikomey, Ekkehard, E-mail: dikomey@uke.de [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Raabe, Annette [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany)

2013-02-01

Purpose: To study whether the promoter polymorphism (C-509T) affects transforming growth factor {beta}1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Methods and Materials: Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. Results: The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Conclusion: Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

The human oxytocin gene promoter is regulated by estrogens.

Science.gov (United States)

Richard, S; Zingg, H H

1990-04-15

Gonadal steroids affect brain function primarily by altering the expression of specific genes, yet the specific mechanisms by which neuronal target genes undergo such regulation are unknown. Recent evidence suggests that the expression of the neuropeptide gene for oxytocin (OT) is modulated by estrogens. We therefore examined the possibility that this regulation occurred via a direct interaction of the estrogen-receptor complex with cis-acting elements flanking the OT gene. DNA-mediated gene transfer experiments were performed using Neuro-2a neuroblastoma cells and chimeric plasmids containing portions of the human OT gene 5'-glanking region linked to the chloramphenicol acetyltransferase gene. We identified a 19-base pair region located at -164 to -146 upstream of the transcription start site which is capable of conferring estrogen responsiveness to the homologous as well as to a heterologous promoter. The hormonal response is strictly dependent on the presence of intracellular estrogen receptors, since estrogen induced stimulation occurred only in Neuro-2a cells co-transfected with an expression vector for the human estrogen receptor. The identified region contains a novel imperfect palindrome (GGTGACCTTGACC) with sequence similarity to other estrogen response elements (EREs). To define cis-acting elements that function in synergism with the ERE, sequences 3' to the ERE were deleted, including the CCAAT box, two additional motifs corresponding to the right half of the ERE palindrome (TGACC), as well as a CTGCTAA heptamer similar to the "elegans box" found in Caenorhabditis elegans. Interestingly, optimal function of the identified ERE was fully independent of these elements and only required a short promoter region (-49 to +36). Our studies define a molecular mechanism by which estrogens can directly modulate OT gene expression. However, only a subset of OT neurons are capable of binding estrogens, therefore, direct action of estrogens on the OT gene may be

Lack of direct evidence for natural selection at the candidate thrifty gene locus, PPARGC1A.

Science.gov (United States)

Cadzow, Murray; Merriman, Tony R; Boocock, James; Dalbeth, Nicola; Stamp, Lisa K; Black, Michael A; Visscher, Peter M; Wilcox, Phillip L

2016-11-15

The gene PPARGC1A, in particular the Gly482Ser variant (rs8192678), had been proposed to be subject to natural selection, particularly in recent progenitors of extant Polynesian populations. Reasons include high levels of population differentiation and increased frequencies of the derived type 2 diabetes (T2D) risk 482Ser allele, and association with body mass index (BMI) in a small Tongan population. However, no direct statistical tests for selection have been applied. Using a range of Polynesian populations (Tongan, Māori, Samoan) we re-examined evidence for association between Gly482Ser with T2D and BMI as well as gout. Using also Asian, European, and African 1000 Genome Project samples a range of statistical tests for selection (F ST , integrated haplotype score (iHS), cross population extended haplotype homozygosity (XP-EHH), Tajima's D and Fay and Wu's H) were conducted on the PPARGC1A locus. No statistically significant evidence for association between Gly482Ser and any of BMI, T2D or gout was found. Population differentiation (F ST ) was smallest between Asian and Pacific populations (New Zealand Māori ≤ 0.35, Samoan ≤ 0.20). When compared to European (New Zealand Māori ≤ 0.40, Samoan ≤ 0.25) or African populations (New Zealand Māori ≤ 0.80, Samoan ≤ 0.66) this differentiation was larger. We did not find any strong evidence for departure from neutral evolution at this locus when applying any of the other statistical tests for selection. However, using the same analytical methods, we found evidence for selection in specific populations at previously identified loci, indicating that lack of selection was the most likely explanation for the lack of evidence of selection in PPARGC1A. We conclude that there is no compelling evidence for selection at this locus, and that this gene should not be considered a candidate thrifty gene locus in Pacific populations. High levels of population differentiation at this locus and the

Recent Trends of Polymer Mediated Liposomal Gene Delivery System

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Shyamal Kumar Kundu

2014-01-01

Full Text Available Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole.

Association of RAC1 Gene Polymorphisms with Primary End-Stage Renal Disease in Chinese Renal Recipients.

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Yani Liu

Full Text Available RAC1 gene could influence susceptibility to renal failure by altering the activity and expression of Rac1, which is a member of the Rho family of small GTP-binding proteins. In clinical practice, renal transplantation provides the optimal treatment for people with end-stage renal disease (ESRD. The objective of this present study was to determine whether the RAC1 gene polymorphisms were associated with primary ESRD susceptibility in Chinese renal recipients.Six single nucleotide polymorphisms (SNPs of RAC1 gene, including rs836488 T>C, rs702482 A>T, rs10951982 G>A, rs702483 A>G, rs6954996 G>A, and rs9374 G>A, were genotyped in 300 renal transplant recipients (cases and 998 healthy Chinese subjects (controls by using TaqMan SNP genotyping assay. Allele, genotype, and haplotype frequencies of the six SNPs were compared between cases and controls. Odds ratios (OR and 95% confidence intervals (CI were calculated in logistic regression models to evaluate the associations of the six SNPs with ESRD risk.The genotype distributions for the six SNPs in controls were consistent with Hardy-Weinberg equilibrium (P > 0.05. Association analysis revealed that three SNPs were significantly associated with ESRD risk. Positive associations with ESRD risk were found for the rs836488, rs702482, and rs702483 in the co-dominant model (minor allele homozygotes versus major allele homozygotes; specifically, the frequencies of the minor allele homozygotes and the minor allele for the three SNPs were higher in the cases than in the controls. In addition, these three SNPs also had associations with increased ESRD risk under the additive model (P 0.05. In haplotype analysis, carriers with "C-T-G-G-G-G" haplotype had a significantly higher risk of ESRD compared with the most common haplotype "T-A-G-A-G-G" (P = 0.011, OR = 1.46, 95% CI = 1.09-1.94.This study suggested that polymorphisms of RAC1 gene might influence the susceptibility to ESRD in Chinese Han population. Further

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

International Nuclear Information System (INIS)

Mizoshiri, N.; Kishida, T.; Yamamoto, K.; Shirai, T.; Terauchi, R.; Tsuchida, S.; Mori, Y.; Ejima, A.; Sato, Y.; Arai, Y.; Fujiwara, H.; Yamamoto, T.; Kanamura, N.; Mazda, O.; Kubo, T.

2015-01-01

Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

2015-11-27

Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

A SCN9A gene-encoded dorsal root ganglia sodium channel polymorphism associated with severe fibromyalgia

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Vargas-Alarcon Gilberto

2012-02-01

Full Text Available Abstract Background A consistent line of investigation suggests that autonomic nervous system dysfunction may explain the multi-system features of fibromyalgia (FM; and that FM is a sympathetically maintained neuropathic pain syndrome. Dorsal root ganglia (DRG are key sympathetic-nociceptive short-circuit sites. Sodium channels located in DRG (particularly Nav1.7 act as molecular gatekeepers for pain detection. Nav1.7 is encoded in gene SCN9A of chromosome 2q24.3 and is predominantly expressed in the DRG pain-sensing neurons and sympathetic ganglia neurons. Several SCN9A sodium channelopathies have been recognized as the cause of rare painful dysautonomic syndromes such as paroxysmal extreme pain disorder and primary erythromelalgia. The aim of this study was to search for an association between fibromyalgia and several SCN9A sodium channels gene polymorphisms. Methods We studied 73 Mexican women suffering from FM and 48 age-matched women who considered themselves healthy. All participants filled out the Fibromyalgia Impact Questionnaire (FIQ. Genomic DNA from whole blood containing EDTA was extracted by standard techniques. The following SCN9A single-nucleotide polymorphisms (SNP were determined by 5' exonuclease TaqMan assays: rs4371369; rs4387806; rs4453709; rs4597545; rs6746030; rs6754031; rs7607967; rs12620053; rs12994338; and rs13017637. Results The frequency of the rs6754031 polymorphism was significantly different in both groups (P = 0.036 mostly due to an absence of the GG genotype in controls. Interestingly; patients with this rs6754031 GG genotype had higher FIQ scores (median = 80; percentile 25/75 = 69/88 than patients with the GT genotype (median = 63; percentile 25/75 = 58/73; P = 0.002 and the TT genotype (median = 71; percentile 25/75 = 64/77; P = 0.001. Conclusion In this ethnic group; a disabling form of FM is associated to a particular SCN9A sodium channel gene variant. These preliminary results raise the possibility that

Contribution of Myostatin gene polymorphisms to normal variation in lean mass, fat mass and peak BMD in Chinese male offspring

Institute of Scientific and Technical Information of China (English)

Hua YUE; Miao LI; Yu-juan LIU; Song-hua WU; Zhen-lin ZHANG; Jin-wei HE; Hao ZHANG; Chun WANG; Wei-wei HU; Jie-mei GU; Yao-hua KE; Wen-zhen FU; Yun-qiu HU

2012-01-01

Myostatin gene is a member of the transforming growth factor-β (TGF-β) family that negatively regulates skeletal muscle growth.Genetic polymorphisms in Myostatin were found to be associated with the peak bone mineral density (BMD) in Chinese women.The purpose of this study was to investigate whether Myostatin played a role in the normal variation in peak BMD,lean mass (LM),and fat mass (FM) of Chinese men.Methods:Four hundred male-offspring nuclear families of Chinese Han ethnic group were recruited.Anthropometric measurements,includingthe peak BMD,body LM and FM were measured using dual-energy X-ray absorptiometry (DXA).The single nucleotide polymorphisms (SNPs) studied were tag-SNPs selected by sequencing.Both rs2293284 and +2278G＞A were genotyped using TaqMan assay,and rs3791783 was genotyped with PCR-restriction fragment length polymorphism (RFLP) analysis.The associations of the SNPs with anthropometfic variations were analyzed using the quantitative transmission disequilibrium test (QTDT).Results:Using QTDT to detect within-family associations,neither single SNP nor haplotype was found to be associated with peak BMD at any bone site.However,rs3791783 was found to be significantly associated with fat mass of the trunk (P＜0.001).Moreover,for within-family associations,haplotypes AGG,AAA,and TGG were found to be significantly associated with the trunk fat mass (all P＜0.001).Conclusion:Our results suggest that genetic variation within Myostatin may play a role in regulating the variation in fat mass in Chinese males.Additionally,the Myostatin gene may be a candidate that determines body fat mass in Chinese men.

Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

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Yang Lin

2013-02-01

Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

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Zhang Li

2009-08-01

Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease.

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Shen, Jin-Song; Meng, Xing-Li; Yokoo, Takashi; Sakurai, Ken; Watabe, Kazuhiko; Ohashi, Toya; Eto, Yoshikatsu

2005-05-01

Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright (c) 2005 John Wiley & Sons, Ltd.

The Scope of Gene Patent Protection and the TRIPS Agreement

DEFF Research Database (Denmark)

Sommer, Tine

2007-01-01

The Scope of Gene Patent Protection and the TRIPS Agreement - An Exclusively Nondiscriminatory Approach?   Gene patenting in Europe has provoked much debate both before and since the adoption of Directive 98/44/EC on the legal protection of biotechnological inventions. Some of the major points...... of discussion have been focused on the scope of protection (e.g. purpose-bound protection) and gene patents being subject to a specific DNA regime on patent rights. The Directive can be interpreted as favouring such a solution, but so far the European Commission has decided neither to support nor reject...

Over-Expression of Dopamine D2 Receptor and Inwardly Rectifying Potassium Channel Genes in Drug-Naive Schizophrenic Peripheral Blood Lymphocytes as Potential Diagnostic Markers

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Ágnes Zvara

2005-01-01

Full Text Available Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3 was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2 and the inwardly rectifying potassium channel (Kir2.3 were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

Decoding Gene Patents in Australia

OpenAIRE

Denley, Adam; Cherry, James

2015-01-01

Patents directed to naturally occurring genetic material, such as DNA, RNA, chromosomes, and genes, in an isolated or purified form have been granted in Australia for many years. This review provides scientists with a summary of the gene patent debate from an Australian perspective and specifically reviews how the various levels of the legal system as they apply to patents—the Australian Patent Office, Australian courts, and Australian government—have dealt with the issue of whether genetic m...

A mechanistic explanation of popularity: genes, rule breaking, and evocative gene-environment correlations.

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Burt, Alexandra

2009-04-01

Previous work has suggested that the serotonergic system plays a key role in "popularity" or likeability. A polymorphism within the 5HT-sub(2A) serotonin receptor gene (-G1438A) has also been associated with popularity, suggesting that genes may predispose individuals to particular social experiences. However, because genes cannot code directly for others' reactions, any legitimate association should be mediated via the individual's behavior (i.e., genes-->behaviors-->social consequences), a phenomenon referred to as an evocative gene-environment correlation (rGE). The current study aimed to identify one such mediating behavior. The author focused on rule breaking given its prior links to both the serotonergic system and to increased popularity during adolescence. Two samples of previously unacquainted late-adolescent boys completed a peer-based interaction paradigm designed to assess their popularity. Analyses revealed that rule breaking partially mediated the genetic effect on popularity, thereby furthering our understanding of the biological mechanisms that underlie popularity. Moreover, the present results represent the first meaningfully explicated evidence that genes predispose individuals not only to particular behaviors but also to the social consequences of those behaviors. (c) 2009 APA, all rights reserved.

A De Novo Whole GCK Gene Deletion Not Detected by Gene Sequencing, in a Boy with Phenotypic GCK Insufficiency

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N. H. Birkebæk

2011-01-01

Full Text Available We report on a boy with diabetes mellitus and a phenotype indicating glucokinase (GCK insufficiency, but a normal GCK gene examination applying direct gene sequencing. The boy was referred for diabetes mellitus at 7.5 years old. His father, grandfather and great grandfather suffered type 2 DM. Several blood glucose profiles showed (BG of 6.5–10 mmol/L L. After three years on neutral insulin Hagedorn (NPH in a dose of 0.3 IU/kg/day haemoglobin A1c (HbA1c was 6.8%. Treatment was changed to sulphonylurea 750 mg a day, and after 4 years HbA1c was 7%. At that time a multiplex ligation-dependent amplification gene dosage assay (MLPA was done, revealing a whole GCK gene deletion. Medical treatment was ceased, and after one year HbA1c was 6.8%. This case underscores the importance of a MLPA examination if the phenotype of a patient is strongly indicative of GCK insufficiency and no mutation is identified using direct sequencing.

Mi-1.2, an R gene for aphid resistance in tomato, has direct negative effects on a zoophytophagous biocontrol agent, Orius insidiosus.

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Pallipparambil, Godshen R; Sayler, Ronald J; Shapiro, Jeffrey P; Thomas, Jean M G; Kring, Timothy J; Goggin, Fiona L

2015-02-01

Mi-1.2 is a single dominant gene in tomato that confers race-specific resistance against certain phloem-feeding herbivores including aphids, whiteflies, psyllids, and root-knot nematodes. Few prior studies have considered the potential non-target effects of race-specific resistance genes (R genes), and this paper evaluates the compatibility of Mi-mediated resistance in tomato with a beneficial zoophytophagous predator, Orius insidiosus (Say). In addition to preying on aphids and other pests, this piercing-sucking insect also feeds from the xylem, epidermis, and/or mesophyll, and oviposits within plant tissues. Comparison of O. insidiosus confined to isogenic tomato plants with and without Mi-1.2 revealed that immatures of O. insidiosus had lower survival on resistant plants even when the immatures were provisioned with prey that did not feed on the host plant. Molecular gut content analysis confirmed that adults and immatures of O. insidiosus feed on both resistant (Mi-1.2+) and susceptible (Mi-1.2-) genotypes, and bioassays suggest that resistance does not affect oviposition rates, plant sampling, or prey acceptance by O. insidiosus adults. These results demonstrate a direct negative impact of R-gene-mediated host plant resistance on a non-target beneficial species, and reveal that Mi-mediated resistance can impact organisms that do not feed on phloem sap. Through laser capture microdissection and RT-PCR, Mi-1.2 transcripts were detected in the epidermis and mesophyll as well as the phloem of tomato plants, consistent with our observations that Mi-mediated resistance is active outside the phloem. These results suggest that the mode of action and potential ecological impacts of Mi-mediated resistance are broader than previously assumed. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

T helper-2 cytokine/regulatory T-cell gene polymorphisms and their relation with risk of psoriasis in a South Indian Tamil cohort.

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Indhumathi, Sundar; Rajappa, Medha; Chandrashekar, Laxmisha; Ananthanarayanan, Palghat Hariharan; Thappa, Devinder Mohan; Negi, Vir Singh

2017-02-01

Psoriasis is known to be associated with an up-regulation of T-helper (Th)-1 & Th-17 cytokines and a relative down-regulation of Th-2 and T-regulatory (T-reg) cytokines. Certain allelic variants of these cytokine genes may alter Th1/Th17 and Th2/T-reg balance and may be associated with the risk of psoriasis. Hence we aimed to determine the association of IL-4 (rs2243250), IL-10 (rs1800871 and rs1800896) and FOXP3 (rs3761548) gene polymorphisms with risk of psoriasis in South Indian Tamils. A total of 360 cases of psoriasis and 360 healthy controls were recruited. The polymorphism in IL-4 (rs2243250) & IL-10 (rs1800871) were typed by ARMS-PCR and IL-10 (rs1800896) & FOXP3 (rs3761548) were typed by TaqMan 5'allele discrimination assay. We observed that IL-4 (rs2243250) had a reduced risk of psoriasis, while the IL-10 (rs1800871) conferred an increased susceptibility to psoriasis, as compared with controls. However, IL-10 (rs1800896) and FOXP3 (rs3761548) gene polymorphisms were not associated with psoriasis risk. The plasma IL-4 levels was not different between the cases and controls, however the heterozygous CT genotype demonstrated significant high IL-4 levels. Plasma IL-10 levels were significantly increased in cases compared to controls, however none of the genotypes were associated with the plasma IL-10 levels. Our results suggest that IL-4 (rs2243250) polymorphism is protective against psoriasis, while IL-10 (rs1800871) polymorphism confers increased risk of psoriasis in South Indian Tamils. Detection of these genetic variants as predictive risk factors may lead to the selection of patient-tailored therapy to maximize the effectiveness of therapy. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Staphylococcus aureus Isolates Carrying Panton-Valentine Leucocidin Genes: Their Frequency, Antimicrobial Patterns, and Association With Infectious Disease in Shahrekord City, Southwest Iran.

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Shariati, Laleh; Validi, Majid; Hasheminia, Ali Mohammad; Ghasemikhah, Reza; Kianpour, Fariborz; Karimi, Ali; Nafisi, Mohammad Reza; Tabatabaiefar, Mohammad Amin

2016-01-01

A diversity of virulence factors work together to create the pathogenicity of Staphylococcus aureus. These factors include cell surface components that promote adherence to surfaces as well as exoproteins such as Panton-Valentine leukocidin (PVL), encoded by the luk-PV genes, that invade or bypass the immune system and are toxic to the host, thereby enhancing the severity of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to determine the frequency of PVL-positive MRSA strains by real-time PCR and their antibiotic susceptibility patterns by phenotypic test. In total, 284 Staphylococcus isolates, identified by phenotypic methods from clinical samples of Shahrekord University Hospitals, Shahrekord, Iran, were tested for nuc, mecA, and PVL genes by TaqMan real-time PCR. The antibiotic susceptibility patterns of PVL-containing MRSA strains were determined via the disk diffusion method. In total, 196 isolates (69%) were nuc positive (i.e., S. aureus); of those isolates, 96 (49%) were mecA positive (MRSA). Eighteen (18.8%) of the 96 MRSA positive and 3 (3%) of the 100 methicillin-susceptible Staphylococcus aureus (MSSA) strains were PVL positive. PVL-positive MRSA strains were mostly recovered from tracheal specimens. Eight PVL-positive MRSA strains were resistant to all the tested antibiotics except vancomycin. A significant correlation (P = 0.001) was found between the mecA positivity and the presence of luk-PV genes. Community acquired (CA)-MRSA is becoming a public health concern in many parts of the world, including Asian countries. The variable prevalence of luk-PV-positive MRSA isolates in different regions and their rather high frequency in pneumonia necessitate the application of rapid diagnostic methods such as real-time PCR to improve treatment effectiveness.

[Association of T174M polymorphism of the angiotensinogen gene with the higher risk of cerebral stroke in women].

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Stetskaia, T A; Bushueva, O Iu; Bulgakova, I V; Vialykh, E K; Shuteeva, T V; Biriukov, A E; Ivanov, V P; Polonikov, A V

2014-01-01

To study the association of M235T (rs699) and T174M (rs4762) polymorphisms of the angiotensinogen (AGT) gene with the risk of cerebral stroke (CS) in the Russians of the Central Chernozem Region. A total of 638 DNA samples obtained from 353 patients with CS and 285 sex- and age-matched healthy individuals were examined. The polymorphisms were genotyped by polymerase chain reaction (T174M) and TaqMan allelic discrimination (M235T) assays. Heterozygous AGT 174TM genotype carriers were found to be at a higher risk for CS (odd ratio (OR) = 1.52; 95% confidence interval (CI), 1.08-2.15; p = 0.02). A gender-stratified analysis showed that the mutant 174M allele (OR = 1.86; 95% CI, 1.14-3.03, p = 0.01) and variant 174TM and 174MM genotypes (OR = 1.86; 95% CI, 1.09-3.20; p = 0.02) were associated with the higher risk of cerebral stroke in women. The association of AGT T174M polymorphism with the risk of CS was first found; but the higher risk of the disease in the carriers of variant alleles and genotypes was observed in the women only.

Genome-wide identification of KANADI1 target genes.

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Paz Merelo

Full Text Available Plant organ development and polarity establishment is mediated by the action of several transcription factors. Among these, the KANADI (KAN subclade of the GARP protein family plays important roles in polarity-associated processes during embryo, shoot and root patterning. In this study, we have identified a set of potential direct target genes of KAN1 through a combination of chromatin immunoprecipitation/DNA sequencing (ChIP-Seq and genome-wide transcriptional profiling using tiling arrays. Target genes are over-represented for genes involved in the regulation of organ development as well as in the response to auxin. KAN1 affects directly the expression of several genes previously shown to be important in the establishment of polarity during lateral organ and vascular tissue development. We also show that KAN1 controls through its target genes auxin effects on organ development at different levels: transport and its regulation, and signaling. In addition, KAN1 regulates genes involved in the response to abscisic acid, jasmonic acid, brassinosteroids, ethylene, cytokinins and gibberellins. The role of KAN1 in organ polarity is antagonized by HD-ZIPIII transcription factors, including REVOLUTA (REV. A comparison of their target genes reveals that the REV/KAN1 module acts in organ patterning through opposite regulation of shared targets. Evidence of mutual repression between closely related family members is also shown.

Species-independent MicroRNA Gene Discovery

KAUST Repository

Kamanu, Timothy K.

2012-12-01

MicroRNA (miRNA) are a class of small endogenous non-coding RNA that are mainly negative transcriptional and post-transcriptional regulators in both plants and animals. Recent studies have shown that miRNA are involved in different types of cancer and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. miRNA genes are classified into different groups (miRNA families). This study reports about 19,000 unknown miRNA genes in nine species whereby approximately 15,300 predictions were computationally validated to contain at least one experimentally verified functional miRNA product. The predictions are based on a novel computational strategy which relies on miRNA family groupings and exploits the physics and geometry of miRNA genes to unveil the hidden palindromic signals and symmetries in miRNA gene sequences. Unlike conventional computational miRNA gene discovery methods, the algorithm developed here is species-independent: it allows prediction at higher accuracy and resolution from arbitrary RNA/DNA sequences in any species and thus enables examination of repeat-prone genomic regions which are thought to be non-informative or ’junk’ sequences. The information non-redundancy of uni-directional RNA sequences compared to information redundancy of bi-directional DNA is demonstrated, a fact that is overlooked by most pattern discovery algorithms. A novel method for computing upstream and downstream miRNA gene boundaries based on mathematical/statistical functions is suggested, as well as cutoffs for annotation of miRNA genes in different miRNA families. Another tool is proposed to allow hypotheses generation and visualization of data matrices, intra- and inter-species chromosomal distribution of miRNA genes or miRNA families. Our results indicate that: miRNA and mi

Terapia gênica Gene therapy

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Nance Beyer Nardi

2002-01-01

Full Text Available Terapia gênica é um procedimento médico que envolve a modificação genética de células como forma de tratar doenças. Os genes influenciam praticamente todas as doenças humanas, seja pela codificação de proteínas anormais diretamente responsáveis pela doença, seja por determinar suscetibilidade a agentes ambientais que a induzem. A terapia gênica é ainda experimental, e está sendo estudada em protocolos clínicos para diferentes tipos de doenças. O desenvolvimento de métodos seguros e eficientes de transferência gênica para células humanas é um dos pontos mais importantes na terapia gênica. Apesar do grande esforço dirigido na última década para o aperfeiçoamento dos protocolos de terapia gênica humana, e dos avanços importantes na pesquisa básica, as aplicações terapêuticas da tecnologia de transferência gênica continuam ainda em grande parte teóricas. O potencial da terapia gênica é muito grande, devendo ainda causar grande impacto em todos os aspectos da medicina.Gene therapy is a medical intervention that involves modifying the genetic material of living cells to fight disease. Genes influence virtually every human disease, either by encoding for abnormal proteins, which are directly responsible for the disease, or by causing a susceptibility to environmental agents which induce it. Gene therapy is still experimental, and is being studied in clinical trials for many different types of diseases. The development of safe and effective methods of implanting normal genes into the human cell is one of the most important technical issues in gene therapy. Although much effort has been directed in the last decade toward improvement of protocols in human gene therapy, and in spite of many considerable achievements in basic research, the therapeutic applications of gene transfer technology still remain mostly theoretical. The potential for gene therapy is huge and likely to impact on all aspects of medicine.

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

Science.gov (United States)

Entcheva, P; Liebl, W; Johann, A; Hartsch, T; Streit, W R

2001-01-01

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes

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2010-01-01

Background Horizontal gene transfer (HGT) is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants. Results In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR) survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR) were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT)-PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes. Conclusions This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native mitochondrial copies suggests

Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes

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Hao Weilong

2010-12-01

Full Text Available Abstract Background Horizontal gene transfer (HGT is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants. Results In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT-PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes. Conclusions This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native

Consensus strategy in genes prioritization and combined bioinformatics analysis for preeclampsia pathogenesis.

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Tejera, Eduardo; Cruz-Monteagudo, Maykel; Burgos, Germán; Sánchez, María-Eugenia; Sánchez-Rodríguez, Aminael; Pérez-Castillo, Yunierkis; Borges, Fernanda; Cordeiro, Maria Natália Dias Soeiro; Paz-Y-Miño, César; Rebelo, Irene

2017-08-08

Preeclampsia is a multifactorial disease with unknown pathogenesis. Even when recent studies explored this disease using several bioinformatics tools, the main objective was not directed to pathogenesis. Additionally, consensus prioritization was proved to be highly efficient in the recognition of genes-disease association. However, not information is available about the consensus ability to early recognize genes directly involved in pathogenesis. Therefore our aim in this study is to apply several theoretical approaches to explore preeclampsia; specifically those genes directly involved in the pathogenesis. We firstly evaluated the consensus between 12 prioritization strategies to early recognize pathogenic genes related to preeclampsia. A communality analysis in the protein-protein interaction network of previously selected genes was done including further enrichment analysis. The enrichment analysis includes metabolic pathways as well as gene ontology. Microarray data was also collected and used in order to confirm our results or as a strategy to weight the previously enriched pathways. The consensus prioritized gene list was rationally filtered to 476 genes using several criteria. The communality analysis showed an enrichment of communities connected with VEGF-signaling pathway. This pathway is also enriched considering the microarray data. Our result point to VEGF, FLT1 and KDR as relevant pathogenic genes, as well as those connected with NO metabolism. Our results revealed that consensus strategy improve the detection and initial enrichment of pathogenic genes, at least in preeclampsia condition. Moreover the combination of the first percent of the prioritized genes with protein-protein interaction network followed by communality analysis reduces the gene space. This approach actually identifies well known genes related with pathogenesis. However, genes like HSP90, PAK2, CD247 and others included in the first 1% of the prioritized list need to be further

Do GnRH analogues directly affect human endometrial epithelial cell gene expression?

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Zhang, Xiaomei; Bocca, Silvina Maria; Franchi, Anahí ; Anderson, Sandra; Kaur, Mandeep; Bajic, Vladimir B.; Oehninger, Sergio Carlos

2010-01-01

were: (i) to study the modulatory effect of GnRH analogues by RT-PCR [in the absence and presence of E2 and P4, and cyclic adenosine monophos-phate (cAMP)] on mRNA expression of genes modulated during the window of implantation in GnRH analogues

Polymorphisms of the GR and HSD11B1 genes influence body mass index and weight gain during hormone replacement treatment in patients with Addison's disease.

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Molnár, Ágnes; Kövesdi, Annamária; Szücs, Nikolette; Tóth, Miklós; Igaz, Péter; Rácz, Károly; Patócs, Attila

2016-08-01

Glucocorticoid substitution is essential in patients with chronic primary adrenocortical insufficiency (Addison's disease) and both over-treatment and inadequate dosage have deleterious effects. Individual sensitivity to glucocorticoids is partly genetically determined. To test the hypothesis whether the well-characterized SNPs of the GR and HSD11B1 genes may modulate the individual sensitivity to exogenous glucocorticoids and may influence clinical and/or laboratory parameters and the glucocorticoid substitution dosage in patients with Addison's disease. 68 patients with primary adrenocortical insufficiency were involved. Clinical and laboratory data, as well as the dosage of the hormone replacement therapy were collected. Peripheral blood DNA was isolated, and the GR and HSD11B1 SNPs were examined using allele-specific PCR or Taqman assay on Real Time PCR. The allele frequency of the GR N363S polymorphism was higher in patients compared to the control group and the disease appeared significantly earlier in patients harbouring the GR A3669G compared to noncarriers. These patients had higher ACTH level measured at the time of diagnosis. Homozygous BclI carriers had higher body mass index (BMI) and lower total hydrocortisone equivalent supplementation dose needed than heterozygous or noncarriers. The BMI and weight gain during hormone replacement therapy were also higher in carriers of the HSD11B1 rs4844880 treated with glucocorticoids other than dexamethasone. The BclI polymorphism of the GR gene and the rs4844880 of the HSD11B1 gene may contribute to weight gain and may affect the individual need of glucocorticoid substitution dose in these patients. © 2016 John Wiley & Sons Ltd.

Regulating Hypothalamus Gene Expression in Food Intake: Dietary Composition or Calorie Density?

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Mi Jang

2017-01-01

Full Text Available BackgroundThe proportion of saturated fatty acids/unsaturated fatty acids in the diet seems to act as a physiological regulation on obesity, cardiovascular diseases, and diabetes. Differently composed fatty acid diets may induce satiety of the hypothalamus in different ways. However, the direct effect of the different fatty acid diets on satiety in the hypothalamus is not clear.MethodsThree experiments in mice were conducted to determine whether: different compositions of fatty acids affects gene mRNA expression of the hypothalamus over time; different types of fatty acids administered into the stomach directly affect gene mRNA expression of the hypothalamus; and fat composition changes in the diet affects gene mRNA expression of the hypothalamus.ResultsThe type of fat in cases of purified fatty acid administration directly into the stomach may cause changes of gene expressions in the hypothalamus. Gene expression by dietary fat may be regulated by calorie amount ingested rather than weight amount or type of fat.ConclusionTherefore, the calorie density factor of the diet in regulating hypothalamic gene in food intake may be detrimental, although the possibility of type of fat cannot be ruled out.

Characterisation of genome-wide PLZF/RARA target genes.

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Salvatore Spicuglia

Full Text Available The PLZF/RARA fusion protein generated by the t(11;17(q23;q21 translocation in acute promyelocytic leukaemia (APL is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear.We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.

The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

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Alberto Elías-Villalobos

2015-08-01

Full Text Available Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

Difference between age-related macular degeneration and polypoidal choroidal vasculopathy in the hereditary contribution of the A69S variant of the age-related maculopathy susceptibility 2 gene (ARMS2).

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Yanagisawa, Suiho; Kondo, Naoshi; Miki, Akiko; Matsumiya, Wataru; Kusuhara, Sentaro; Tsukahara, Yasutomo; Honda, Shigeru; Negi, Akira

2011-01-01

To investigate whether the A69S variant of the age-related maculopathy susceptibility 2 gene (ARMS2) has a different hereditary contribution in neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). We initially conducted a comparative genetic analysis of neovascular AMD and PCV, genotyping the ARMS2 A69S variant in 181 subjects with neovascular AMD, 198 subjects with PCV, and 203 controls in a Japanese population. Genotyping was conducted using TaqMan technology. Results were then integrated into a meta-analysis of previous studies representing an assessment of the association between the ARMS2 A69S variant and neovascular AMD and/or PCV, comprising a total of 3,828 subjects of Asian descent. The Q-statistic test was used to assess between-study heterogeneity. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using a fixed effects model. The genetic effect of the A69S variant was stronger in neovascular AMD (allelic summary OR=3.09 [95% CI, 2.71-3.51], fixed effects parchitecture of this phenotypically heterogeneous disorder.

Theranostic Imaging of Cancer Gene Therapy.

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Sekar, Thillai V; Paulmurugan, Ramasamy

2016-01-01

Gene-directed enzyme prodrug therapy (GDEPT) is a promising therapeutic approach for treating cancers of various phenotypes. This strategy is independent of various other chemotherapeutic drugs used for treating cancers where the drugs are mainly designed to target endogenous cellular mechanisms, which are different in various cancer subtypes. In GDEPT an external enzyme, which is different from the cellular proteins, is expressed to convert the injected prodrug in to a toxic metabolite, that normally kill cancer cells express this protein. Theranostic imaging is an approach used to directly monitor the expression of these gene therapy enzymes while evaluating therapeutic effect. We recently developed a dual-GDEPT system where we combined mutant human herpes simplex thymidine kinase (HSV1sr39TK) and E. coli nitroreductase (NTR) enzyme, to improve therapeutic efficiency of cancer gene therapy by simultaneously injecting two prodrugs at a lower dose. In this approach we use two different prodrugs such as ganciclovir (GCV) and CB1954 to target two different cellular mechanisms to kill cancer cells. The developed dual GDEPT system was highly efficacious than that of either of the system used independently. In this chapter, we describe the complete protocol involved for in vitro and in vivo imaging of therapeutic cancer gene therapy evaluation.

Interactions between urinary 4-tert-octylphenol levels and metabolism enzyme gene variants on idiopathic male infertility.

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Yufeng Qin

Full Text Available Octylphenol (OP and Trichlorophenol (TCP act as endocrine disruptors and have effects on male reproductive function. We studied the interactions between 4-tert-Octylphenol (4-t-OP, 4-n- Octylphenol (4-n-OP, 2,3,4-Trichlorophenol (2,3,4-TCP, 2,4,5-Trichlorophenol (2,4,5-TCP urinary exposure levels and polymorphisms in selected xenobiotic metabolism enzyme genes among 589 idiopathic male infertile patients and 396 controls in a Han-Chinese population. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS was used to measure alkylphenols and chlorophenols in urine. Polymorphisms were genotyped using the SNPstream platform and the Taqman method. Among four phenols that were detected, we found that only exposure to 4-t-OP increased the risk of male infertility (P(trend = 1.70×10(-7. The strongest interaction was between 4-t-OP and rs4918758 in CYP2C9 (P(inter = 6.05×10(-7. It presented a significant monotonic increase in risk estimates for male infertility with increasing 4-t-OP exposure levels among men with TC/CC genotype (low level compared with non-exposed, odds ratio (OR = 2.26, 95% confidence intervals (CI = 1.06, 4.83; high level compared with non-exposed, OR = 9.22, 95% CI = 2.78, 30.59, but no associations observed among men with TT genotype. We also found interactions between 4-t-OP and rs4986894 in CYP2C19, and between rs1048943 in CYP1A1, on male infertile risk (P(inter = 8.09×10(-7, P(inter = 3.73×10(-4, respectively.We observed notable interactions between 4-t-OP exposure and metabolism enzyme gene polymorphisms on idiopathic infertility in Han-Chinese men.

Single nucleotide polymorphisms of the angiotensin-converting enzyme (ACE) gene are associated with essential hypertension and increased ACE enzyme levels in Mexican individuals.

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Martínez-Rodríguez, Nancy; Posadas-Romero, Carlos; Villarreal-Molina, Teresa; Vallejo, Maite; Del-Valle-Mondragón, Leonardo; Ramírez-Bello, Julian; Valladares, Adan; Cruz-López, Miguel; Vargas-Alarcón, Gilberto

2013-01-01

To explore the role of the ACE gene polymorphisms in the risk of essential hypertension in Mexican Mestizo individuals and evaluate the correlation between these polymorphisms and the serum ACE levels. Nine ACE gene polymorphisms were genotyped by 5' exonuclease TaqMan genotyping assays and polymerase chain reaction (PCR) in 239 hypertensive and 371 non- hypertensive Mexican individuals. Haplotypes were constructed after linkage disequilibrium analysis. ACE serum levels were determined in selected individuals according to different haplotypes. Under a dominant model, rs4291 rs4335, rs4344, rs4353, rs4362, and rs4363 polymorphisms were associated with an increased risk of hypertension after adjusting for age, gender, BMI, triglycerides, alcohol consumption, and smoking. Five polymorphisms (rs4335, rs4344, rs4353, rs4362 and rs4363) were in strong linkage disequilibrium and were included in four haplotypes: H1 (AAGCA), H2 (GGATG), H3 (AGATG), and H4 (AGACA). Haplotype H1 was associated with decreased risk of hypertension, while haplotype H2 was associated with an increased risk of hypertension (OR = 0.77, P = 0.023 and OR = 1.41, P = 0.004 respectively). According to the codominant model, the H2/H2 and H1/H2 haplotype combinations were significantly associated with risk of hypertension after adjusted by age, gender, BMI, triglycerides, alcohol consumption, and smoking (OR = 2.0; P = 0.002 and OR = 2.09; P = 0.011, respectively). Significant elevations in serum ACE concentrations were found in individuals with the H2 haplotype (H2/H2 and H2/H1) as compared to H1/H1 individuals (P = 0.0048). The results suggest that single nucleotide polymorphisms and the "GGATG" haplotype of the ACE gene are associated with the development of hypertension and with increased ACE enzyme levels.

Text mining in cancer gene and pathway prioritization.

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Luo, Yuan; Riedlinger, Gregory; Szolovits, Peter

2014-01-01

Prioritization of cancer implicated genes has received growing attention as an effective way to reduce wet lab cost by computational analysis that ranks candidate genes according to the likelihood that experimental verifications will succeed. A multitude of gene prioritization tools have been developed, each integrating different data sources covering gene sequences, differential expressions, function annotations, gene regulations, protein domains, protein interactions, and pathways. This review places existing gene prioritization tools against the backdrop of an integrative Omic hierarchy view toward cancer and focuses on the analysis of their text mining components. We explain the relatively slow progress of text mining in gene prioritization, identify several challenges to current text mining methods, and highlight a few directions where more effective text mining algorithms may improve the overall prioritization task and where prioritizing the pathways may be more desirable than prioritizing only genes.

Genes essential for phototrophic growth by a purple alphaproteobacterium: Genes for phototrophic growth

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Yang, Jianming [Key Lab of Applied Mycology, College of Life Sciences, Qingdao Agricultural University, Qingdao Shandong Province People' s Republic of China; Department of Microbiology, University of Washington, Seattle WA USA; Yin, Liang [Department of Microbiology, University of Washington, Seattle WA USA; Lessner, Faith H. [Department of Biological Sciences, University of Arkansas, Fayetteville AR USA; Nakayasu, Ernesto S. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Payne, Samuel H. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Fixen, Kathryn R. [Department of Microbiology, University of Washington, Seattle WA USA; Gallagher, Larry [Department of Genome Sciences, University of Washington, Seattle WA USA; Harwood, Caroline S. [Department of Microbiology, University of Washington, Seattle WA USA

2017-07-24

Anoxygenic purple phototrophic bacteria have served as important models for studies of photophosphorylation. The pigment-protein complexes responsible for converting light energy to ATP are relatively simple and these bacteria can grow heterotrophically under aerobic conditions, thus allowing for the study of mutants defective in photophosphorylation. In the past, genes responsible for anoxygenic phototrophic growth have been identified in a number of different bacterial species. Here we systematically studied the genetic basis for this metabolism by using Tn-seq to identify genes essential for the anaerobic growth of the purple bacterium Rhodopseudomonas palustris on acetate in light. We identified 171 genes required for growth in this condition, 35 of which are annotated as photosynthesis genes. Among these are a few new genes not previously shown to be essential for phototrophic growth. We verified the essentiality of many of the genes we identified by analyzing the phenotypes of mutants we generated by Tn mutagenesis that had altered pigmentation. We used directed mutagenesis to verify that the R. palustris NADH:quinone oxidoreductase complex IE is essential for phototrophic growth. As a complement to the genetic data, we carried out proteomics experiments in which we found that 429 proteins were present in significantly higher amounts in cells grown anaerobically in light compared to aerobically. Among these were proteins encoded by subset of the phototrophic growth-essential genes.